Accepted Manuscript

Circulating and Tissue-resident CD4+ T Cells With Reactivity to Intestinal

Microbiota Are Abundant in Healthy Individuals and Function is Altered During
Inflammation

Ahmed N. Hegazy, Nathaniel R. West, Michael J.T. Stubbington, Emily Wendt, Kim
I.M. Suijker, Angeliki Datsi, Sebastien This, Camille Danne, Suzanne Campion,
Sylvia H. Duncan, Benjamin M.J. Owens, Holm H. Uhlig, Andrew McMichael,
Andreas Bergthaler, Sarah A. Teichmann, Satish Keshav, Fiona Powrie

PII: S0016-5085(17)35979-6
DOI: 10.1053/j.gastro.2017.07.047
Reference: YGAST 61339

To appear in: Gastroenterology

Accepted Date: 25 July 2017

Please cite this article as: Hegazy AN, West NR, Stubbington MJT, Wendt E, Suijker KIM, Datsi A,
This S, Danne C, Campion S, Duncan SH, Owens BMJ, Uhlig HH, McMichael A, Oxford IBD Cohort
Investigators, 
 Bergthaler A, Teichmann SA, Keshav S, Powrie F, Circulating and Tissue-resident CD4+
T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function is
Altered During Inflammation, Gastroenterology (2017), doi: 10.1053/j.gastro.2017.07.047.

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ACCEPTED MANUSCRIPT

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 ACCEPTED MANUSCRIPT

Manuscript Number: GASTRO-D-16-01275R1

Title: Circulating and Tissue-resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are
Abundant in Healthy Individuals and Function is Altered During Inflammation

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Authors: Ahmed N. Hegazy1,2,10, Nathaniel R. West1,2,10, Michael J. T. Stubbington3,4, Emily

Wendt1, Kim I. M. Suijker2, Angeliki Datsi1, Sebastien This1, Camille Danne2, Suzanne Campion5,

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Sylvia H. Duncan6, Benjamin M. J. Owens1, Holm H. Uhlig1,7, Andrew McMichael5, Oxford IBD

SC
Cohort Investigators8, Andreas Bergthaler9, Sarah A. Teichmann3,4, Satish Keshav1, Fiona

Powrie1,2

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Affiliations:
1
AN
Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, Experimental
M
Medicine Division, John Radcliffe Hospital, University of Oxford, UK
2
Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and
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Musculoskeletal Sciences, University of Oxford, UK

TE

3
European Molecular Biology Laboratory-European Bioinformatics Institute, Hinxton, UK
4
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
EP

5
Nuffield Department of Medicine Research Building, University of Oxford, Oxford, UK
6
Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, UK
C

7
Department of Paediatrics, University of Oxford, Oxford, UK
AC

8
Individual investigators are listed in the acknowledgement section
9
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna,

Austria
10
Co-first authors

Grant support: ANH was supported by an EMBO long-term fellowship and a Marie Curie

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 ACCEPTED MANUSCRIPT

fellowship. NRW was supported by a CRI Irvington Post-doctoral Fellowship. BMJO was

supported by an Oxford-UCB Pharma Postdoctoral Fellowship. MJTS and SAT were supported by

ERC grant ThSWITCH and ThDEFINE (260507). SC and AM were supported by the Center for

HIV/AIDS Vaccine Immunology and Immunogen Discovery (grant UM1-AI100645). The Rowett

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Institute of Nutrition and Health receives financial support from the Scottish Government Rural and

Environmental Sciences and Analytical Services (SG-RESAS). Foundation Louis Jeantet,

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Wellcome Trust (Investigator award 095688/Z/11/Z), and ERC (ERC/HN/2013/21) supported FP

SC
and this project. HHU is supported by the Crohn’s & Colitis Foundation of America (CCFA), The

Leona M. and Harry B. Helmsley Charitable Trust. SD receive financial support from the Scottish

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Government Rural and Environmental Sciences and Analytical Services (RESAS).

AN
Abbreviations: Antigen presenting cells (APCs), Brefeldin A (BFA), Carboxyfluorescein

succinimidyl ester (CFSE), Central memory (CM), Chemokine ligand (CCL), Chemokine receptor
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(CCR), Crohn’s disease (CD), Effector memory (EM), Fluorescence minus one (FMO),

Gastrointestinal tract (GIT), GATA-binding factor-3 (GATA-3), Inflammatory bowel disease

D

(IBD), Interferon-gamma (IFN-γ), Interleukin (IL-), Lamina propria mononuclear cells (LPMCs),
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Lipopolysaccharide (LPS), Magnetic cell separation (MACS), Major histocompatibility complex

(MHC), Peripheral blood mononuclear cell (PBMC), Phytohaemagglutinin (PHA), RAR-related

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orphan receptor gamma t (RORγt), Regulatory T cells (Treg), Staphylococcus enterotoxin B (SEB),

T cell receptor (TCR), T helper (Th), T-box-expressed-in-T-cells (T-bet), Tumour necrosis factor
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alpha (TNF-α), Ulcerative colitis (UC), Violet proliferation dye (VPD).

AC

Corresponding author: Prof. Fiona Powrie, FRS, Kennedy Institute of Rheumatology, University of

Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK

Email: Fiona.Powrie@kennedy.ox.ac.uk; Tel: +44 (0)1865 612 659

Disclosures: These authors disclose the following: F.P. has received research support or

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 ACCEPTED MANUSCRIPT

consultancy fees from Eli Lilly, Merck, GSK, Janssen, Compugen, UCB, and MedImmune. S.K.

has received consulting fees and research support from ChemoCentryx Inc. and GSK in the past.

The remaining authors declare no conflict of interest. HHU has project collaboration with Eli Lilly

and UCB Pharma related to this project. Dr Keshav has provided consultancy services for a number

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of pharmaceutical and healthcare companies including Abbvie, Actavis Allergan, Astra-Zeneca,

Boehringer Ingelheim, ChemoCentryx, Dr Falk Pharma, Ferring, Gilead, GSK, Merck, Mitsubishi

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Tanabe Pharma, Pharmacosmos, Pfizer, Takeda, and Vifor Pharma, and received research support

SC
from Abbvie, ChemoCentryx, GSK, and Merck.

Transcript Profiling: None

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Writing Assistance: None

AN
Author Contributions: ANH, NRW designed, performed, and analysed experiments. ANH, NRW,

and FP conceived and designed the project, interpreted data, and wrote the manuscript. MJTS
M
analysed TCR sequencing data. EW, KS, AD, ST, SC, BMJO, CD, SHD, AB were involved in

acquisition of data, data analysis and interpretation of data. SAT, SK, HHU, AM provided essential
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materials and were involved in data interpretation and discussions. Oxford IBD Cohort
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Investigators provided IBD patient samples and ethical approval for the project. The Oxford IBD

Cohort Investigators are: Dr. Carolina Arancibia, Dr. Adam Bailey, Dr. Ellie Barnes, Dr. Beth
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Bird-Lieberman, Dr. Oliver Brain, Dr. Barbara Braden, Dr. Jane Collier, Dr. James East, Dr. Lucy

Howarth, Dr. Satish Keshav, Dr. Paul Klenerman, Dr. Simon Leedham, Dr. Rebecca Palmer, Dr.
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Fiona Powrie, Dr. Astor Rodrigues, Dr. Alison Simmons, Dr. Peter Sullivan, Dr. Simon Travis, Dr.
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Holm Uhlig.

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Abstract (250 words):

Background & Aims: Interactions between commensal microbes and the immune system are
tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in
humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We
measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal
microbes and determined their clonal diversity. We also assessed their functional phenotypes and

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effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in
patients with chronic intestinal inflammation.

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Methods: We collected samples of peripheral blood mononuclear cells (PBMC) and intestinal
tissues from healthy individuals (controls, n=13–30) and patients with inflammatory bowel diseases
(IBD, total n=119; 59 with UC and 60 with Crohn’s disease). We used 2 independent assays

SC
(CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal
bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp.
lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus

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obeum, Salmonella typhimurium and Clostridium difficile) to quantify, expand, and characterize
microbe-reactive CD4+ T cells. We sequenced T-cell receptor vβ genes in expanded microbe-
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reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-
reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and
gene expression patterns were measured by flow cytometry and quantitative PCR.
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Results: Circulating and gut-resident CD4+ T cells from controls responded to bacteria at
frequencies of 40–4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T
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cells were mainly of a memory phenotype, present in PBMCs and intestinal tissue, and had a
diverse T-cell receptor vβ repertoire. These cells were functionally heterogeneous, produced barrier
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protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A
(IL17A), interferon gamma, and tumor necrosis factor. In patients with IBD, microbiota-reactive
CD4+ T cells were reduced in the blood compared to intestine; T-cell responses we detected had an
increased frequency of IL17A production compared to responses of T cells from blood or intestinal
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tissues of controls.

Conclusions: In an analysis of PBMC and intestinal tissues from patients with IBD vs controls, we
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found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire,
and does not necessarily indicate disrupted interactions between immune cells and the commensal
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microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing

barrier-protective cytokines and providing a large pool of T cells that react to pathogens.

KEY WORDS: IFNG, TNF, immune regulation; activation

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Introduction

Vast numbers of microbes populate the gastrointestinal (GI) tract and contribute to digestion,

epithelial barrier integrity, and the development of appropriately educated mucosal immunity1.

Intestinal immune responses are tightly regulated to allow protective immunity against pathogens

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while limiting responses to dietary antigens and innocuous microbes. The ‘mucosal firewall’

prevents systemic dissemination of microbes by confining microbial antigens to the gut-associated

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lymphoid tissue (GALT)2. In the GALT, dendritic cells drive regulatory T cell differentiation in

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response to dietary antigens and commensal bacteria3. Nevertheless, vast numbers of potentially

commensal-reactive effector and memory T cells populate intestinal mucosae4. Recent evidence

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suggests that in mice, tolerance to commensal-derived antigens may be lost during

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pathogen-induced epithelial damage and subsequent transient exposure to commensals5,6. In

humans, circulating memory T cells recognise peptides derived from gut bacteria and can
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cross-react to pathogens, which may confer immunological advantage during subsequent new

infections7,8. While this process may be beneficial during homeostasis, deranged responses to
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commensals may promote inflammatory conditions such as inflammatory bowel disease (IBD).
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IBD (including Crohn’s disease (CD) and ulcerative colitis (UC)) results from a prolonged

disturbance of gut homeostasis, the precise aetiology of which is uncertain. One hypothesis is that,
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in genetically susceptible individuals, IBD may be triggered by intestinal dysbiosis that promotes

aberrant immune stimulation9. Indeed, in mouse models of colitis, intestinal microbiota promote
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inflammation in part by stimulating microbiota-reactive CD4+ T cells5,10. Whether this drives IBD
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in humans, however, remains unknown.

Although CD4+ T cell responses to intestinal bacteria are known to occur in humans11-13,

several aspects of this topic are largely uncharacterised, including (a) the frequency of human T

cells in the gut and periphery that are reactive to phylogenetically distinct intestinal microbes; (b)

the T cell receptor diversity and clonotype sharing of these T cells; (c) the functional phenotype of

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gut microbe-reactive T cells and their impact on tissue-resident cell populations; and (d) how

microbe-reactive T cells change during chronic intestinal inflammation. To address this knowledge

gap, we extensively characterised CD4+ T cell responses to intestinal microbiota in healthy

individuals and IBD patients.

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Using two independent assays, we observed that for almost all enteric bacteria examined,

bacteria-reactive CD4+ T cells were present at a frequency of 40 to 500 per million CD4+ T cells in

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adult peripheral blood. Bacteria-reactive T cells were also prevalent in the gut mucosa, with

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prominent enrichment for proteobacteria-reactivity. Microbiota-responsive T cells showed a diverse

TCRVβ repertoire and potently stimulated inflammatory responses by intestinal epithelial and

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stromal cells. Intriguingly, T cells from IBD patients displayed a normal spectrum of microbial

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responses, but expressed high amounts of IL-17A, consistent with increased amounts of

Th17-polarising cytokines in inflamed intestinal tissue. Collectively, these data demonstrate that
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microbiota-reactive CD4+ T cells are prevalent and normal constituents of the human immune

system that are functionally altered during IBD pathogenesis.

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Materials and Methods

Human Samples and Cell Isolation. Leukoreduction chambers from healthy individuals were

obtained from the National Blood Service (Bristol, UK). Peripheral EDTA blood samples were

obtained from IBD patients attending the John Radcliffe Hospital Gastroenterology unit or from

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healthy in-house volunteers. IBD patients (total, n=119; UC, n=59; CD, n=60) diagnosed by

endoscopic, histological and radiological criteria were recruited for the study. Healthy volunteers

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(n=30) without any known underlying acute or chronic pathological condition served as control

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donors. Demographic and clinical characteristics of IBD patients are summarized in Supplementary

Tables 5, 6, and 7. All donors provided informed, written consent. The NHS Research Ethics

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System provided ethical approval (reference numbers 09/H0606/5 for IBD patients and 11/YH/0020

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for controls; OCHRe ref 15/A237 for cord blood samples). For details regarding cell isolation, see

Supplemental Experimental Procedures.

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CD154-based Detection of Antigen-Specific T Cells. CD154 detection was done as previously

described14,15. Briefly, cells were plated at 5x106/cm2 for 7-12h with heat-inactivated bacteria. 5
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µg/ml brefeldin A (BFA, Sigma Aldrich) was added at 2 h. After 8-12 h, cells were harvested and
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treated as described in the Intracellular Cytokine, CD154 And Transcription Factor Staining section

(see below). For MACS enrichment of CD4+CD154+ T cells, see Supplemental Experimental
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Procedures.

Antigen-specific Recall Response (CFSE dilution assay) And T Cell Culture. Memory CD4+
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CD45RO+ CD45RA- T cells were enriched from PBMC with untouched memory CD4+ T cell
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enrichment kit (Miltenyi Biotec), sorted to >97% purity on a FACS ARIA III (BD) using CD45RA

and CD45RO expression, and were labelled with CFSE or Violet proliferation dye (VPD,

Invitrogen). CD14+ monocytes were isolated from PBMC using anti-CD14 microbeads (Miltenyi

Biotec), irradiated (45 Gy) and then pre-incubated for 3 h with bacterial lysates before T-cell

co-culture. T cells were co-cultured with the irradiated autologous monocytes at a ratio of 2:1 for 5-7

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days. Cells were cultured in RPMI-1640 supplemented with 2 mM glutamine, 1% (v/v) non-essential

amino acids, 1% (v/v) sodium pyruvate, penicillin (50 U/ml), streptomycin (50 mg/ml; all from

Invitrogen) and 5% (v/v) human serum (National Blood Service, Bristol, UK). CD14+ monocytes

were irradiated (45 Gy) and then pre-incubated for 3 h with bacterial lysates before T-cell co-culture.

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For MHCII blockade, 10 µg/ml of a pan-HLA class-II blocking antibody (HLA-DR, DP, DQ;

(Tü39)) was added 30 minutes before T-cell co-culture. T cell lines were generated by sorting

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CFSElow ICOShigh CD4+ T cells after seven days of stimulation and expanding them with IL-2 (300

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U/ml) and anti-CD3/CD28 beads (beads/T cell ratio, 1:4, Dynals) for 10-14 days. Supernatants were

collected from 1x106 CD4+ T cells stimulated for 24h with PMA (5 ng/ml) and ionomycin (500

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ng/ml; Sigma).

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Flow Cytometry and Cell Sorting. PBMCs and LPMCs were stained according to standard

protocols. For details, see Supplemental Experimental Procedures.

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Intracellular Cytokine, CD154, And Transcription Factor Staining. For intracellular cytokine

staining, cells were stained with fixable viability dye eFluor® 780 (eBioscience) and surface
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markers, fixed with 2% formaldehyde (Merck), and stained for cytokines in MACS buffer
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containing 0.05% saponin (Sigma-Aldrich). Transcription factor expression was analysed using the

FoxP3 staining buffer set (eBioscience) according to manufacturer’s instructions.

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Stimulation of Intestinal Cell Lines. CCD18Co (non-transformed human colon fibroblasts,

ATCC), and LIM1863 (human colon epithelial cells; a kind gift of Dr. Robert Whitehead, Ludwig
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Institute for Cancer Research) cells were cultured in humidified incubators with 5% CO2 at 37°C in
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DMEM or RPMI media (Sigma) with 10% FCS (Sigma) and 100 U penicillin/0.1 mg/ml

streptomycin. Cells were stimulated with 5% T cell supernatants for 24h. Cytokine neutralization

was achieved by supernatant pre-incubation for 1-2h with 10 µg/ml anti–IL17A (eBio64CAP17),

anti–IFN-γ (B27), anti–TNF-α (Remicade), and anti–IL22 (IL22JOP).

Statistics. Statistical analyses were performed with GraphPad Prism v6.0 for Macintosh

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(GraphPad Software). Statistically significant p values were indicated as follows: ns, not significant;

* P≤0.05; ** P≤ 0.01; *** P≤0.001; **** P≤0.0001. Tests are specified in figure legends.

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Results

Healthy adults possess circulating memory CD4+ T cells that are reactive to intestinal

microbiota

CD154 (also known as CD40 ligand) is rapidly upregulated by CD4+ T cells following antigen

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stimulation, irrespective of their differentiation phenotype, MHC alleles, or the precise nature of the

antigenic epitope14,15. We therefore used CD154 to detect naïve and memory CD4+ T cell responses

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among peripheral blood mononuclear cells (PBMCs) after stimulation with heat-inactivated bacteria

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(Figure 1A). Seven aerobic and anaerobic intestinal bacterial species representing the four

dominant gut phyla were chosen: Escherichia coli, Lactobacillus acidophilus, Bifidobacterium

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animalis subsp. lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis,

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and Ruminococcus obeum (Supplementary Figure 1A and Supplementary Table S1). These

bacteria are common in the healthy intestine but are altered in relative abundance during
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inflammation16,17. Furthermore, we analysed responses to Salmonella typhimurium and Clostridium

difficile due to their association with IBD18,19. T cell responses to the above bacteria were compared
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to those against well characterized barrier surface-related microbes that drive robust Th17 responses
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(Staphylococcus aureus and Candida albicans) or strong Th1 responses (Mycobacterium

tuberculosis)20,21. The presence of a large pool of M. tuberculosis-reactive memory Th1 cells in

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non-exposed individuals has previously been documented. The responses in healthy controls are

directed towards non-tuberculous mycobacteria (NTMs) rather than towards MTB20,22,23. The
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superantigen Staphylococcus enterotoxin B (SEB) was used as a positive stimulation control.

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Stimulation with enteric bacteria reproducibly induced detectable numbers of CD4+CD154+

T cells in the peripheral blood. CD4+ T cells reactive to S. aureus, C. albicans, and M. tuberculosis

were generally more abundant (Figure 1B and Supplementary Figure 1B, C).

Activation markers CD69 and ICOS were upregulated on activated antigen-reactive

CD4+CD154+ T cells (Supplementary Figure 1D). Responses were MHCII-dependent

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(Supplementary Figure 1E), and LPS failed to induce CD154 expression, confirming that CD154

upregulation was not a consequence of non-specific microbial responses (Figure 1B and

Supplementary Figure 1C). Based on CD154+ cell frequencies, we calculated that enteric

bacteria-reactive CD4+ T cells were present at precursor frequencies of 40 to 500 cells per 106

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circulating CD4+ T cells for almost all enteric bacteria surveyed (Figure 1C and Supplementary

Figure 1F).

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The newborn gut is primarily colonised with maternal vaginal and faecal bacteria after

birth24. To understand whether T cell reactivity to microbes develops after birth, we compared

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CD154 expression in umbilical cord blood with adult blood after enteric bacteria stimulation. As

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expected, appreciable responses to microbiota were observed only in adult blood. However,

CD4+CD154+ T cell frequencies after SEB stimulation were similar between adult and cord blood

(Figure 1D and Supplementary Figure 1G).

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Human memory T cells downregulate the naïve marker CD45RA and produce cytokines

more efficiently than naïve T cells25,26. In healthy individuals, the majority of bacteria-reactive
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CD4+ T cells had a memory phenotype (over 80% on average), indicating that they had been primed
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in vivo (Figure 1E, F and Supplementary Figure 2A, B). Microbiota-reactive

CD4+CD154+CD45RA- T cells expressed high amounts of TNF-α and IL-2 when compared to
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CD4+CD154+CD45RA+ T cells (Figure 1G and Supplementary Figure 2C). Therefore, the

circulating pool of memory CD4+ T cells contains numerous microbiota-reactive cells that arise
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after birth and produce cytokines including TNF-α and IL-2.

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Circulating microbiota-reactive CD4+ T cells express surface molecules that permit mucosal

trafficking

Memory T cells express numerous adhesion molecules and chemokine receptors to access

different tissues under steady-state and inflammatory conditions4,27,28. For example, α4β7 integrin

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and CCR9 regulate T cell migration to distinct parts of the gut. Blockade of α4β7 integrin has

shown clinical efficacy for treating IBD, whereas CCR9 blockade yielded mixed results29,30. To

identify homing receptors expressed by bacteria-reactive CD4+ T cells, we enriched CD4+CD154+ T

cells using magnetic beads to visualise rare enteric-bacteria-reactive T cells, and analysed them by

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flow cytometry (Figure 2A). Microbiota-reactive T cells had a central memory phenotype, with

over 60% expressing high levels of CCR7 (Figure 2B). Furthermore, 5–10% of CD4+CD154+ T

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cells expressed the gut-homing surface markers integrin β7 and CCR9 (Figure 2C, D). Relative to

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total memory CD4+ T cells and CD4+CD154- T, enteric bacteria-reactive T cells had high

expression of the mucosa-homing receptors CCR4 and CCR6 (above 60%), low expression of

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CCR10, and comparable expression of CXCR3 and CCR2 (Figure 2D and Supplementary Figure

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2D). Microbiota-reactive CD4+ T cells also expressed high amounts of CD161, a marker enriched

on Th17 cells (Figure 2D, E). The majority of memory CD4+CD154+ T cells co-expressed CCR7,
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CCR4, CD161, and CCR6 in various combinations, some of which also expressed integrin β7

(Figure 2E, pie chart). Therefore, circulating microbiota-reactive CD4+ T cells are equipped with
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several homing receptors that promote mucosal access.

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When comparing gut microbiota-reactive CD4+ T cells with those reactive to non-enteric

organisms (including S. aureus-, M. tuberculosis-, and C. albicans), enteric bacteria-reactive T cells

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were partially enriched only in CCR4 expression (Figure 2F). Thus, the homing receptor phenotype

of enteric bacteria-reactive T cells is consistent with that of T cells reactive to a broad diversity of
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mucosal microbes.
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Microbiota-reactive CD4+ T cells are enriched in gut tissue

The gut harbours over 3x1010 CD4+ T cells, but their specificity is unknown4,5. We therefore

estimated the abundance of human microbiota-reactive CD4+ T cells in the gut by examining

non-inflamed colon specimens using the CD154 assay (Figure 3A). Lamina propria CD4+ T cells

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showed a dominant EM and CM phenotype and expressed both tissue-resident and gut-related

markers, with 80% of cells being CD69+ (Supplementary Figure 3A).

We next stimulated lamina propria mononuclear cells (LPMCs) with microbial lysates or

SEB. We combined intracellular CD154 detection with TNF-α staining to increase assay

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sensitivity, as lamina propria CD4+ T cells expressed low amounts of CD154 without stimulation

(Supplementary Figure 3B,C). Compared with peripheral blood frequencies of unrelated donors,

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there were similar frequencies of S. aureus and SEB reactivity, and reduced M.

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tuberculosis-reactivity in the gut. However, gut CD4+ T cells were enriched in reactivity towards

intestinal bacteria and C. albicans (Figure 3B and Supplementary Figure 3D). Bacteria-reactive

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cells comprised 150–4000 cells per 106 gut-resident memory CD4+ T cells for all enteric bacteria

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tested. Given that peripheral blood contained 40–500 bacteria-reactive cells per 106 memory CD4+

T cells (for each bacteria tested), this suggests that bacteria-reactive T cells are 3–8 fold more
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frequent in gut tissue, as compared to those in circulation. The strong enrichment of S. typhimurium

and E. coli-reactivity in the gut was confirmed by assessing CD154 and TNF-α expression in CD4+
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T cells from donor-matched blood and intestinal tissue (Figure 3C). Since the gut harbours up to
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3x1010 memory T cells (versus 5–10x109 in blood)4, many of which are bacteria-reactive, the

absolute number of gut-resident microbiota-reactive CD4+ T cells is likely to be at least 10 times

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greater than that in peripheral blood.

Gut-resident bacteria-reactive (CD154+TNF-α+) T cells produced high amounts of IFN-γ,

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IL-17A, and IL-2, while production of IL-22, GM-CSF, and IL-4 was generally low (Figure 3D
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and Supplementary Figure 3E,F). Interestingly, lamina propria T cells showed increased IL-17A

expression and reduced IFN-γ production relative to cells with similar reactivity in peripheral blood

(Figure 3D and Supplementary Figure 3E).

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Enteric bacteria-reactive CD4+ T cells are clonally diverse

To assess the clonal diversity of circulating bacteria-reactive memory CD4+ T cells, we expanded

CFSE-labelled CD4+ T cells using whole bacteria and autologous irradiated monocytes as APCs

(Supplementary Figure 4A)21. S. aureus-, M. tuberculosis-, and SEB-reactive T cells served as

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controls. Antigen-reactive T cells proliferated in an MHCII-dependent manner and were readily

detectible after 3–6 days (Figure 4A and Supplementary Figure 4B–D). Proliferating cells

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expressed several activation markers including ICOS, CD25, and OX40 (Figure 4A and

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Supplementary Figure 4B,E,F). Consistent with the CD154 assay, S. aureus, M. tuberculosis, and

SEB strongly induced T cell proliferation (Figure 4A,B and Supplementary Figure 3B,C).

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Flow cytometry analysis revealed a diverse TCRVβ repertoire in bacteria-reactive T cells,

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similar to polyclonal stimulation with phytohaemagglutinin (PHA) but different to stimulation with

SEB, which is known to activate a restricted Vβ repertoire (Figure 4C)31. To directly assess the
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clonal diversity of bacteria-reactive CD4+ T cells, we isolated CFSElow bacteria-reactive memory T

cells and assessed TCRVβ clonotypes by multiplex PCR and deep sequencing. 150–800 clonotypes
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were detected for each reactivity (Supplementary Figure 4G). The largest clonal diversity was
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detected among E. coli- and S. typhimurium- reactive cells, consistent with frequencies observed in

the CD154 assay (see Figure 1B,C and Supplementary Figure 1B,F). While closely related
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species (e.g. E. coli versus S. typhimurium) had 3–8% overlap in T cell clonotypes, little clonotype

sharing was observed between T cells reactive to more distantly related bacteria (Supplementary
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Figure 3H). Indeed, E. coli- and B. animalis-reactive CD4+ T cell lines were strongly restimulated
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when cultured with autologous monocytes loaded with E. coli or B. animalis lysates, respectively.

In contrast, E. coli-reactive T cells responded weakly to the closely related S. typhimurium, while B.

animalis-reactive T cells responded weakly to L. acidophilus, F. prausnitzii, and C. difficile

(Supplementary Figure 5A). These data confirm the low degree of cross-reactivity predicted from

TCRVβ sequencing.

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Microbiota-reactive memory CD4+ T cells are functionally heterogeneous and produce

barrier-promoting cytokines

To functionally characterise circulating microbiota-reactive memory cells, we analysed CFSElow

cells using flow cytometry after stimulation with enteric bacteria for 6 days. Enteric

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microbiota-reactive cells produced Th1- and Th17-related cytokines including IFN-γ, IL-17A, and

IL-22, but only low amounts of the Th2 cytokine IL-4, comparable to cells reactive towards S.

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aureus or C. albicans (Figure 4D and Supplementary Figure 5B,C). In contrast, memory T cells

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reactive towards SEB, M. tuberculosis, influenza vaccine components, or tetanus toxoid showed a

polarized Th1 profile with low expression of IL-17A (Figure 4D and Supplementary Figure

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5B,C). Boolean gating revealed a high degree of functional heterogeneity in expanded

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microbiota-reactive memory T cells, with frequent co-expression of IL-17A, IL-22, and IFN-γ

(Figure 4E). Bacteria-reactive cells co-expressed the transcription factors RORγt and T-bet, which
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are characteristic of Th17 and Th1 cells, respectively (Figure 4F,G and Supplementary Figure

5D,E). Intriguingly, a subset of CD4+ T cells reactive to F. prausnitzii, L. acidophilus, or B.

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animalis produced the immunoregulatory cytokine IL-10 in addition to IFN-γ and IL-17A
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(Supplementary Figure 5F). Thus, unlike T cells that are reactive towards M. tuberculosis or

vaccine antigens, enteric microbiota-reactive T cells are functionally distinct and produce
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barrier-promoting and immunoregulatory cytokines.

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Microbiota-reactive memory T cells promote intestinal stromal and epithelial cell activation
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During periods of epithelial damage and exposure to commensals, activation of microbiota-reactive

memory T cells could promote protective immune responses. To assess their tissue-modulating

capabilities, cell-free supernatants of microbiota-reactive memory T cells were used to stimulate

CCD18Co intestinal myofibroblasts and LIM1863 colonic epithelial cells. CCD18Co and LIM1863

cells were then assessed for expression of various immune response genes that were selected a

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priori to represent responses to major T cell-derived cytokines. Both cell types responded by

expressing several cytokine and chemokine genes known to be induced by IL-17A (including IL1B,

CSF2, IL6, CXCL1, and CXCL8), as well as IFN-γ-inducible genes including CXCL9, CXCL10, and

CXCL11 (Figure 5A,B)32. Conversely, supernatants from SEB-stimulated memory T cells (which

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produce little IL-17A) mainly induced IFN-γ-dependent genes. Thus, stimulation of

non-hematopoietic intestinal cells by microbiota-reactive T cells may promote recruitment and

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activation of myeloid cell populations to facilitate pathogen control and tissue repair.

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We next assessed the effects of individual cytokines in E. coli-reactive T cell supernatants

using combinations of neutralizing antibodies. This experiment revealed distinct IFN-γ- and IL-

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17A/TNF-α–dependent groups of response genes in both intestinal epithelial cells and fibroblasts.

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IFN-γ blockade strongly reduced expression of several chemokine genes including CXCL9,

CXCL10, CXCL11, CCL2, and CCL7 (IFN-γ-dependent module; Figure 5C,D). Intriguingly, single
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blockade of IL-17A, IL-22, or TNF-α did not affect stromal or epithelial cell activation (Figure

5C,D). However, combined blockade of IL-17A and TNF-α influenced a large number of genes
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including CSF2, IL1B, TNF, CXCL1, CXCL8, CXCL5, CXCL6, and CCL20 (IL-
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17A/TNF-α-dependent module). Triple blockade of IFN-γ, IL-17A, and TNF-α completely

inhibited stromal and epithelial cell activation. IL-22 blockade did not affect cytokine or chemokine
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production, but attenuated induction of the anti-microbial peptide REG3G in LIM1863 cells. Given
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that the products of T cell-stimulated stromal and epithelial cells are highly expressed in the
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inflamed mucosa of IBD patients (Figure 5E and 7D), this signature might reflect the activation of

microbiota-reactive T cells following epithelial disruption, a key feature of IBD.

Microbiota-reactive CD4+ T cells in inflamed intestinal tissue show a Th17-skewed phenotype

in IBD patients

IBD is thought to arise in part from aberrant adaptive immune responses to microbiota9. Human

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CD4+ T cells in IBD have been functionally characterised mainly by polyclonal stimulation33-35.

Therefore, we evaluated microbiota-reactive CD4+ T cell responses in IBD patients using the

CD154 detection approach. Circulating microbiota-reactive CD4+ T cell frequencies were decreased

in IBD patients compared with healthy donors, which might reflect their selective recruitment to the

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inflamed gut (Figure 6A and Supplementary Figure 6A). However, intestinal memory CD4+ T

cells from IBD patients did not display reciprocally higher frequencies of microbial specificity

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(Figure 6B and Supplementary Figure 6B). We next calculated the frequency of memory CD4+

T cells in inflamed mucosae using flow cytometry. Memory CD4+ T cells were present at higher

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frequencies in inflamed tissue from IBD patients compared to tissue from matched non-lesional

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sites of IBD patients and healthy controls (Figure 6C). These findings were confirmed using a

previously published bioinformatics approach known as CIBERSORT in an independent cohort36

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(Supplementary Figure 6C). Based on both approaches, memory CD4+ T cells are typically 2–4
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fold more frequent in inflamed tissue from IBD patients compared to tissue from healthy controls.

Thus, because inflamed tissue contains a higher abundance of memory CD4+ T cells than healthy
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mucosa, it can be inferred that gut-resident microbiota-reactive CD4+ T cells are similarly enriched
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in patients with active IBD (Figure 6C, Supplementary Figure 6C).

To evaluate functional alterations in microbiota-reactive CD4+ T cells in IBD, intracellular

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CD154 detection was combined with cytokine analysis. Compared with healthy controls, circulating

microbiota-reactive CD4+ T cells from IBD patients displayed increased IL-17A and IL-2
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production, but decreased expression of IFN-γ (Figure 7A and Supplementary Figure 6D, E).
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Interestingly, increased IL-17A production was observed in all enteric bacteria-reactive responses,

but not in S. aureus, M. tuberculosis, or SEB responses (Figure 7A and Supplementary Figure

6E,F). These changes were observed in both CD and UC and were independent of disease activity

or therapy (Supplementary Figure 6G). However no difference in IL-10 production was observed

between healthy donors and IBD patients (Supplementary Figure 6H). IFN-γ and IL-17A

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co-expression is thought to identify pathogenic CD4+ T cells in mouse colitis models37, so we

assessed their co-expression in E. coli-reactive memory CD4+ T cells. Compared with controls, IBD

patients displayed significantly increased frequencies of IL-17A+IFN-γ- cells and a marginal

increase in IL-17A+IFN-γ+ cells, while the IL-17A-IFN-γ+ fraction was significantly reduced

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(Figure 7B). E. coli-reactive CD4+ T cells from inflamed intestinal tissue showed an increase in IL-

17A single producers similar to that seen in peripheral blood (Figure 7C).

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Because the Th17-inducing cytokines IL1B, IL6, and IL23A were highly enriched in the

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inflamed intestinal tissue of IBD patients (Figure 7D), we reasoned that they might promote Th17

polarization of bacteria-reactive T cells. Indeed, treatment of microbiota-reactive CD4+ T cells from

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healthy donors and IBD patients with IL-1β, IL-6, or IL-23 for one week during stimulation with E.

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coli, S. typhimurium, L. acidophilus, or B. animalis (CFSE dilution assay) resulted in a 1.5–2-fold

increase in IL-17A production (Figure 7E and Supplementary Figure 6I).

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Taken together, these experiments demonstrate that circulating and gut-resident

microbiota-reactive CD4+ T cells express increased frequencies of IL-17A in IBD. Intestinal tissues
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from patients with active IBD express gene modules driven by Th1/Th17-derived cytokines,
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suggesting that bacteria-reactive memory cells could contribute to the tissue response.
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Discussion

The gastrointestinal tract harbours a large and diverse population of commensal bacteria, and how

the immune system interacts with them is subject to intense investigation. Here we used two

different methodologies to characterise microbiota-reactive CD4+ T cell frequencies and phenotypes

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in the blood and intestinal tissue of healthy individuals and those with IBD. For each bacterial strain

tested, the healthy CD4+ T cell repertoire contains reactive cells at a frequency of 40–4000 per

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million, consistent with other antigen-reactive memory T cells38. Microbiota-reactive CD4+ T cells

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were mainly of a memory phenotype, present in both blood and gut tissue, had a diverse TCRVβ

repertoire, and showed little clonotype sharing. Notably, microbiota-reactive CD4+ T cells were

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functionally heterogeneous in terms of homing receptor expression and effector functions and could

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stimulate intestinal cells via production of IL-17A, IFN-γ, IL-22, and TNF-α. Moreover,

microbiota-reactive CD4+ T cells were recruited to sites of inflammation and showed increased IL-
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17A production in patients with IBD.

Characterizing T cell responses to bacteria is technically challenging due to their complex

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antigenic make-up. We therefore used the CD154 and CFSE dilution assays, both of which exploit
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microbial complexity to provide large numbers of antigens. The combination of CFSE dilution and

TCRVβ sequencing allowed us to quantify clonotype heterogeneity and sharing between different
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bacteria-reactive T cells. Given the phylogenetic similarity of several bacteria used in this study, the

paucity of clonotype sharing was surprising. Nevertheless, enteric bacteria-reactive T cells could be
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cross-reactive to other antigens not assessed in this study, and may have been primed during
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immune responses to other targets39. High interclonal and intraclonal functional heterogeneity in

human CD4+ T cell responses to microbes and vaccines was recently observed20. However,

clonotype sharing between different microbial stimuli has not previously been studied and requires

further investigation.

Microbiota-reactive CD4+ T cells showed substantial phenotypic and functional

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heterogeneity. The majority of circulating enteric bacteria-reactive CD4+ T cells co-expressed

chemokine receptors including CCR4, CCR6, and CCR7, while a smaller fraction expressed the

gut-related homing receptors α4β7 and CCR9. These receptors promote access to secondary

lymphoid organs and various mucosal tissues, including the intestine4,28. Furthermore, circulating

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and gut-resident microbiota-reactive T cells displayed both Th17 and Th1 characteristics, and in

some cases produced IL-1040. Gut-resident cells showed a clear Th17 bias when compared to

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circulating populations, which was more pronounced in IBD.

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Based on our observations, we can speculate that continuous sampling of luminal antigens

by intestinal DCs causes low-level stimulation of gut-resident CD4+ T cells to produce cytokines

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that support epithelial integrity, barrier function, and intestinal homeostasis41,42. Indeed, cytokine

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production by commensal-reactive CD4+ T cells might play a more significant role in supporting

gut homeostasis than previously thought (Supplementary Figure 7). However, this homeostatic
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circuit might be disrupted in IBD due to dysbiotic changes and/or perturbed myeloid cell activity,

causing inappropriate T cell activation and a pathogenic imbalance of cytokine production9.

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While IL-17A is frequently cited as a pathogenic cytokine, it is also critical for promoting
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mucosal barrier function and protection from pathogens32. Absence of IL-17A was recently shown

to increase epithelial injury and compromise barrier function in mouse models of colitis43,44. Indeed,
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IL-17A is a critical driver of neutrophil recruitment, and its absence could therefore exacerbate

mucosal inflammation by facilitating bacterial invasion and dispersal45. Notably, blockade of IL-
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17A in CD caused disease exacerbation despite being well tolerated and therapeutically effective in
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psoriasis46. Thus, IL-17A likely plays a key tissue-protective role in humans, suggesting that the

increased Th17 polarization of microbiota-reactive T cells in IBD patients could reflect an effort to

bolster tissue integrity.

Host-microbial homeostasis depends on minimising contact between microorganisms and

mucosal surfaces via the combined action of epithelial cells, mucus, IgA, antimicrobial peptides,

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and immune cells1,2. Active immune responses to gut flora have been linked to disease9. However,

this concept should be revisited in light of our current findings and the observation that healthy

individuals generate antibody responses to commensals47,48. At least two plausible mechanisms

could explain the genesis of these microbiota-reactive responses. First, mucosal dendritic cells

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constantly survey the luminal microenvironment and thereafter migrate to secondary lymphoid

tissues to initiate B and T cell responses49,50. Second, during GI infections in mice, immune

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responses against commensals and pathogens are induced in parallel51. Thus, continuous luminal

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sampling of intestinal microbiota and periodic epithelial breaches during gastrointestinal infections

might provide a plethora of memory T cells with potential reactivity towards newly encountered

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pathogens7,8. Therefore, contrary to the notion that they promote inflammatory pathology, acquired

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commensal-reactive T cell responses may be essential to promote barrier function and IL-10

mediated immune regulation, two cornerstones of intestinal homeostasis.

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Acknowledgments

We thank Helen Ferry, Jenny Middleton, Sam Bullers, Sebastian Rogatti Granados, Priya

Siddhanathi, James Chivenga, Ngonidzashe Charumbira, Jennifer Hollis, Linda Holden, Fiona

Goddard, Karen Doig, Nicole Stoesser, Nicole Gordon and Claire Pearson for FACS sorting,

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technical support, patient sample collection and lab management. We thank Harry Flint, Carolina V.

Arancibia-Cárcamo, Emily Thornton, Tobias Schwerd, David Danko, Arnold Han, Mark Davis, M.

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Hussein Al-Mossawi, Paul Bowness, and all laboratory members for valuable support and

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discussions. We also thank Thomas Penz from the Biosequencing Facility at CeMM. We

acknowledge the Oxford Radcliffe and GI Biobanks and the Oxford IBD cohort study supported by

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the NIHR Oxford Biomedical Research Centre (grant no. HBRWAE04 Task HB81.G). We thank

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all volunteers and patients who took part in this study.
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References

1. Littman DR, Pamer EG. Role of the Commensal Microbiota in Normal and Pathogenic Host

Immune Responses. Cell Host & Microbe 2011;10:311–323.

2. Macpherson AJ, Slack E, Geuking MB, et al. The mucosal firewalls against commensal

PT
intestinal microbes. Semin Immunopathol 2009;31:145–149.

3. Honda K, Littman DR. The microbiome in infectious disease and inflammation. Annu Rev

RI
Immunol 2012;30:759–795.

SC
4. Farber DL, Yudanin NA, Restifo NP. Human memory T cells: generation,

compartmentalization and homeostasis. Nat Rev Immunol 2014;14:24–35.

U
5. Belkaid Y, Bouladoux N, Hand TW. Effector and memory T cell responses to commensal

6.
AN
bacteria. Trends Immunol 2013;34:299–306.

Littman DR, Pamer EG. Role of the commensal microbiota in normal and pathogenic host
M
immune responses. Cell Host Microbe 2011;10:311–323.

7. Su LF, Kidd BA, Han A, et al. Virus-specific CD4(+) memory-phenotype T cells are
D

abundant in unexposed adults. Immunity 2013;38:373–383.

TE

8. Campion SL, Brodie TM, Fischer W, et al. Proteome-wide analysis of HIV-specific naive

and memory CD4 +T cells in unexposed blood donors. J Exp Med 2014;211:1273–1280.
EP

9. Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel

disease. Nature 2011;474:298–306.

C

10. Cong Y, Brandwein SL, McCabe RP, et al. CD4+ T cells reactive to enteric bacterial
AC

antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response

and ability to transfer disease. J Exp Med 1998;187:855–864.

11. Duchmann R, Kaiser I, Hermann E, et al. Tolerance exists towards resident intestinal flora

but is broken in active inflammatory bowel disease (IBD). Clin Exp Immunol 1995;102:448–

455.

23
 ACCEPTED MANUSCRIPT

12. Duchmann R, May E, Heike M, et al. T cell specificity and cross reactivity towards

enterobacteria, bacteroides, bifidobacterium, and antigens from resident intestinal flora in

humans. Gut 1999;44:812–818.

13. Ergin A, Syrbe U, Scheer R, et al. Impaired peripheral Th1 CD4+ T cell response to

PT
Escherichia coli proteins in patients with Crohn's disease and ankylosing spondylitis. J Clin

Immunol. 2011;31:998–1009.

RI
14. Chattopadhyay PK, Yu J, Roederer M. A live-cell assay to detect antigen-specific CD4+ T

SC
cells with diverse cytokine profiles. Nat Med 2005;11:1113–1117.

15. Frentsch M, Arbach O, Kirchhoff D, et al. Direct access to CD4+ T cells specific for defined

U
antigens according to CD154 expression. Nat Med 2005;11:1118–1124.

16.
AN
Duncan SH, Louis P, Flint HJ. Cultivable bacterial diversity from the human colon. Lett

Appl Microbiol 2007;44:343–350.

M
17. Kostic AD, Xavier RJ, Gevers D. The microbiome in inflammatory bowel disease: current

status and the future ahead. Gastroenterology 2014;146:1489–1499.

D

18. Bosca-Watts MM, Tosca J, Anton R, et al. Pathogenesis of Crohn's disease: Bug or no bug.
TE

World J Gastrointest Pathophysiol 2015;6:1–12.

19. Gradel KO, Nielsen HL, Schønheyder HC, et al. Increased short- and long-term risk of
EP

inflammatory bowel disease after salmonella or campylobacter gastroenteritis.

Gastroenterology 2009;137:495–501.
C

20. Becattini S, Becattini S, Latorre D, et al. Functional heterogeneity of human memory CD4+
AC

T cell clones primed by pathogens or vaccines. Science 2015;347:400–406.

21. Zielinski CE, Mele F, Aschenbrenner D, et al. Pathogen-induced human TH17 cells produce

IFN-γ or IL-10 and are regulated by IL-1β. Nature 2012;484:514–518.

22. Black GF, Weir RE, Chaguluka SD, et al. Gamma Interferon Responses Induced by a Panel

of Recombinant and Purified Mycobacterial Antigens in Healthy, Non-Mycobacterium bovis

24
 ACCEPTED MANUSCRIPT

BCG-Vaccinated Malawian Young Adults. Clin and Vaccine Immunol 2003;10:602–611.

23. Lindestam Arlehamn CS, Paul S, Mele F, et al. Immunological consequences of intragenus

conservation of Mycobacterium tuberculosis T-cell epitopes. Proc Natl Acad Sci U S A

2015;112:E147–55.

PT
24. Matamoros S, Gras-Leguen C, Le Vacon F, et al. Development of intestinal microbiota in

infants and its impact on health. Trends Microbiol 2013;21:167–173.

RI
25. Appay V, van Lier RAW, Sallusto F, et al. Phenotype and function of human T lymphocyte

SC
subsets: consensus and issues. Cytometry A 2008;73:975–983.

26. Mascher B, Schlenke P, Seyfarth M. Expression and kinetics of cytokines determined by

U
intracellular staining using flow cytometry. J Immunol Methods 1999;223:115–121.

27.
AN
Moser B, Loetscher P. Lymphocyte traffic control by chemokines. Nat Immunol

2001;2:123–128.
M
28. Mueller SN, Gebhardt T, Carbone FR, et al. Memory T Cell Subsets, Migration Patterns, and

Tissue Residence. Annu Rev Immunol 2013;31:137–161.

D

29. Keshav S, Wendt E. CCR9 antagonism: potential in the treatment of Inflammatory Bowel
TE

Disease. CEG 2015;Volume 8:119–130.

30. Neurath MF. New targets for mucosal healing and therapy in inflammatory bowel diseases.
EP

Mucosal immunol 2014;7:6–19.

31. Kappler J, Kotzin B, Herron L, et al. V beta-specific stimulation of human T cells by

C

staphylococcal toxins. Science 1989;244:811–813.

AC

32. Ouyang W, Kolls JK, Zheng Y. The Biological Functions of T Helper 17 Cell Effector

Cytokines in Inflammation. Immunity 2008;28:454–467.

33. Annunziato F, Annunziato F, Cosmi L, et al. Phenotypic and functional features of human

Th17 cells. J Exp Med 2007;204:1849–1861.

34. Kleinschek MA, Boniface K, Sadekova S, et al. Circulating and gut-resident human Th17

25
 ACCEPTED MANUSCRIPT

cells express CD161 and promote intestinal inflammation. J Exp Med 2009;206:525–534.

35. Globig A-M, Hennecke N, Martin B, et al. Comprehensive Intestinal T Helper Cell Profiling

Reveals Specific Accumulation of IFN-γ+IL-17+Coproducing CD4+ T Cells in Active

Inflammatory Bowel Disease. Inflamm Bowel Dis 2014;20:2321–2329.

PT
36. Newman AM, Liu CL, Green MR, et al. Robust enumeration of cell subsets from tissue

expression profiles. Nat Methods 2015;12:453–457.

RI
37. Shale M, Schiering C, Powrie F. CD4(+) T-cell subsets in intestinal inflammation. Immunol

SC
Rev 2013;252:164–182.

38. Bacher P, Scheffold A. New technologies for monitoring human antigen-specific T cells and

U
regulatory T cells by flow-cytometry. Curr Opin in Pharmacol 2015;23:17–24.

39.
AN
Mason D. A very high level of crossreactivity is an essential feature of the T-cell receptor.

Immunol Today 1998;19:395–404.

M
40. Sallusto F. Heterogeneity of Human CD4 +T Cells Against Microbes. Annu Rev Immunol

2016;34:317–334.
D

41. Blaschitz C, Raffatellu M. Th17 cytokines and the gut mucosal barrier. J Clin Immunol
TE

2010;30:196–203.

42. Sonnenberg GF, Fouser LA, Artis D. Border patrol: regulation of immunity, inflammation
EP

and tissue homeostasis at barrier surfaces by IL-22. Nat Immunol 2011;12:383–390.

43. Maxwell JR, Zhang Y, Brown WA, et al. Differential Roles for Interleukin-23 and
C

Interleukin-17 in Intestinal Immunoregulation. Immunity 2015;43:739–750.

AC

44. Lee JS, Tato CM, Joyce-Shaikh B, et al. Interleukin-23-Independent IL-17 Production

Regulates Intestinal Epithelial Permeability. Immunity 2015;43:727–738.

45. Smith AM, Rahman FZ, Hayee B, et al. Disordered macrophage cytokine secretion underlies

impaired acute inflammation and bacterial clearance in Crohn's disease. J Exp Med

2009;206:1883–1897.

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46. Yang J, Sundrud MS, Skepner J, et al. Targeting Th17 cells in autoimmune diseases. Trends

Pharmacol Sci 2014;35:493–500.

47. Palm NW, de Zoete MR, Cullen TW, et al. Immunoglobulin A Coating Identifies

Colitogenic Bacteria in Inflammatory Bowel Disease. Cell 2014;158:1000–1010.

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48. Zeng MY, Cisalpino D, Varadarajan S, et al. Gut Microbiota-Induced Immunoglobulin G

Controls Systemic Infection by Symbiotic Bacteria and Pathogens. Immunity 2016;44:647–

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658.

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49. Rescigno M, Urbano M, Valzasina B, et al. Dendritic cells express tight junction proteins and

penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001;2:361–367.

U
50. Macpherson AJ. Induction of Protective IgA by Intestinal Dendritic Cells Carrying

51.
AN
Commensal Bacteria. Science 2004;303:1662–1665.

Hand TW, Santos Dos LM, Bouladoux N, et al. Acute Gastrointestinal Infection Induces
M
Long-Lived Microbiota-Specific T Cell Responses. Science 2012;337:1553–1556.
D

Author names in bold designate shared co-first authorship

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C EP
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Figure Legends

Figure 1. Healthy adults possess circulating memory CD4+ T cells that are reactive to

intestinal microbiota.

PBMCs were stimulated with heat-inactivated bacteria for 8-12h, and bacteria-reactive CD4+ T

PT
cells were detected by intracellular CD154 expression.

(A) Experimental setup.

RI
(B) CD154+ frequencies among peripheral CD4+ T cells in adults after short-term stimulation with

SC
heat-inactivated bacteria (n=30 independent donors).

(C) Estimated microbiota-reactive cells per million CD4+ T cells in adult blood (n=30). The

U
numbers of microbiota-reactive T cells were calculated based on the frequencies of CD4+CD154+ T

AN
cells. Background (no microbe stimulation) was subtracted from bacterial stimulations. Significance

calculated in relation to LPS stimulation.

M
(D) Frequencies (± SEM) of CD154+ cells among CD4+ T cells in adult (n=30) or cord blood (n=3)

after short-term stimulation with heat-inactivated bacteria.

D

(E) Expression of CD45RA on CD4+CD154+ T cells, showing four representative stimulations of

TE

the same donor.

(F) Mean (± SEM) frequencies of memory cells within CD4+CD154+ T cells. Each symbol
EP

represents an antigen-reactive population from one individual (n=19-30).

(G) Expression of CD45RA, TNF-α, and IL-2 by CD4+CD154+ T cells after short-term PBMC
C

stimulation with bacterial lysates. Statistics: panels B, C, and F, one-way ANOVA with Sidak’s
AC

multiple comparison test; panel D, Mann-Whitney test.

Figure 2. Circulating microbiota-reactive CD4+ T cells express several surface molecules that

promote mucosal trafficking.

CD4+CD154+ T cells were analysed by flow cytometry after magnetic enrichment following

28
 ACCEPTED MANUSCRIPT

short-term PBMC stimulation with heat-inactivated bacteria.

(A) Experimental setup and enrichment efficiency after stimulation with SEB.

(B) Representative CD45RA and CCR7 expression on enriched CD4+CD154+ T cells. Central

memory T cell frequencies are shown (right bar graph). Frequencies (± SEM) of 4 independent

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experiments are depicted with n=8 independent donors.

(C) Heat map depicting mean frequencies of surface marker and chemokine receptor expression on

RI
enriched CD4+CD154+ T cells (n=5-8 independent donors).

(D) Surface marker and chemokine receptor expression frequencies among CD4+CD154+ T cells

SC
after short-term stimulation with B. animalis. Data representative of 5-8 independent donors.

U
(E) Co-expression of surface molecules with CCR6 after short-term stimulation of CD4+CD154+

CD45RA- T cells with B. animalis (left panel). Boolean gating analysis shows each possible
AN
combination of CCR7, CCR4, CCR6, and CD161 expression (right panel).
M
(F) Surface marker and chemokine receptor expression frequencies among total memory CD4+ T

cells and CD4+CD154+ T cells after short-term stimulation with B. animalis, S. aureus, M.
D

tuberculosis, C. albicans, or SEB. Frequencies (± SEM) of 4 independent experiments are depicted

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with n=5-8 independent donors. Statistics: panel F, One-way ANOVA with Sidak’s multiple

comparison test.
EP

Figure 3. Microbiota-reactive CD4+ T cells are enriched in gut tissue

C

LPMCs were isolated from non-inflamed and tumour-free surgical specimens from colorectal
AC

cancer patients.

(A) Experimental setup. LPMCs were stimulated with heat-inactivated bacteria for 8-12h and

assessed for intracellular expression of cytokines and CD154.

(B) Estimated microbiota-reactive cells per million CD4+ T cells in adult blood and intestinal tissue

from unrelated donors, based on CD154 staining (n=17 for control mucosa; n=25-31 for blood).

29
 ACCEPTED MANUSCRIPT

(C) Matched LPMCs and PBMCs were stimulated with heat-inactivated E. coli (blue symbols) or S.

typhimurium (red symbols), analysed for CD154 expression, and compared with respect to the

estimated microbiota-reactive cells per million CD4+ T cells. Connected dots represent matched

samples.

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(D) Frequencies (±SEM) of IL-17A, IFN-γ and IL-22 production by CD154+TNF-α+ memory

CD4+ T cells isolated from LPMCs or PBMCs from unrelated donors after stimulation with

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heat-inactivated bacteria (n=10-23 donors). Significance calculated between the respective

SC
stimulations in control mucosa and peripheral blood. Statistics: panels B and D, Mann-Whitney test;

panel C, paired t-test.

U
AN
Figure 4. Microbiota-reactive memory CD4+ T cells are clonally diverse and functionally

heterogeneous
M
Memory CD4+ T cells were labelled with CFSE or VPD (violet proliferation dye-450) and

stimulated with heat-inactivated bacteria in the presence of autologous monocytes.

D

(A) CFSE profiles and ICOS expression on days 3 and 6 of stimulation in a representative donor.
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(B) Percentage of CFSElow CD4+ T cells of each individual donor (n=18).

(C) Pie charts showing TCRVβ expression by proliferating VPDlow cells measured by Vβ antibody
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staining on day 7 of stimulation. Average of three independent donors is depicted. TCRVβ usage of

the different reactivities of three independent donors is summarized in Supplementary Table 2.

C

(D) Mean (± SEM) cytokine production frequencies of proliferating CFSElow cells and non-
AC

activated CFSEhigh cells after PMA/ionomycin stimulation (n=12-13 independent donors).

(E) Boolean gating analysis showing each possible combination of IL-17A, IFN-γ, and IL-22

production by CFSElow proliferating cells. Data from 9 independent donors.

(F, G) RORγt and T-bet expression in proliferating CFSElow cells measured by intracellular staining

on day 7 of stimulation. (G) Boolean gating analysis showing each possible combination of RORγt,

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 ACCEPTED MANUSCRIPT

T-bet, and GATA-3 production by CFSElow proliferating cells. Data from 3 independent donors.

Statistics: panel B, one-way ANOVA with Sidak’s multiple comparison test; panel D, one-way

ANOVA with Bonferroni’s multiple comparison test.

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Figure 5. Microbiota-reactive memory T cells promote intestinal stromal and epithelial cell

activation.

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Healthy donor memory CD4+ T cells from peripheral blood were labelled with CFSE and

SC
stimulated with heat-inactivated bacteria in the presence of autologous monocytes.

CD4+CFSElowICOShighcells were FACS-sorted on day 7 and expanded for 10-14 days with anti-

U
CD3/CD28 beads. Expanded cells were stimulated at equal numbers with PMA/ionomycin for 24h

to produce conditioned supernatants.

AN
(A, B) Cell-free supernatants from different T cell specificities was used to stimulate CCD18Co
M
intestinal myofibroblasts and LIM1863 colon epithelial cells. Gene expression in stimulated cells

was measured by qPCR and normalised to control treatment (media containing PMA/ionomycin
D

alone). Results of independent stimulations were pooled together into the following categories:
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Proteobacteria-reactive T cells (S. typhimurium- and E. coli-reactive); Actinobacteria-reactive T

cells (B. animalis-reactive); Firmicutes-reactive T cells (F. prausnitzii- and L. acidophilus-reactive).

EP

Data are from three independent T cell donors.

(C, D) Supernatants from E. coli-reactive CD4+ T cells were used to stimulate CCD18Co (C) or
C

LIM1863 (D) cells. Supernatants were pre-treated with one or more cytokine-neutralizing
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antibodies as indicated. Gene expression was median-normalized, log2 transformed, and plotted as a

heat map. Data representative of 2–3 independent experiments.

(E) Q-PCR analysis of mucosal biopsies from the Oxford IBD cohort, categorised by endoscopic

assessment of disease activity. Demographic and clinical characteristics of IBD patients are

summarized in Supplementary Table 5. Statistics: panels A, B, and E, One-way ANOVA with

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 ACCEPTED MANUSCRIPT

Sidak’s multiple comparison test.

Figure 6. Abundance of circulating and gut-resident enteric bacteria-reactive CD4+ T cells in

IBD

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(A) PBMCs from healthy donors or IBD patients were stimulated with the indicated

heat-inactivated bacteria and analyzed for CD154 expression. Frequencies (± SEM) of CD154+

RI
cells among CD4+ T cells are depicted (n=30-38). Demographic and clinical characteristics of IBD

SC
patients are summarized in Supplementary Table 6.

(B) LPMCs were isolated from inflamed surgical specimens from IBD patients or non-inflamed and

U
tumour-free surgical specimens from colorectal cancer patients. Isolated LPMCs were stimulated

AN
with the indicated heat-inactivated bacteria and analysed for CD154 expression. Frequencies (±

SEM) of CD154+ cells among CD4+ T cells are depicted (n=10-20).

M
(C) LPMCs from endoscopic intestinal biopsies were obtained from healthy controls or matched

lesional and non-lesional sites of IBD patients from an independent Oxford cohort (n=12 controls
D

and 17 IBD). Dots represent frequencies of CD45RA−CD4+ memory T cells among total live
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LPMCs from different donors; connected dots represent matched biopsies. Demographic and

clinical characteristics of IBD patients are summarized in Supplementary Table 7. Statistics: panels
EP

A, B and C, Mann-Whitney test; panel C, one-way ANOVA with Sidak’s multiple comparison test.
C

Figure 7. Microbiota-reactive CD4+ T cells show a Th17-skewed phenotype in IBD patients.

AC

(A-B) PBMCs isolated from healthy donors and IBD patients were stimulated with heat-inactivated

bacteria or SEB and analysed for intracellular CD154 and cytokine expression.

(A) Frequencies (± SEM) of IL-17A, IFN-γ, and IL-22 expression in CD154+TNF-α+ memory

CD4+ T cells (n=23-33 independent donors). Demographic and clinical characteristics of IBD

patients are summarized in Supplementary Table 6.

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(B) Frequencies (± SEM) of IL-17A and IFN-γ co-expression in CD154+TNF-α+ memory CD4+ T

cells after short-term stimulation with heat-inactivated bacteria (n=23-34 independent donors).

(C) LPMCs from inflamed IBD surgical specimens or non-inflamed and tumour-free surgical

specimens from colorectal cancer patients were stimulated with heat-inactivated E. coli. Boolean

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gating shows each possible combination of IL-17A, IFN-γ, and IL-22 production by

CD154+TNF-α+ memory CD4+ T cells (n=6 and n=7 independent donors for IBD and controls,

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respectively).

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(D) Q-PCR analysis of IL1B, IL6, and IL23A in intestinal mucosal specimens categorised by

endoscopic assessment of disease activity. Demographic and clinical characteristics of IBD patients

U
are summarized in Supplementary Table 5.

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(E) CD4+CD45RO+CD45RA-CD25-CD8- memory CD4+ T cells were isolated from healthy donor

blood, labelled with CFSE, and stimulated with autologous monocytes pulsed with B. animalis in
M
the presence or absence of the indicated cytokines. Data represent mean (± SEM) fold changes in

IL-17A or IFN-γ expression frequencies relative to cells expanded without cytokines. Statistics:
D

panels A, B and C, Mann-Whitney test; panels D and E, one-way ANOVA with Sidak’s multiple
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comparison test.
C EP
AC

33
 ACCEPTED MANUSCRIPT

A Firmicutes
Bacteroidetes
Proteobacteria
E E. coli B. animalis
Actinobacteria 100 100

+ Bacterial lysate
Multicolor 80 80

8-16h
Intracellular flow cytometry 60 94
6
60 88
12
stain (Detection of
+BFA surface molecules) 40 40

% of maximum
PBMCs
20 20

B 10
**** *** ** * ** * ** ** ** * ****
0
0 10

F. prausnitzii
2
10
3
10
4
10
5
0
0 10 2

SEB
10 3 10 4 10 5
CD154+ of CD4+ cells (%)

PT
100 100

1 80 80

93 7 42
60 60 58

0.1 ns
40 40

20 20

RI
0.01 0 0
0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5

0.001 CD45RA
CD154+ CD4+ T cells
0.0001 Total CD4+ T cells

SC
0.00001
F **** **** **** **** **** **** **** **** **** **** ns
B. usn lus

lb sis
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S

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80

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50
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4
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30
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20
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3.31 0.77 3.09 0.99

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EP

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10 10

D 0.08
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*** 0.03
B. animalis L. acidophilus F. prausnitzii
** 0.03 *** 0.015 **
10
0
2

30.9 65
10
0
2

57.7 38.2
2 3 4 5 3 4 5
0 10 10 10 10 0 10 10 10

7.6 2.63 7.6 2.63

B. animalis

10 5 10 5

0.06
C

0.02 0.02 0.010 10 4 10

4
CD154+ of CD4+ cells (%)

0.04 10 3 10 3
AC

10 2 10 2
CD45RA

0.01 0.01 0.005 0 0

0.02 21.3 68.4 29.2 60.5

0 10 2 10 3 10 4 10 5 0 10 3 10 4 10 5
F. prausnitzii

0 0 0 0.000 10
5 4.46 1.79 10
5 6.25 0

SEB S. aureus M. tuberculosis C. albicans 10

4 10 4

8 ns 0.15
*** 0.15
*** 0.15 ** 10 3 10 3

2
10 2 10

6 0 0

0.10 0.10 0.10 23.2 70.5 43.8 50

3 4 5
10 2 10 3 10 4 10 5 0 10 10 10
4
0

10 5 36.5 3.93 10
5
32.1 8.31
0.05 0.05 0.05 4 4

2
10 10
SEB

10
3
10 3

0 0 0 0 10 2 10
2

Adult blood
0 0

26.7 32.9 25.1 34.5

Cord blood 0 10 2 10 3 10 4 10 5 0 10
3
10
4
10
5

TNF-α IL-2

Figure 1
 ACCEPTED MANUSCRIPT
Bacterial lysate
+αCD40 CD4+ CD154+ T cells
A +αCD28
8-16h
CD154
surface CD154
Multicolor B Total CD4+ T cells
Naive
flow cytometry
5

CCR7+ of CD45RA- CD4

stain
10

magnetic

CD45RA
enrichment (Detection of 10 4
100
PBMCs chemokine receptors
and surface molecules)
SEB, gated on CD3 cells + 10 3
80

T cells (%)
0

Original fraction Positive fraction negative fraction 60

EM CM
10 5 10 5 10 5
4.8 49 0.5 0 10 2 10 3 10 4 10 5
40
10 4 10 4 10 4
CCR7
CD154

10 5
20
10 3 10 3 10 3

CD45RA
10 4
0

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D Total CD4+ T cells
C
10 5 8.25 0.8 8.96 0.13 6.65 2.44 8.08 1.01

SC
R β7

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C R3

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81.6 9.34 89.8 1.09 20.7 70.2 5.47 85.4

U
60 B. animalis Integrin β7 CCR2 CCR4 CCR6
40

20
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F. prausnitzii
AN 10 5

10

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4
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0
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M
91.7 0.8 91.6 0.74 80.9 10.1 21.4 69.5
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CCR9 CCR10 CXCR3 CD161

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D

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ns ns ns ns ns
20
ns ns ns ns ns
80
ns ns ns ns ns
CD4+ CD154+
E CD4+ CD154+ CD45RA- T cells memory T cells 15 15 60
TE

Total CD4+ CD45RA- T cells

10 5 83.3 10.1 90.6 2.27 10 10 40
4
10
Percentage of CD154+ CD4+ T cells

Integrin β7 5 5 20
CCR4
0 CCR6
CCR7
EP

CD161 0 0 0
6.37 0.19 6.5 0.6 CCR4 CCR6 CCR7
100 100 ns ns ns ns ** 100 ns ns ns ns ns
CCR6

n=4 ns ** * * ***
Integrin β7 CCR9 80 80 80
10 5 15 78.5 16.1 77.4 20.6 73
60 60 60
C

10 4

40 40 40
AC

20 20 20
0

1.24 5.33 0.33 6.23 3.14 3.28 0 0 0

0 10 3 10 4 10 5 0 10
3
10
4
10
5
0 10 2 10 3 10 4 10 5
CCR10 CXCR3 CXCR5 CD161
20 50 ns ns ns ** ns 20 ns ns ns ns ns 80
CCR4 CCR7 CD161 ns ns ns ns * * * ns ns ns * ***
40
15 15 60
30
10 10 40
20
5 5 20
10

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Figure 2
 ACCEPTED MANUSCRIPT

A Firmicutes
Bacteroidetes
Proteobacteria
D 60
IL-17A
Actinobacteria

****
****
***

***
ns ns ns ns ns ns

*
+ Bacterial lysate
50
Intracellular Multicolor
8-16h stain flow cytometry
+BFA (Detection of intracellular 40
LPMCs cytokines and
PBMCs surface molecules)
30
B 20

PT
E. coli B. animalis L. acidophilus
6000 400 1500 ns
**** * 10

Cytokine+ of TNF-α+ CD154+ CD4+ cells (%)

(0.057)
5000
300
4000 1000 0

RI
3000 200 IFN-γ
2000 500 100
Microbiota-reactive cells/106 CD4+ cells

****
****

****
100 90 ns ns ns

**
**

**
1000

*
80

SC
0 0 0
70
F. prausnitzii B. vulgatus R. intestinalis R. obeum 60
300 250 250 1600 50
** ** **** 1400
**
250 40
200 200 1200 30

U
200 1000
150 150 20
150 800
100 100
10
600
100
50
0
50

0
50

0
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200
0 15
0

Control mucosa
IL-22

SEB S. aureus M. tuberculosis C. albicans Peripheral blood

M
40000 ns 2500 ns 1000 **** 2500 *** ns ns ns ns ns ns ns ns

*
*
10
2000 800 2000
30000
1500 600 1500
D

20000
1000 400 1000 5
10000
500 200 500
TE

0 0 0 0 0
Control mucosa (n=17)
B. aus ilus

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00

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20

30

40

50

Microbiota-reactive cells
/106 CD4+ cells

Figure 3
 ACCEPTED MANUSCRIPT

A 0
no microbe
5
0.56
E. coli B. animalis L. acidophilus
0.17 0.27 39.6
SEB B

CFSElow of CD4+ CD3+ cells (%)

5 10 5 5 5
10 10 10 10

100

*
****

***

****
4

0.62
4 10 4 4 4

0.14 0.5 0.49 2.4

10 10 10 10

Day 3 10 3 10
3
10 3 10 3 10 3

80
0 0 0 0 0

0.06
60
ICOS

0.14 0.12 0.1 0.9

2 3 4 5
2 3 4 5 0 10 10 10 10 2 3 4 5 2 3 4 5 2 3 4 5
0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10

0.03 19.5 13.6 12.1 65.1

3.27
1.31

0.7
1.45
10 5 10 5 10 5 10 5 10 5

40
0.1
4 4 4 4
10 10 4 10 10 10

Day 6 10 3 10
3 10 3 10 3 10 3

10
2
10
2
10
2
10
2
20
0
0 0 0 0

0.22 1.14 1.81 4.38 21

PT
0 10
3
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4
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4
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3
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4
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RI
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IL-17A IFN-γ

****
****
***
****

****
***
***
*
****
ns
SC
100 100
CD4+ cells

80 80
L. acidophilus F. prausnitzii SEB

Cytokine+ of CFSElow CD4+ cells (%)

60 60

U
40 40

TRBV9
TRBV5-6
TRBV20-1
TRBV5-5
TRBV28
TRBV4-1,2,3
TRBV29-1
TRBV4-3
TRBV5-1
TRBV12-3,4
AN 20

0
20

0
TRBV3-1 TRBV25-1 TRBV10-3 TRBV6-5,6,9 TRBV6-2
TRBV6-6 TRBV27 TRBV14 TRBV19 TRBV18 IL-22 IL-4
50 25
M
TRBV30 TRBV11-2 TRBV2 TRBV13 Unkown TRBV

ns

ns

ns
ns
ns
ns
ns
**
*
*
40 20

E B. animalis SEB
30 15
D

E. coli
20 10
TE

10 5

0 0
L. acidophilus F. prausnitzii
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B
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FS
C

IL-17A + + + + - - - -
IFN-γ + + - - + + - -
IL-22 + - + - + - + -
G E. coli B. animalis SEB
C

F
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SEB E. coli B. animalis

10
5
1 0.1 10 5 17.1 0.37 10 5 5.8 0.7
10 4 10 4 10 4
RORγt

3 3
10 10 10 3

10 2 10 2 10 2
RORγt + + + + - - - -
0 0 0
T-bet + + - - + + - -
72.6 26.3 31.5 51 15.5 78 GATA-3 + - + - + - + -
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5

10 5 46.3 0.8 10
5
18.6 0.35 10 5 9.35 0.5
4
10 10 4 10 4
T-bet

10 3
3
10 10 3

10 2
2
10 10 2

0 0 0

27.2 25.7 29.9 51.1 11.9 78.3

10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5
2 3 4 5
0 0 0 10 10 10 10

CFSE

Figure 4
 ACCEPTED MANUSCRIPT
A IL1B CSF2 IL6 CXCL1 CXCL5 CXCL8
25 **** 15 ** 15 *** ** 15 **** ** 15 **** ** 20 **** **
Expression relative to
**** ns

control treated (log2)

ns ns * ns* ** ns *
20 **** * * *
15
10 10 10 10
15
10
10
5 5 5 5
5
5

0 0 0 0 0 0
CXCL9 CXCL10 CXCL11
30 **** **** ****
Expression relative to

ns 15 ns 15
control treated (log2)

ns
ns ns ns
ns ns ns
Control treated
20 10 10
SEB expanded T cells

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Proteobacteria-reactive T cells
10 5 5
Actinobacteria-reactive T cells
Firmicutes-reactive T cells

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0 0 0

B TNF CXCL1 CXCL5 CXCL8 CXCL9 CXCL10 CXCL11

****
ICAM1
15 ns
* 10 ns **
ns 15 ns ** 8 ns ns* 25 **** ns
ns 20 ****
ns
ns 25 ns ns
ns 6 **** ns ns
* *
Expression relative to

ns
control treated (log2)

ns ns
* * * *

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8 20 20
6 15
10 10 4
6 15 15
4 10
4 10 10
5 5 2
2 5
2 5 5

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0 0 0 0 0 0 0 0

CαIFN-γ AN D αIFN-γ
+ - - - + + + - - - + + - + + - + - - - + + + - - - + + - + + -
αIL-22 - + - - + - - + + - + - + + + - αIL-22 - + - - + - - + + - + - + + + -

trol
trol

αIL-17A - - + - - + - + - + + + + - + - αIL-17A - - + - - + - + - + + + + - + -

con
con

αTNF-α - - - + - - + - + + - + + + + - αTNF-α - - - + - - + - + + - + + + + -
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CXCL2 TNF
CSF2 CXCL5
CXCL3 CXCL2
IL1B CXCL1
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CXCL5 CXCL3
CXCL6 MUC1
CXCL8 CXCL8
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CCL3 IL6
IL6 REG3G
CXCL1 CXCL9
ICAM1 CXCL10
CXCL10 CXCL11
CXCL11 ICAM1
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CXCL9 CCL2
CCL7 -3 -2 -1 0 1 2 3 LIM1863
CCL2
-3 -2 -1 0 1 2 3 CCD18co IL-17A-/TNF-α-dependent module
C

IFN-γ-dependent module

CXCL2 CSF2 CXCL3 CXCL5 CXCL6 CXCL8 CXCL1

E
AC

ns ns ns ns ns ns ns
10 **** ns 10 **** ns 10 **** ns 20 **** ns 15 **** ns 15 **** * 10 **** ns
Expression relative to control median (log2)

8 8 8 15 8
10 10
6 6 6 10 6
4 4 4 5 5 5 4
2 2 2 0 2
0 0
0 0 0 -5 0
-2 -2 -2 -10 -5 -5 -2
CXCL10 CXCL11 CXCL9
ns ns ns
10 ** ns 10 ** ns 10 ** ns
8 8 8
Controls (n=9)
Active IBD, lesional site (n=30)
6 6 6
Active IBD, uninvolved site (n=20)
4 4 4 IBD, remission (n=14)
2
0
2
0
2
0 Figure 5
-2 -2 -2
 ACCEPTED MANUSCRIPT

A PBMCs C
E. coli B. animalis L. acidophilus **

CD4+ CD45RA- of total live cells (%)

0.06 0.03 **** 0.03 *** 45
****
**
40
0.04 0.02 0.02
35

0.02 0.01 0.01 30

25 ns
0.00 0.00 0.00
20
F. prausnitzii B. vulgatus R. intestinalis R. obeum
CD154+ of CD4+ cells (%)

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0.015 *** 0.015 *** 0.008 **** 0.008 *** 15

0.006 0.006 10
0.010 0.010
0.004 0.004 5

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0.005 0.005 0
0.002 0.002
Controls (n=13)
0.000 0.000 0.000 0.000 Active IBD, lesional site (n=15)

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SEB S. aureus M. tuberculosis C. albicans Active IBD, uninvolved site (n=15)
** ns * *
8 0.15 0.10 0.08

6 0.08
0.06
0.10
0.06

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4 0.04
0.04
0.05
2 0.02
0.02

0
Control (n=30)
0.00 0.00 AN
0.00

IBD (n=38)
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LPMCs
B 0.5
E. coli
ns 0.04
B. animalis
ns 0.03
L. acidophilus
ns
D

0.4
0.03
0.02
0.3
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0.02
0.2
0.01
0.01
0.1
CD154+ TNF-α+ of CD4+ cells (%)

0.0 0.00 0.00

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F. prausnitzii B. vulgatus R. intestinalis R. obeum

0.025 ns 0.04 ns 0.020 ns 0.025 ns

0.020 0.020
0.03 0.015
0.015 0.015
C

0.02 0.010
0.010 0.010
0.01 0.005
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0.005 0.005

0.000 0.00 0.000 0.000

SEB S. aureus M. tuberculosis C. albicans

4 ns 0.15 ns 0.020 ns 0.25 ns

0.20
3 0.015
0.10
0.15
2 0.010
0.10
0.05
1 0.005
0.05

0 0.00 0.000 0.00

Control mucosa (n=17-20)
Inflamed mucosa (n=10-15)

Figure 6
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A 70 IL-17A
E. coli
100 IFN-γ 40 IL-22
B E. coli
50 IL-17A IFN-γ 60 IL-17A IFN-γ 80 IL-17A IFN-γ
+ - + + - +

**** ** ns
60
90 **** ns
70 **
80 40 50
Cytokine+ of TNF-α+ CD154+ CD4+ cells (%)
30 60

Cytokine+ of TNF-α+ CD154+ CD4+ cells (%)

50 70
40
40 60 30 50
50 20 30 40
30 40 20 30
20 30 20
10 20
20 10
10 10

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10 10
0 0 0 0 0 0
SEB SEB
70 IL-17A 100 IFN-γ IL-22 50 IL-17A IFN-γ 60 IL-17A IFN-γ 80 IL-17A IFN-γ
+ - + + - +
40

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ns ns ns ns ns ns
90
60 70
80 40 50
50 30 60
70
40
60 30 50
40

SC
50 20 30 40
30 40 20 30
30 20
20 10 20
20 10
10 10
10 10

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0 0 0 0 0 0
Controls (n=23) Controls (n=23)
IBD (n=33) IBD (n=34)

C E. coli D
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IL1B IL6 IL23A
Expression relative to control

10 10 10
ns ns ns
M
**** ns **** ns **** ns
8 8 8
median (log2)

* 6 6 6
D

4 4 4
2 2 2
Control mucosa Inflamed mucosa
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0 0 0
TNF-α + + + + + + + +
-2 -2 -2
IL-17A + + + + - - - -
Controls (n=9)
IFN-γ + + - - + + - - Active IBD, lesional site (n=30)
IL-22
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+ - + - + - + - Active IBD, uninvolved site (n=20)

IBD, remission (n=14)

E IL-17A IFN-γ
C

2.5 2.5
** ns ns * ns ns ns ns
(relative to control)

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2.0 2.0
Cytokine+

1.5 1.5

1 1

0.5 0.5

0 0
IL-1β − + − − + − + − − +

IL-6 − − + − + − − + − +
IL-23 − − − + + − − − + +

Figure 7
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Supplemental Information

Induction of Local and Systemic CD4+ T-cell Responses to Intestinal Microbes in

Healthy Individuals and Alterations in Patients With Inflammatory Bowel Diseases

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Ahmed N. Hegazy*, Nathaniel R. West*, Michael J. T. Stubbington, Emily Wendt, Kim

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Suijker, Angeliki Datsi, Sebastien This, Camille Danne, Suzanne Campion, Sylvia Duncan,

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Benjamin M. J. Owens, Holm H. Uhlig, Andrew McMichael, Oxford IBD Cohort

Investigators, Andreas Bergthaler, Sarah A. Teichmann, Satish Keshav, Fiona Powrie#

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*
Co-first authors
#
Corresponding author
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Supplemental Figure Legends

Supplementary Figure 1. Bacterial strains used in the study and detection of enteric

bacteria-specific memory CD4+ T cells (related to Figure 1)

(A) Bacterial species are listed according to their family and phylum classification. Coloured

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circles relate the bacteria to their gaseous requirement.

(B) Frequencies of CD154+ cells among peripheral CD4+ T cells in adults after short-term

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stimulation with heat-inactivated bacteria. Dots represent different donors (n=30).

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(C) Representative flow cytometric plots of different stimulations showing CD154 expression

against CD4 within CD3+ cells after short-term stimulation with heat-inactivated bacteria.

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Frequencies of CD154 within CD4+ T cells are depicted.

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(D) Representative plot of CD69 and ICOS expression on CD4+CD154+ T cells after

short-term stimulation with heat-inactivated S. typhimurium.

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(E) PBMCs were stimulated with the indicated heat-inactivated bacteria in presence or

absence of MHC-II blocking antibodies and analysed for CD154 expression. Percentage of
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reduction in CD154+ frequencies after MHCII blockade compared to control treated is

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depicted. Frequencies (± SEM) of 3 independent experiments is depicted (n=5-6 independent

donors).
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(F) Estimated microbiota-reactive cells per million CD4+ T cells in adult blood (n=30). The

numbers of microbiota-reactive T cells were calculated based on the frequencies of

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CD4+CD154+ T cells. Background (no microbe stimulation) was subtracted from bacterial
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stimulations. Significance calculated in relation to LPS stimulation.

(G) Frequencies ± SEM of CD154+ cells among CD4+ T cells in adult (n=30) or cord blood

(n=3) after short-term stimulation with heat-inactivated bacteria.

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Statistics: panels E and G, Mann-Whitney test; C, and F, panel F, one-way ANOVA with

Sidak’s multiple comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001;

**** P≤0.0001.

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Supplementary Figure 2. Detection and characterisation of microbiota-specific memory

CD4+ T cells (related to Figure 1 and 2)

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(A) Frequencies of CD154+ cells among naïve and memory peripheral CD4+ T cells in adults

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after short-term stimulation with heat-inactivated bacteria. Dots represent different donors

(n=30).

(B) Percentage of memory cells within CD4+CD154+ T cells. Each symbol represents an

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antigen-reactive population from one individual; the line indicates the mean of experiments

performed independently with blood obtained at different times (n=19-30). Significance

calculated in relation to total CD4+ T cells.

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(C) Percentages of cytokine-expressing cells among memory CD4+CD45RA-CD154+ T cells
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are shown. Frequencies (± SEM) of 27 independent donors are depicted.

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(D) Surface marker and chemokine receptor expression frequencies among CD4+

CD45RA- CD154+ and CD4+ CD45RA- CD154- T cells after short-term stimulation with B.
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animalis. Frequencies within CD4+ CD45RA- CD154+ or CD4+ CD45RA- CD154- T cells are

depicted.
C

Statistics: panel A, Mann-Whitney tests; panels B, One-way ANOVA with Sidak’s multiple
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comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001.

Supplementary Figure 3. Detection and characterisation of microbiota-specific CD4+ T

cells within gut-resident memory T cells (related to Figure 3)

(A) Representative flow cytometry plots showing CD45RA, Integrin β7, CD69, CCR7, and

CD127 expression in gut-resident CD3+CD4+ T cells. Data representative of ten independent

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donors.

(B, C) Gating strategy for identification of enteric bacteria-specific memory CD4+ T cells in

LPMCs and PBMCs after stimulation with heat-inactivated E. coli in a representative donor.

(D) LPMCs were stimulated with heat-inactivated bacteria and analysed for CD154

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expression. Estimated gut resident microbiota-reactive cells per million CD4+ T cells

compared with adult blood is depicted (n=17 for control mucosa; n=25-31 for blood).

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(E, F) Cytokine production by CD4+CD154+TNF-α+ T cells after short-term stimulation with

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the indicated heat-inactivated bacteria. Frequencies (± SEM) of 10-23 independent donors.

Significance calculated between the respective stimulations in control mucosa and peripheral

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blood.

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Statistics: panel D, E and F, Mann-Whitney test; ns, not significant; *P≤0.05; ** P≤0.01; ***

P≤0.001; **** P≤0.0001.

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Supplementary Figure 4. Expansion and detection of microbiota-specific memory CD4+

T cells (related to Figure 4)

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Total memory CD4+ T cells were isolated, labelled with CFSE and cultured with autologous
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irradiated monocytes in the presence or absence of the indicated heat-inactivated bacteria and

blocking antibodies to MHCII.

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(A) Experimental setup.

(B) Shown are the CFSE profiles and ICOS expression on days 3 and 6 of stimulation in a
C

representative donor.
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(C) Frequencies (± SEM) of CFSElow proliferating cells after stimulation with S. aureus and

M. tuberculosis (n=5-21). Data representative of 5-10 independent experiments. Each dot

represents an independent donor.

(D) Frequencies (± SEM) of CFSElow proliferating CD4+ T cells in presence or absence of

anti–MHC-II. Data representative of two independent experiments.

(E, F) CFSE profiles and expression of CD25 and OX40 on day 6 of stimulation in a

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representative donor. (F) Geometric mean and frequencies (± SEM) of ICOS, CD25, and

OX40 expression on CFSElowCD4+ T cells on day 6.

(G) Number of unique TCRβ clonotypes detected in bacteria-reactive CD4+ T cells. TCRVβ

sequencing was performed from 3 independent donors. Each circle represents an independent

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donor. TCRVβ usage of the different reactivities of three independent donors is summarized

in Supplementary Table 3.

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(H) Heat map showing the frequency of shared clonotypes between different bacteria-reactive

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CD4+ T cell responses. SEB and PHA expanded memory T cells were used as controls. Data

from 3 independent donors. The CDR3 sequences are summarized in Supplementary Table 4.

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Statistics: panels C, D and F, One-way ANOVA with Sidak’s multiple comparison test; ns,

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not significant; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001.

Supplementary Figure 5. Cross-reactivity, cytokine production and transcription factor

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expression by antigen-reactive memory CD4+ T cells (related to Figure 5)

(A) CD154+ memory CD4 T cells from PBMCs were sorted after short-term stimulation with
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E. coli and B. animalis lysates and expanded them for 10-14 days with CD3/CD28 and IL-2.
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The E. coli Nissle- and B. animalis-reactive T cell lines were CFSE-labelled and then co-

incubated with autologous monocytes loaded with the various bacterial lysates. Frequencies
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(± SEM) of CFSElow proliferating cells after 5 days of stimulation with the indicated bacteria

are shown. Data from 3-6 independent donors.

C

(B-F) Total memory CD4+ T cells were isolated, labelled with CFSE and cultured with
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autologous irradiated monocytes in the presence of the indicated heat-inactivated bacteria or

antigens.
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(B, C) Production of cytokines by proliferating CFSE cells measured by intracellular

staining after PMA/Ionomycin stimulation on day 7 of primary stimulation with monocytes

and heat-inactivated bacteria. Frequencies (± SEM) of 3-13 independent donors are depicted.

Each dot represents an independent donor.

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(D, E) RORγt, T-bet and GATA-3 expression on day 7 of primary stimulation with monocytes

and influenza seasonal vaccination or heat-inactivated S. aureus in a representative donor.

(E) Boolean gating analysis showing each possible combination of RORγt T-bet and

GATA-3 production by CFSElow proliferating cells. Data generated from 3 independent

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donors.

(F) Production of IL-10 by expanded CFSElow CD4+ cells after PMA/Ionomycin stimulation.

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Frequencies (± SEM) and IFN-γ / IL-17A co-expression within IL-10+ and IL-10- cells are

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depicted from 5-8 independent donors.

Statistics: panels A, C, One-way ANOVA with Sidak’s multiple comparison test; panel F,

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Kruskal-Wallis test with Dunn’s multiple comparison; ns, not significant; *** P≤0.001; ****

P≤0.0001. AN
Supplementary Figure 6. Functional characteristics of enteric bacteria-reactive CD4+ T
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cells in IBD (related to Figure 7)

(A) PBMCs isolated from healthy donors or IBD patients were stimulated with the indicated
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heat-inactivated bacteria and analysed for CD154 expression. Frequencies (± SEM) of

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reactive CD154+ cells among CD4+ T cells in peripheral blood are depicted (n=30-38).

Demographic and clinical characteristics of IBD patients are summarized in Supplementary

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Table 6.

(B) LPMCs were isolated from inflamed surgical specimens from IBD patients or
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non-inflamed and tumour-free surgical specimens from colorectal cancer patients. Isolated
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LPMCs were stimulated with the indicated heat-inactivated bacteria and analysed for CD154

expression. Frequencies (± SEM) of reactive CD154+ TNF-α+ cells among CD4+ T cells in

peripheral blood are depicted (n=10-20).

(C) Microarray analysis of intestinal biopsies obtained at endoscopy (n=6 controls, 24 UC and

37 CD; Gene Expression Omnibus entry GSE16879). The relative abundance of memory

CD4+ T cells in control and inflamed IBD tissue was estimated in silico using CIBERSORT

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(see Supplementary Methods for details).

(D-F) PBMCs isolated from healthy and IBD patients were stimulated with the indicated

heat-inactivated bacteria or SEB and analysed for CD154 expression and intracellular

cytokine expression. Demographic and clinical characteristics of IBD patients are

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summarized in Supplementary Table 6.

(D) IL-2 expression in CD154+ TNF-α+ memory CD4+ T cells after stimulation with the

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indicated heat-inactivated bacteria or SEB. Frequencies (± SEM) of 23-33 independent

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donors. Each dot represent an independent donor.

(E, F) IL-17A, IFN-γ, IL-22, and IL-2 expression in CD154+ TNF-α + memory CD4+ T cells

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after stimulation with the indicated heat-inactivated bacteria. Each dot represents an

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independent donor. Demographic and clinical characteristics of IBD patients are summarized

in Supplementary Table 6.
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(G) IL-17A expression in CD154+ TNF-α + memory CD4+ T cells after stimulation with S.

typhimurium. IBD patients were categorised by disease phenotype (UC, CD), disease activity
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according to clinical notes, and current treatment.

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(H) Total memory CD4+ T cells were isolated from healthy controls or IBD patients blood,

labelled with CFSE and cultured with autologous irradiated monocytes in the presence of the
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indicated heat-inactivated bacteria or antigens. Production of IL-10 by expanded CFSElow

CD4+ cells after PMA/Ionomycin stimulation on day 7 of stimulation. Frequencies (± SEM)

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are depicted from 9-11 independent donors.

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(I) CD4+CD45RO+CD45RA-CD25-CD8- memory CD4+ T cells were isolated from healthy

controls or IBD patients blood, labelled with CFSE, and stimulated with autologous

monocytes pulsed with B. animalis in the presence or absence of the indicated cytokines. Data

represent mean (± SEM) fold changes in IL-17A or IFN-γ expression frequencies relative to

cells expanded without cytokines.

Statistics: panels A-E and G-H, Mann-Whitney test; panel F, One-way ANOVA with Sidak’s

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multiple comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001; ****

P≤0.0001.

Supplementary Figure 7. Microbiota-specific memory T cell responses in intestinal

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homeostasis and inflammation

Microbiota-specific memory CD4+ T cells are abundant in peripheral blood and mucosa.

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Luminal sampling of intestinal microbiota by dendritic cells and repetitive epithelial breaches

after birth might induce CD4+ T cell responses targeted towards intestinal bacteria. Enteric

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bacteria-specific CD4+ T cells produce barrier-protective cytokines such as IL-17A, IFN-γ

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and IL-22. Furthermore, certain commensal-specific CD4+ T cells also produce IL-10. We

speculate that the cytokine production by commensal-specific CD4+ T cells might play a
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significant role in supporting gut homeostasis and the mutualistic relationship with intestinal
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microbiota. In inflammation, epithelial break down and leakage leads to increased availability

of luminal antigens and bacterial stimuli, which activate the innate system and the expression
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of Th17 inducing cytokines (IL-1β, IL-6 and IL-23). Recruited microbiota-specific memory
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CD4+ T cells accumulate in inflamed mucosa and are polarised towards more IL-17A

production. Activated memory cells express IL-17A, TNF-α, and IFN-γ, which stimulate
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intestinal cells such as stromal and epithelial cells to express various chemokine ligands and

cytokines. This may promote recruitment and activation of myeloid cell populations.
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Supplemental Tables

Supplementary Table 1. Table S1. Enteric bacterial species and control species used in the

study (related to Figure 1).

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Supplementary Table 2. T cell receptor (TCR) Vβ usage in expanded microbiota-reactive T

cells measured by Vβ antibody panel from Beckman Coulter (related to Figure 3).

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Supplementary Table 3. T cell receptor (TCR) Vβ usage in expanded microbiota-reactive T

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cells using multiplex PCR assay and deep sequencing (related to Figure 3).

Supplementary Table 4. CDR3 sequences of microbiota-reactive memory CD4+ T cells

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(related to Figure 3).

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Supplementary Table 5. Clinical characteristics of Oxford cohort patients assessed in this

study for gene expression analysis. (related to Figure 5E and 7D).

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Supplementary Table 6. Clinical characteristics of Oxford cohort patients assessed in this

study for the analysis of microbiota-specific CD4 T cells. (related to Figure 7A, B, and
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Supplementary 6A-H).
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Supplementary Table 7. Clinical characteristics of Oxford cohort patients assessed in this

study for cell accumulation in the mucosa. (related to Figure 3B, C and Figure 7C).
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Supplemental Experimental Procedures

Human Samples and Cell Isolation. Peripheral blood mononuclear cells (PBMCs) were

isolated by Ficoll-Paque (GE Healthcare Life Sciences) density gradient centrifugation,

resuspended in PBS with 2mM EDTA and 0.02% BSA, and further processed. Gut specimens

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were obtained from patients with IBD undergoing surgery for severe, chronically active, or

complicated disease. Control gut specimens from macroscopically healthy areas were

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collected from colorectal cancer patients as non-inflammatory controls. One intestinal pinch

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biopsy was obtained from healthy donors (Colorectal cancer screening, or other non-IBD

related conditions) or IBD patients during routine endoscopy, from lesional, and non-lesional

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sites, attending the John Radcliffe Hospital (Oxford, UK). Inflammation status of biopsies

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was binarized into either inflamed or uninflamed categories based on endoscopic assessment.

Further information about the analysed IBD patients can be found in Supplementary Table
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5, Supplementary Table 6, and Supplementary Table 7.

LPMCs were isolated as described1. In brief, mucosa was dissected and washed in 1 mM DTT
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at room temperature for 15 min to remove mucus. Specimens were washed three times with
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0.75 mM EDTA at 37°C for 45 min to detach epithelial crypts and digested overnight with

0.1 mg/ml collagenase D (Roche). Cells were centrifuged for 30 min in a Percoll gradient and
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collected at the 40–60% interface. All solutions were supplemented with antibiotics

(penicillin/streptomycin, 40 µg/ml gentamicin, and 0.025 µg/ml amphotericin B).

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Flow Cytometry. Cells were stained with the following monoclonal antibodies as described2:
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CD4-BV510 (OKT4), CD4 PE-Dazzle (RPA-T4), CD3-PE-CF594 or –BV510 (UCHT1),

CD45RA-BV711 (HI100), CD45RO-BV570 (UCHL1), CD161-eF450 (HP3G10), CCR7-PE

(G034H7), CCR2-APC (K036C2), CCR6-BV605 (G034E3), CCR9-PE-Cy7 (L053E8),

CXCR3-PE/Cy5 (1C6/CXCR3), CCR4-PE-Cy7 (1G1), CCR10-PE (314305), Integrin

7-FITC/APC (FIB504), CD69-PE-Cy7 (FN50), IL-2-BV650 (MQ1-17H12), TNF- -PB

(MAb11), CD154-FITC/PE-Cy5 (24-31), IL-17A BV711 (eBio64DEC17), IL-22-PE-Cy7

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(22URTI), IFN-γ-AF700 (B27), GM-CSF-APC (BVD2-21C11), IL-4-PE (8D4-8)

(eBioscience, Biolegend and Becton Dickinson). FACS panels contained up to 12

fluorochromes. Compensation beads were used for compensations (BDBioscience). TCRVβ

usage was assessed using the IOTest Beta Mark TCR Vbeta Repertoire Kit (Beckman

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Coulter). Samples were acquired on FACS LSRFortessa and FACSLSRII (Becton

Dickinson); ≥2x105 memory CD4+ T cells were acquired. Data were analysed with FlowJo

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(Tree Star) and SPICE. For analysis of cytokine expression by microbiota-specific T cells, a

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minimum of 20 CD4+ CD154+ TNF-α + T cells was used; donors with lower events were

excluded.

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CD154 Enrichment of Antigen-specific T Cells. For MACS enrichment of CD4+CD154+ T

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cells, CD154 Enrichment and Detection Kit was used (Miltenyi Biotec). Briefly, cells were

plated at 5x106/cm2 for 7-12 h with heat-inactivated bacteria in the presence of anti-CD40
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blocking antibody (HB14) and anti-CD28 stimulation antibody (CD28.6). Anti-CD40

blockade prevents down-regulation of CD154, while CD28 co-stimulation optimizes

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induction of CD154 expression. For MHCII blockade, 10 µg/ml of a pan-HLA class-II

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blocking antibody (HLA-DR, DP, DQ; (Tü39)) was added 30 minutes before bacterial

stimulation. Cord and adult blood analysis was performed identically. Antigen-presenting cell
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(APCs) abundance is similar in cord and adult blood3,4.

Cross Reactivity Assay. CD154+ CD4+ T cells were isolated using MACS enrichment and
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FACS sorting from total PBMCs after stimulation with E. coli Nissle and B. animalis lysates
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as described above. CD154+ CD4+ T cells were expanded for 10-14 days with IL-2 and anti-

CD3/CD28 beads (beads/T cell ratio, 1:4, Dynals). The expanded T cell lines were washed in

IL-2 free medium and incubated for 12h without IL-2 in RPMI-1640 supplemented with 2

mM glutamine, 1% (v/v) non-essential amino acids, 1% (v/v) sodium pyruvate, penicillin (50

U/ml), streptomycin (50 mg/ml; all from Invitrogen) and 5% (v/v) human serum (National

Blood Service, Bristol, UK). The expanded T cell lines were labelled with CFSE, and then

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were co-incubated with autologous monocytes loaded with various bacterial lysates. T cells

were co-cultured with the irradiated autologous monocytes at a ratio of 2:1 for 5-7 days. The

CFSE dilution was measured at the end of the culture. Autologous CD14+ monocytes were

isolated from PBMC using anti-CD14 microbeads (Miltenyi Biotec) and frozen until the T

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cell co-culture. Monocytes were thawed down, irradiated (45 Gy) and then pre-incubated for

3 h with bacterial lysates before T-cell co-culture.

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Intracellular Cytokine Analysis. For intracellular cytokine staining, cells were restimulated

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with PMA (5 ng/ml) and ionomycin (500 ng/ml; Sigma) or bacterial lysates. 5 µg/ml brefeldin

A (Sigma-Aldrich) was added at 2 h. After 4-12 h, cells were stained with fixable viability

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dye eFluor® 780 (eBioscience) and surface markers, fixed with 2% formaldehyde (Merck),

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and stained for cytokines in buffer containing 0.05% saponin (Sigma-Aldrich). In some

assays, cytokine analysis was combined with intracellular CD154 detection as described5,6.
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Transcription Factors Staining. Transcription factor expression was analysed using the

FoxP3 staining buffer set (eBioscience) according to manufacturer’s instructions. Briefly,

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cells were stained with fixable viability dye eFluor® 780 (eBioscience) and surface markers,
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fixed with 1x Fixation/Permeabilization buffer, followed by antibody staining and washing in

1x permeabilization buffer. Tbet-Pacific Blue (4B10) and GATA-3-Alexa-647 (TWAJ) were

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from eBioscience. RORγt-PE (Q21-559) was from BDBioscience.

Preparation of Bacterial Lysates. Different bacteria were cultured in their respective

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optimal media for 16h at 37 °C, washed in sterile PBS and heat-inactivated at 65 °C for 1
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hour, followed by three freeze-thaw cycles. Extremely oxygen sensitive bacteria were

provided by Sylvia Duncan, University of Abeerden, after heat-inactivation. Suspensions

were centrifuged at max speed for 15 min and supernatants collected. Protein concentration

was quantified using Nanodrop (Thermo Fisher Scientific). The following bacterial strains

were used: Bacteroides vulgatus (Bv 1447), Bifidobacterium animalis subsp. lactis Bi-07,

Clostridium difficile (OXF1003, Toxin AB-), E. coli (Nissle 1917), Faecalibacterium

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prauznitzii (A2-165), Lactobacillus acidophilus (NCFM), Roseburia intestinalis (M50/1),

Ruminococcus obeum (A2-162), Salmonella enterica serovar typhimurium (NCTC 12023),

Staphylococcus aureus (NCTC 6571). Further information regarding the bacterial stains is in

Supplementary Table 1. Bacterial lysates were titrated in CFSE dilution assay and an

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optimal concentration was used (5-15 µg/ml). Tetanus Toxoid (Calbiochem) and influenza

seasonal vaccine (OPTAFLU, Novartis) were used at 5 µg/ml. Heat-killed Mycobacterium

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tuberculosis (H37Ra) and Candida albicans were from InvivoGen. Ultrapure LPS-EB from

E. coli 0111:B4 (InvivoGen) was used as a stimulation control. SEB was used at 1 µg/ml

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(Sigma).

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RNA Extraction, cDNA Synthesis and qPCR. Tissue was homogenized using a motor with

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sterile RNase/DNase-free disposable pestles (both VWR) in RLT buffer (QIAGEN). Cells

were lysed directly in RLT buffer. RNA was isolated using RNeasy Mini kit (QIAGEN) or
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Quick-RNATM MiniPrep kit (Zymo Research, Irvine, USA) followed by cDNA preparation

using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with random
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hexamers. Q-PCR was performed using a CFX96 (Bio-Rad) or ViiA7 384-well real-time PCR
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system (Applied Biosystems) with TaqMan assays (Life Technologies) and PrecisionPLUS

Mastermix (Primerdesign). Expression levels were normalized to a house-keeping (hk) gene

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(RPLP0) and expressed as 2^-(CTgene-CThk). Heat maps were made using Cluster 3.0 and

Java TreeView (Michael Eisen, Stanford University).

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CIBERSORT Analysis. To calculate enrichment of cell populations using CIBERSORT7, we

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analyzed the GSE16879 dataset on default settings. For each sample, relative expression of

PTPRC (CD45, representing relative leukocyte content) was calculated separately using

median-normalized microarray data. This value was then multiplied by CIBERSORT cell

type scores (e.g. proportion of memory CD4+ T cells in the total leukocyte fraction) to

estimate cell type enrichment levels. Finally, for cell types of interest, fold differences

between IBD and control specimens were calculated to estimate relative cell type abundance

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in active IBD lesions versus healthy tissue.

β Sequencing and Analysis. Memory T cells (3.6x106) were cultured with autologous
TCRVβ

monocytes pulsed with heat-inactivated bacteria from three healthy donors. Expanded

CFSElow ICOS+CD4+ T cells were sorted into DNase/RNase free water with BSA (10 mg/ml)

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in 96 well plates (100 cells/well) and stored at -80°C. TCRVβ sequence analysis was obtained

by a series of three nested PCR reactions as described previously8. Reverse transcription and

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preamplification were performed with a One-Step RT-PCR kit (Qiagen) using multiplex PCR

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with multiple Vβ region primers and a Cβ region primer. After the first reaction, an aliquot

was used for the second PCR using a set of multiple internally nested TCRVβ primers and

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internally nested Cβ region primer with HotStarTaq DNA polymerase kit (Qiagen). In the

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final PCR reaction, an aliquot of the second PCR was used and amplification was performed

using barcoding primers containing the common 23 base sequence (incorporated into the
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second set of Vβ primers) and a third internally nested Cβ primer and Illumina Pair-End

primers. After the third PCR reaction, each PCR product should have a unique set of barcodes
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incorporated that specifies plate, row and column and have Illumina Paired-End sequences
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that enabled sequencing on the Illumina MiSeq platform. The PCR products were combined

at equal proportion by volume, run on a 1.2% agarose gel, and a band around 350 to 380 bp
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was excised and gel purified using a Qiaquick gel extraction kit (Qiagen). Barcoded products

were sequenced on the Illumina MiSeq platform.

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Sequence reads were trimmed using 'Trim Galore' software

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(http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with default settings.

Reads were demultiplexed using a custom Python script according to the presence of index

sequences. A further custom script then split reads from each well according to the presence

of primer sequences used in the PCR amplification. These reads were then analysed by

MICXR v1.6 (ref to http://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3364.html)

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using settings appropriate for the anticipated TCR locus of origin (A or B). MIXCR provided

CDR3 sequences along with counts of the number of times each sequence was observed.

Detected CDR3 sequences were filtered to exclude those with ≤10 counts per well. There

were 48 wells per donor of SEB-stimulated or PHA-stimulated cells whilst there were

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96-wells of bacterially-stimulated cells. Furthermore, some wells did not contain any valid

TCR sequences after filtering for low read counts suggesting that amplification or sequencing

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had failed for those wells. To permit comparisons of the number of TCR sequences between

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plates with differing numbers of successful wells, counts were scaled to the theoretical

maximum of 96 wells.

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Supplemental References

1. Geremia A, Arancibia-Cárcamo CV, Fleming MPP, et al. IL-23-responsive innate

lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011;208:1127–

1133.

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2. Brodie T, Brenna E, Sallusto F. OMIP-018: Chemokine receptor expression on human T

helper cells. Cytometry A 2013.

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3. Marodi L, Leijh PCJ, Furth RV. Characteristics and Functional Capacities of Human

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Cord Blood Granulocytes and Monocytes. Pediatr Res 1984;18:1127–1131.

4. Ebba Sohlberg SS-HKBES-E. Cord blood monocyte subsets are similar to adult and

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show potent peptidoglycan-stimulated cytokine responses. Immunology 2011;133:41.

5. AN
Frentsch M, Arbach O, Kirchhoff D, et al. Direct access to CD4+ T cells specific for

defined antigens according to CD154 expression. Nat Med 2005;11:1118–1124.

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6. Bacher P, Schink C, Teutschbein J, et al. Antigen-Reactive T Cell Enrichment for Direct,

High-Resolution Analysis of the Human Naive and Memory Th Cell Repertoire. The
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Journal of Immunology 2013;190:3967–3976.

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7. Newman AM, Liu CL, Green MR, et al. Robust enumeration of cell subsets from tissue

expression profiles. Nat. Methods 2015;12:453–457.

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8. Han A, Glanville J, Hansmann L, et al. Linking T-cell receptor sequence to functional

phenotype at the single-cell level. Nat Biotechnol 2014;32:684–692.

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Supplementary Table 1. Enteric bacterial species and control species used in the study
Bacterial species Strain Family Phylum Kingdom Phylum Gram Growth Changes Refere
Designation abundance stain characteristics during IBD on nce
(of the species) Phylum level

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Bacteroides vulgatus Bv 1447 Bacteroidaceae Bacteroidetes Bacteria ~20-40% Negative Obligate ê 1, 2, 3,
anaerobe 4
Bifidobacterium animalis subsp. Bi-07 Bifidobacteriaceae Actinobacteria Bacteria ~1-10% Positive Obligate ê (at  family   1, 2, 3,

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lactis anaerobe level) 4
Clostridium difficile OXF1003, Clostridiaceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3,

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Toxin AB- anaerobe 4
Escherichia coli Nissle 1917 Enterobacteriaceae Proteobacteria Bacteria ~5-15% Negative Facultative é 1, 2, 3,
anaerobe 4
Faecalibacterium prauznitzii A2-165) Ruminococcaceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3,

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anaerobe 4
ê

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Lactobacillus acidophilus NCFM Lactobacillaceae Firmicutes Bacteria ~50-70% Positive Obligate 1, 2, 3,
anaerobe 4
Roseburia intestinalis M50/1 Lachnospiraceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3,
anaerobe 4

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Ruminococcus obeum A2-162 Lachnospiraceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3,
anaerobe 4

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Salmonella enterica serovar NCTC 12023 Enterobacteriaceae Proteobacteria Bacteria ~5-15% Negative Facultative é 1, 2, 3,
typhimurium anaerobe 4

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Staphylococcus aureus NCTC 6571 Staphylococcaceae Firmicutes Bacteria ~50-70% Positive Facultative not invovled 1, 2, 3,
anaerobe (at family 4
level)
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Mycobacterium tuberculosis H37Ra Mycobacteriaceae Actinobacteria Bacteria ~1-10% Positive Obligate not invovled 1, 2, 3,
aerobe (at family 4
level)
Candida albicans Debaryomycetaceae Saccharomycetes Fungi Facultative é 1, 2, 3,
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(Class) anaerobe 4
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References
1. Morgan XC, Tickle TL, Sokol H, et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 2012;13:R79.
2. Kostic AD, Xavier RJ, Gevers D. The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014;146:1489–1499.
3. Sokol H, Leducq V, Aschard H, et al. Fungal microbiota dysbiosis in IBD. Gut 2016.
4. Gevers D, Kugathasan S, Denson LA, et al. The Treatment-Naive Microbiome in New-Onset Crohn’s Disease. Cell Host Microbe 2014;15:382–392.  
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Supplementary Table 2. T cell receptor (TCR) Vβ usage in expanded microbiota-specific T cells measured by Vβ antibody panel from Beckman Coulter
(related to Figure 3)
Donors Microbe β1
Vβ1 β2
Vβ2 β3
Vβ3 β4
Vβ4 β5.1
Vβ5.1 β5.2
Vβ5.2 β5.3
Vβ5.3 β7.1
Vβ7.1 β7.2
Vβ7.2 β8
Vβ8 β9
Vβ9 β11
Vβ11 β12
Vβ12
TRBV9 TRBV21-1 TRBV28 TRBV29-1 TRBV5-1 TRBV5-6 TRBV5-5 TRBV4-1,2,3 TRBV4-3 TRBV12-3,4 TRBV3-1 TRBV25-1 TRBV10-3
Donor A S. typhimurium 1.98 8.34 3.47 1.61 4.97 0.463 0.451 5.69 0.485 7.15 3.55 0.482 0.525
Donor A E. coli 5.32 9.6 3.8 1.2 7.78 1.32 0.393 0.892 1.25 4.25 1.86 0.968 3.85

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Donor A B. animalis 1.86 11.1 3.87 0.504 8.59 0.301 0.705 0.888 0.135 1.9 3.03 0.272 0.904
Donor A L. acidophilus 2.06 11.7 4.68 0.744 7.18 0.513 0.239 0.647 0.216 2.4 1.94 0.275 1
Donor A F. prausnitzii 1.39 8.16 2.38 0.682 7.13 0.321 0.368 1.12 0.581 2.2 2.82 0.374 0.333

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Donor A C. difficile 0.821 9.46 4.61 1.52 9.56 0.219 0.592 1.43 0 1.54 3.27 0 1.73
Donor A SEB 0.0745 0.0962 25.9 0.418 0.00517 0.446 0.0707 0.0943 0.07 0.165 0.027 0.0528 8.07
Donor A PHA 2.22 13.5 5.99 2.08 1.07 6.21 0.838 0.932 0.673 2.5 3.11 0.44 2.07

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Donor B S. typhimurium 3.39 9.76 5.28 2.27 7.64 0.672 0.721 0.569 0.696 5.5 4.63 0.331 0.88
Donor B E. coli 3.52 11.9 4.32 1.96 7.97 0.714 0.921 1.02 1.56 5 4.22 0.184 0.74
Donor B B. animalis 2.36 4.07 7.63 4.29 7.72 0.461 3.41 1.12 1.48 3.23 1.81 0.575 1.13
Donor B L. acidophilus 8.58 11 3.49 4.26 9.74 0.762 1.81 0.499 2.15 4.19 0.955 0.416 0.949

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Donor B F. prausnitzii 3.06 5.41 6.07 2.15 7.49 0.535 0.748 0.748 1.77 3.85 1.03 0.416 2.57
Donor B C. difficile 1.95 9.02 2.8 1.46 4.5 0.185 0.781 0.588 0.308 1.44 1.43 0.238 13.3

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Donor B SEB 0.226 0.175 29.4 0.939 0.0283 0.434 0.107 0.0555 0.0598 0.274 0.077 0.0443 10
Donor B PHA 0.808 10.1 4.83 0.908 0.698 3.32 0.531 0.363 0.147 1.93 2.64 0.227 1.42
Donor C S. typhimurium 1.52 1.21 4.72 32.9 0.998 0.31 0.0963 0.84 0 9.8 0.357 0.0977 0.13

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Donor C E. coli 1.34 1.22 4.53 26.3 1.28 0.181 0.167 0.642 0 9.4 0.337 0.136 0.256
Donor C B. animalis 1.36 1.61 1.58 1.8 1.92 0.378 0.188 1.2 0.0113 1.08 0.286 0.21 0.216
Donor C L. acidophilus 0.816 5.71 1.26 3.29 1.32 0.333 1.18 1.22 0.00834 2.58 0.943 0.261 0.683

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Donor C F. prausnitzii 0.498 1.51 0.6 1.73 0.585 0.234 0.109 0.269 0.0247 1.07 0.271 0.382 0.222
Donor C C. difficile 0.471 1.09 0.536 0.778 0.615 0.218 0.145 0.563 0.0046 4.63 0.165 0.208 0.258
Donor C
Donor C
SEB
PHA
0.191
2.01
0.155
10.2
19.9
5.36
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1.43
2.43
0.0354
0.963
0.482
6.44
0.114
0.958
0.0692
0.626
0.0188
0.00905
0.234
4.24
0.0615
2.61
0.0577
0.732
8.98
1.87

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Supplementary Table 2. T cell receptor (TCR) Vβ usage in expanded microbiota-specific T cells measured by Vβ antibody panel from Beckman Coulter

Donors Microbe β13.1

Vβ13.1 β13.2
Vβ13.2 β13.6
Vβ13.6 β14
Vβ14 β16
Vβ16 β17
Vβ17 β18
Vβ18 β20
Vβ20 β21.3
Vβ21.3 β22
Vβ22 β23
Vβ23 Unkown Vββ
TRBV6-5,6,9 TRBV6-2 TRBV6-6 TRBV27 TRBV14 TRBV19 TRBV18 TRBV30 TRBV11-2 TRBV2 TRBV13
Donor A S. typhimurium 1.75 1.24 1.45 2.72 1.78 4.92 0.398 0.862 1.09 2.28 0.079 42.265
Donor A E. coli 2.07 1.73 2.09 3.2 0.731 5.7 0.515 4.25 0.901 5.48 0.267 30.583
Donor A B. animalis 0.727 6.54 0.998 1.89 1.09 7.64 0.197 1.98 1.41 15.5 0.955 27.014
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Donor A L. acidophilus 1.07 1.78 0.726 0.627 0.746 14.5 1.29 0.79 1.09 13 0.212 30.575
Donor A F. prausnitzii 1.09 0.556 1.35 1.27 0.896 3.21 0.921 1.39 1.91 19.2 0.463 39.885
Donor A C. difficile 1.09 1.35 1.39 2.07 0.369 2.48 1.13 3.5 1.86 11.9 0.0548 38.0542
Donor A SEB 0.11 5.22 0.333 2.02 0.333 14.6 0.0234 2.73 0.0444 0.354 0.177 38.56553
Donor A PHA 3.16 2.82 1.9 2.95 1.14 4.6 1.55 2.42 2.85 2.26 0.37 32.347
Donor B S. typhimurium 0.912 3.07 3.71 2.06 0.43 5.21 0.335 0.167 3.12 6.83 0.706 31.111
Donor B E. coli 2.47 3.98 2.96 2.71 0.792 5.14 1.41 0.2 3.11 4.82 0.432 27.947

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Donor B B. animalis 1.49 4.37 4.08 1.37 0.421 6.43 0.812 0.559 0.677 4.04 0.118 36.347
Donor B L. acidophilus 2.67 2.82 3.02 2.7 0.148 6.01 0.409 0.36 1.2 6.69 0.076 25.096
Donor B F. prausnitzii 1.07 1.17 4.92 6.59 0.972 8.73 0.237 0.917 0.764 14.1 0.115 24.568
Donor B C. difficile 2.93 0.638 8.27 1.82 0.2 3.29 0.548 0.298 2.81 7.49 1.94 31.766

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Donor B SEB 0.104 7.76 0.724 2.03 0.424 23 0.0317 0.052 0.0674 0.542 0.788 22.657
Donor B PHA 1.94 2 4.05 1.8 0.716 3.37 1.5 0.0536 2.16 0.869 0.223 53.3964
Donor C S. typhimurium 1.05 0.133 0.81 4.43 0.084 0.942 0.0269 0.228 0.796 4.51 0.0442 33.9669

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Donor C E. coli 0.785 0.176 0.878 2.55 0.0657 1.26 0.0797 0.347 0.791 4.57 0.0959 42.6127
Donor C B. animalis 1.05 0.317 0.803 0.52 0.068 2.27 0.222 0.979 0.339 1.3 0.133 80.1597
Donor C L. acidophilus 1.41 0.233 0.415 1.33 0.0705 3 0.193 2.37 0.898 1.06 0.139 69.27716
Donor C F. prausnitzii 0.543 0.52 0.291 1.43 0.0855 3.92 0.0425 1.03 0.33 0.645 0.102 83.5563

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Donor C C. difficile 0.327 0.092 0.138 0.482 0.0694 1.61 0.109 0.735 0.161 0.411 0.106 86.078
Donor C SEB 0.206 3.48 0.345 3.34 0.381 19.9 0.0309 4.82 0.0836 1.04 0.257 34.3879

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Donor C PHA 3.55 1.7 2.16 3.4 1.09 4.67 1.09 3.14 3.11 1.79 0.487 35.36495

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Supplementary Table 3. T cell receptor (TCR) Vβ usage in expanded microbiota specific T cells using multiplex PCR assay and deep sequencing
(related to Figure 3)
S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA β
Vβ
TRBV9 1.617783985 1.462695991 1.669805399 1.107458038 0.883673765 0.521920668 0.397526502 0.700678783 ['1']
TRBV20-1 10.59124767 11.1543723 9.780288763 10.24398685 8.083441982 9.251043841 0.309187279 8.430041603 ['2']
TRBV28 1.0358473 1.346943071 2.209667294 3.910711196 2.013617268 0.417536534 21.37809187 0.569301511 ['3']
TRBV29-1 5.52839851 5.713985057 5.599497803 6.29866759 5.606258149 7.215553236 0.618374558 4.072695424 ['4']
TRBV5-1 8.216945996 5.819214985 4.959196485 3.4781104 2.491670288 6.3282881 0.530035336 4.510619663 ['5.1']
TRBV5-6 0.442271881 0.904977376 0.69052103 0.19034435 0.8402144 0.339248434 0.044169611 0.131377272 ['5.2']
TRBV5-5 0.453910615 0.904977376 0.69052103 0.19034435 0.8402144 0.339248434 0.044169611 0.131377272 ['5.3']

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TRBV4-1 1.0358473 1.062822267 0.715630885 0.103824191 1.115457048 1.957202505 0.088339223 1.401357565 ['7.1']
TRBV4-2 3.852420857 3.335788698 3.992467043 2.993597508 6.156743445 5.414926931 0.706713781 4.050799212 ['7.1']
TRBV4-3 3.86405959 3.388403662 4.017576899 2.993597508 6.156743445 5.401878914 0.706713781 4.13838406 ['7.1', '7.2']
TRBV4-3 3.86405959 3.388403662 4.017576899 2.993597508 6.156743445 5.401878914 0.706713781 4.13838406 ['7.1', '7.2']

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TRBV12-3 2.420856611 2.462380301 1.820464532 2.422564458 0.86918731 0.978601253 0.132508834 1.22618787 ['8']
TRBV12-4 2.420856611 2.462380301 1.820464532 2.422564458 0.86918731 0.978601253 0.132508834 1.22618787 ['8']
TRBV3-1 0.186219739 0.410396717 0.615191463 0.519120955 0.376647834 0.130480167 0 0 ['9']
TRBV25-1 0.034916201 0.021045985 0.037664783 0 0.014486455 0 0.088339223 0 ['11']

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TRBV10-3 0 0.021045985 0 0 0 0 0.088339223 0 ['12']
TRBV6-1 0.477188082 0.820793434 1.858129316 1.69579512 1.839779806 0.247912317 0.618374558 0.262754543 ['13.1']
TRBV6-5 3.526536313 3.893507313 4.268675455 3.928015228 4.389395915 2.518267223 1.192579505 5.517845413 ['13.1']
TRBV6-6 3.479981378 3.809323372 4.18079096 3.8587991 4.244531363 2.466075157 1.192579505 5.386468141 ['13.1', '13.6']
TRBV6-9 0 0 0.012554928 0 0 0.013048017 0 0 ['13.1']

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TRBV6-2 0.53538175 0.894454383 1.305712492 1.055545942 0.782268579 0.874217119 7.155477032 0.634990147 ['13.2']
TRBV6-6 3.479981378 3.809323372 4.18079096 3.8587991 4.244531363 2.466075157 1.192579505 5.386468141 ['13.1', '13.6']
TRBV27 0.151303538 0.168367884 0.640301318 0.46720886 0.970592496 0.013048017 4.06360424 0.021896212 ['14']
TRBV14
TRBV19
TRBV18
1.163873371
0.034916201
0.104748603
0.936546354
0.031568978
0.242028833
0.376647834
0.062774639
0.138104206
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2.145699948
0.034608064
0.346080637
0.608431117
0.173837462
0.217296827
0.365344468
0.013048017
0.130480167
0.132508834
0.574204947
0
1.248084081
0
0.065688636
['16']
['17']
['18']
TRBV30 4.67877095 4.198674103 4.105461394 6.765876449 4.679125018 6.537056367 29.99116608 6.481278739 ['20']
TRBV11-2 1.175512104 1.11543723 2.071563089 3.374286209 0.796755034 0.495824635 0.265017668 0.372235603 ['21.3']
M
TRBV2 0.698324022 1.410081027 0.263653484 0.46720886 0.217296827 0.287056367 0.044169611 0 ['22']
TRBV13 0.058193669 0.073660949 0.025109856 0 0.014486455 0.052192067 0 0.021896212 ['23']
TRBV1 0 0 0 0 0 0 0 0 UNK
TRBV7-1 0 0 0 0 0 0 0 0 UNK
D

TRBV7-2 12.13919926 11.38587814 10.80979284 5.917978889 9.054034478 16.03601253 1.899293286 16.70680972 UNK
TRBV8-1 0 0 0 0 0 0 0 0 UNK
TRBV5-2 0 0 0 0 0 0 0 0 UNK
TE

TRBV6-4 0.034916201 0.168367884 0.025109856 0 0 0 1.01590106 0 UNK

TRBV6-3 0.53538175 0.894454383 1.305712492 1.055545942 0.782268579 0.874217119 7.155477032 0.634990147 UNK
TRBV7-3 4.213221601 3.683047459 4.143126177 4.031839419 4.621179197 2.583507307 1.899293286 3.656667397 UNK
TRBV8-2 0 0 0 0 0 0 0 0 UNK
TRBV5-3 0.244413408 0.199936862 0.050219711 0.017304032 0.014486455 0.03914405 0.309187279 0.043792424 UNK
EP

TRBV10-1 0 0 0 0 0 0 0 0 UNK
TRBV11-1 1.047486034 1.125960223 1.644695543 3.270462018 0.753295669 0.456680585 0.220848057 0.394131815 UNK
TRBV12-1 0 0 0 0 0 0 0 0 UNK
TRBV10-2 0.081471136 0.052614964 0.037664783 0 0.173837462 0 5.344522968 0.153273484 UNK
C

TRBV12-2 0 0 0 0 0 0 0 0 UNK
TRBV7-4 0.046554935 0.11575292 0.025109856 0.051912096 0.02897291 0.052192067 0.750883392 0.10948106 UNK
TRBV5-4 0.675046555 0.926023361 0.502197112 1.401626579 1.434159061 1.696242171 0.176678445 0.131377272 UNK
AC

TRBV7-5 0 0 0 0 0 0 0 0 UNK
TRBV6-7 0 0 0 0 0 0 0 0 UNK
TRBV7-6 1.745810056 2.230874461 1.694915254 1.12476207 1.941184992 3.444676409 1.369257951 3.590978761 UNK
TRBV5-8 0.675046555 0.947069347 0.527306968 1.540058834 1.463131972 1.761482255 0.176678445 0.131377272 UNK
TRBV6-8 0 0 0 0 0 0 0 0 UNK
TRBV7-8 4.166666667 3.314742713 4.369114878 2.595604776 6.634796465 3.405532359 1.766784452 4.92664769 UNK
TRBV7-7 1.641061453 2.051983584 1.556811048 1.12476207 1.839779806 3.314196242 1.369257951 3.284431793 UNK
TRBV5-7 0.40735568 0.820793434 0.665411174 0.19034435 0.724322758 0.326200418 0.044169611 0.10948106 UNK
TRBV7-9 3.514897579 4.082921183 3.892027621 4.499048278 3.215993047 3.001043841 0.35335689 3.70045982 UNK
TRBV11-3 1.198789572 1.199621172 2.084118016 3.374286209 0.796755034 0.495824635 0.265017668 0.416028027 UNK
TRBV12-5 0.162942272 0.242028833 0.11299435 0.103824191 0 0.03914405 0 0.10948106 UNK
TRBV15 2.118249534 1.125960223 0.351537979 0.692161274 0.622917572 1.200417537 0.35335689 1.707904533 UNK
TRBV16 0.046554935 0.021045985 0 0.899809656 0 0 0 0.043792424 UNK
TRBV17 0 0 0 0 0 0 0 0 UNK
TRBV21-1 0 0 0 0 0 0 0 0 UNK
 ACCEPTED MANUSCRIPT

TRBV22-1 0 0 0 0 0 0 0 0 UNK
TRBV23-1 0 0 0 0 0 0 0 0 UNK
TRBV24-1 0.174581006 0.147321898 0.075329567 0.242256446 0.246269738 0.11743215 3.136042403 0.021896212 UNK
TRBV26 0 0 0 0 0 0 0 0 UNK

PT
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U SC
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C EP
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 ACCEPTED MANUSCRIPT

Table S3. T cell receptor (TCR) Vβ usage in expanded microbiota specific T cells using multiplex PCR assays and deep sequencing

Donor 1
S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vββ
TRBV9 0.730868444 0.879443019 0.414421881 0 1.560348784 0.058309038 0.484848485 0.736196319 ['1']
TRBV20-1 7.09372313 9.967020887 14.46332366 12.77573529 10.9683341 9.329446064 0.242424242 7.668711656 ['2']
TRBV28 0.816852966 0.586295346 3.315375052 11.67279412 2.248737953 0.349854227 26.42424242 0.306748466 ['3']
TRBV29-1 9.200343938 7.108831074 9.241607957 8.180147059 11.70261588 11.31195335 0.242424242 4.846625767 ['4']
TRBV5-1 7.308684437 4.067423965 3.895565686 3.125 1.147315282 6.472303207 0.242424242 3.312883436 ['5.1']
TRBV5-6 0.343938091 0.146573837 0.041442188 0.091911765 0.091785223 0 0 0.061349693 ['5.2']

PT
TRBV5-5 0.343938091 0.146573837 0.041442188 0.091911765 0.091785223 0 0 0.061349693 ['5.3']
TRBV4-1 2.708512468 1.026016856 0.994612516 0 0.045892611 1.224489796 0.121212121 0.736196319 ['7.1']
TRBV4-2 2.235597592 4.873580066 4.517198508 0.275735294 7.847636531 5.131195335 0.848484848 5.153374233 ['7.1']
TRBV4-3 2.235597592 4.910223525 4.558640696 0.275735294 7.847636531 5.131195335 0.848484848 5.214723926 ['7.1', '7.2']
TRBV4-3 2.235597592 4.910223525 4.558640696 0.275735294 7.847636531 5.131195335 0.848484848 5.214723926 ['7.1', '7.2']

RI
TRBV12-3 5.54600172 2.161964089 0.414421881 0.091911765 1.147315282 0.699708455 0.121212121 0.552147239 ['8']
TRBV12-4 5.54600172 2.161964089 0.414421881 0.091911765 1.147315282 0.699708455 0.121212121 0.552147239 ['8']
TRBV3-1 0.042992261 0 0 0.091911765 0 0 0 0 ['9']
TRBV25-1 0 0 0 0 0.045892611 0 0.242424242 0 ['11']

SC
TRBV10-3 0 0 0 0 0 0 0 0 ['12']
TRBV6-1 0.171969046 0.036643459 0.165768753 0.091911765 0.321248279 0.233236152 0.363636364 0 ['13.1']
TRBV6-5 3.740326741 5.313301576 1.367592209 6.433823529 0.413033502 1.982507289 0.484848485 3.926380368 ['13.1']
TRBV6-6 3.740326741 5.240014657 1.367592209 6.617647059 0.36714089 1.982507289 0.484848485 3.803680982 ['13.1', '13.6']
TRBV6-9 0 0 0 0 0 0 0 0 ['13.1']

U
TRBV6-2 0.085984523 0.916086479 0.704517199 1.654411765 0.183570445 1.10787172 8 0.920245399 ['13.2']
TRBV6-6 3.740326741 5.240014657 1.367592209 6.617647059 0.36714089 1.982507289 0.484848485 3.803680982 ['13.1', '13.6']
TRBV27 0.386930353 0.293147673 1.036054704 0.183823529 2.8453419 0 4 0 ['14']
TRBV14
TRBV19
TRBV18
TRBV30
2.622527945

0.085984523
2.708512468
0
0.256504214
0
0.109930377
5.386588494
0.331537505
0
0
6.092001658
AN
0.459558824

0.091911765
5.974264706
0
0.413033502
0.045892611
0.183570445
6.05782469
0
0
0
5.189504373
0.121212121

27.15151515
0

0
0.797546012
0
0
7.791411043
['16']
['17']
['18']
['20']
TRBV11-2 0.859845228 0.513008428 1.243265644 0.183823529 1.193207894 0 0.121212121 0.306748466 ['21.3']
M
TRBV2 0.085984523 0.43972151 0.041442188 0 0.091785223 0 0 0 ['22']
TRBV13 0 0 0 0 0 0 0 0 ['23']
TRBV1 0 0 0 0 0 0 0 0 UNK
TRBV7-1 0 0 0 0 0 0 0 0 UNK
TRBV7-2 14.144454 17.0392085 11.1893908 3.952205882 12.80403855 15.62682216 1.454545455 19.57055215 UNK
D

TRBV8-1 0 0 0 0 0 0 0 0 UNK
TRBV5-2 0 0 0 0 0 0 0 0 UNK
TRBV6-4 0.085984523 0 0.041442188 0 0 0 2.181818182 0 UNK
TE

TRBV6-3 0.085984523 0.916086479 0.704517199 1.654411765 0.183570445 1.10787172 8 0.920245399 UNK

TRBV7-3 5.331040413 3.187980945 6.506423539 0 4.726938963 0.758017493 0.96969697 3.190184049 UNK
TRBV8-2 0 0 0 0 0 0 0 0 UNK
TRBV5-3 0.085984523 0.43972151 0 0 0 0.116618076 0.242424242 0 UNK
TRBV10-1 0 0 0 0 0 0 0 0 UNK
EP

TRBV11-1 0.816852966 0.513008428 1.160381268 0.183823529 1.147315282 0 0 0.36809816 UNK

TRBV12-1 0 0 0 0 0 0 0 0 UNK
TRBV10-2 0.042992261 0.036643459 0.041442188 0 0.550711335 0 7.636363636 0.306748466 UNK
TRBV12-2 0 0 0 0 0 0 0 0 UNK
TRBV7-4 0.042992261 0 0 0 0 0 1.212121212 0.18404908 UNK
C

TRBV5-4 0.128976784 0.329791132 0.621632822 0 0.183570445 5.422740525 0.121212121 0 UNK

TRBV7-5 0 0 0 0 0 0 0 0 UNK
TRBV6-7 0 0 0 0 0 0 0 0 UNK
AC

TRBV7-6 1.977644024 0.989373397 3.688354745 4.136029412 1.78981184 3.731778426 0.848484848 4.662576687 UNK
TRBV5-8 0.128976784 0.329791132 0.621632822 0 0.183570445 5.422740525 0.121212121 0 UNK
TRBV6-8 0 0 0 0 0 0 0 0 UNK
TRBV7-8 4.170249355 3.151337486 9.117281392 5.055147059 5.32354291 3.206997085 1.454545455 5.214723926 UNK
TRBV7-7 1.891659501 0.952729938 3.398259428 4.136029412 1.743919229 3.381924198 0.848484848 4.478527607 UNK
TRBV5-7 0.343938091 0.146573837 0.041442188 0.091911765 0.091785223 0 0 0 UNK
TRBV7-9 3.611349957 3.261267864 0.994612516 10.47794118 3.717301514 2.857142857 0.96969697 2.944785276 UNK
TRBV11-3 0.859845228 0.513008428 1.243265644 0.183823529 1.193207894 0 0.121212121 0.36809816 UNK
TRBV12-5 0.042992261 0 0 0 0 0.174927114 0 0.122699387 UNK
TRBV15 1.504729149 1.392451447 0.041442188 0 0 0.174927114 0.121212121 1.840490798 UNK
TRBV16 0 0 0 4.779411765 0 0 0 0 UNK
TRBV17 0 0 0 0 0 0 0 0 UNK
TRBV21-1 0 0 0 0 0 0 0 0 UNK
TRBV22-1 0 0 0 0 0 0 0 0 UNK
TRBV23-1 0 0 0 0 0 0 0 0 UNK
TRBV24-1 0.085984523 0.109930377 0 0 0.137677834 0 1.696969697 0.061349693 UNK
 ACCEPTED MANUSCRIPT

TRBV26 0 0 0 0 0 0 0 0 UNK

PT
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U SC
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C EP
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 ACCEPTED MANUSCRIPT

Table S3. T cell receptor (TCR) Vβ usage in expanded microbiota specific T cells using multiplex PCR assays and deep sequencing

Donor 2
S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vββ
TRBV9 2.232142857 2.699144174 1.598495534 3.249630724 0.166574125 0.30159414 0.295420975 1.194659171 ['1']
TRBV20-1 10.75487013 11.32323897 9.685002351 13.22008863 8.995002776 7.755277897 0.738552437 7.589599438 ['2']
TRBV28 2.353896104 3.324555629 4.37235543 7.311669129 4.664075514 0.258509263 26.44017725 1.475755446 ['3']
TRBV29-1 3.652597403 7.142857143 4.607428303 2.511078287 1.499167129 0.646273158 1.624815362 3.021784961 ['4']
TRBV5-1 11.28246753 5.99078341 5.500705219 5.022156573 7.273736813 4.308487721 0.59084195 5.762473647 ['5.1']
TRBV5-6 0.689935065 1.382488479 2.256699577 0.295420975 2.165463631 0.646273158 0.147710487 0.281096275 ['5.2']

PT
TRBV5-5 0.730519481 1.382488479 2.256699577 0.295420975 2.165463631 0.646273158 0.147710487 0.281096275 ['5.3']
TRBV4-1 0.649350649 0.954575379 1.081335214 0.295420975 2.498611882 1.981904352 0 1.405481377 ['7.1']
TRBV4-2 1.988636364 1.283739302 0.188058298 0.812407681 3.498056635 8.358466178 0.59084195 1.967673928 ['7.1']
TRBV4-3 2.029220779 1.316655695 0.188058298 0.812407681 3.498056635 8.358466178 0.59084195 2.037947997 ['7.1', '7.2']
TRBV4-3 2.029220779 1.316655695 0.188058298 0.812407681 3.498056635 8.358466178 0.59084195 2.037947997 ['7.1', '7.2']

RI
TRBV12-3 0.730519481 1.81040158 2.3977433 4.135893648 1.887840089 2.585092632 0.147710487 3.09205903 ['8']
TRBV12-4 0.730519481 1.81040158 2.3977433 4.135893648 1.887840089 2.585092632 0.147710487 3.09205903 ['8']
TRBV3-1 0.487012987 1.152073733 2.209685002 1.772525849 1.388117712 0.129254632 0 0 ['9']
TRBV25-1 0.081168831 0.032916392 0 0 0 0 0 0 ['11']

SC
TRBV10-3 0 0 0 0 0 0 0.147710487 0 ['12']
TRBV6-1 0.852272727 1.184990125 1.269393512 1.772525849 0.72182121 0.473933649 0.886262925 0.421644413 ['13.1']
TRBV6-5 4.748376623 2.995391705 5.547719793 5.391432792 2.554136591 3.274450668 1.1816839 8.854532677 ['13.1']
TRBV6-6 4.586038961 2.92955892 5.500705219 5.317577548 2.554136591 3.274450668 1.1816839 8.643710471 ['13.1', '13.6']
TRBV6-9 0 0 0 0 0 0 0 0 ['13.1']

U
TRBV6-2 0.527597403 0.658327847 0.329102022 0.664697194 1.054969461 0.043084877 2.954209749 0.281096275 ['13.2']
TRBV6-6 4.586038961 2.92955892 5.500705219 5.317577548 2.554136591 3.274450668 1.1816839 8.643710471 ['13.1', '13.6']
TRBV27 0.081168831 0.032916392 1.034320639 0.960118168 0.166574125 0 3.397341211 0.070274069 ['14']
TRBV14
TRBV19
TRBV18
TRBV30
0.487012987

0.040584416
4.788961039
0
0.855826201
0
0.230414747
4.377880184
0.94029149

0.235072873
0

2.3977433
AN
2.215657312
0.073855244
0.369276219
5.539143279
1.83231538

0.499722376
2.94280955
0
0.215424386
0.043084877
0.344679018
8.746230073
0.443131462
0.295420975

30.42836041
0
1.405481377
0
0.210822207
3.72452565
['16']
['17']
['18']
['20']
TRBV11-2 1.866883117 1.217906517 3.667136812 1.550960118 1.166018878 0.430848772 0.147710487 0.562192551 ['21.3']
M
TRBV2 1.948051948 2.567478604 0.799247767 1.624815362 0.610771793 0.732442913 0 0 ['22']
TRBV13 0.202922078 0.098749177 0 0 0 0 0 0.070274069 ['23']
TRBV1 0 0 0 0 0 0 0 0 UNK
TRBV7-1 0 0 0 0 0 0 0 0 UNK
TRBV7-2 9.699675325 6.978275181 7.663375646 3.249630724 10.77179345 9.392503231 1.920236337 10.11946592 UNK
D

TRBV8-1 0 0 0 0 0 0 0 0 UNK
TRBV5-2 0 0 0 0 0 0 0 0 UNK
TRBV6-4 0 0 0.047014575 0 0 0 0.443131462 0 UNK
TE

TRBV6-3 0.527597403 0.658327847 0.329102022 0.664697194 1.054969461 0.043084877 2.954209749 0.281096275 UNK
TRBV7-3 3.125 3.982883476 1.645510108 5.612998523 2.387562465 3.619129685 2.658788774 3.935347857 UNK
TRBV8-2 0 0 0 0 0 0 0 0 UNK
TRBV5-3 0.162337662 0.131665569 0.094029149 0.073855244 0.055524708 0 0.59084195 0 UNK
TRBV10-1 0 0 0 0 0 0 0 0 UNK
EP

TRBV11-1 1.542207792 1.11915734 2.162670428 1.1816839 1.054969461 0.344679018 0.147710487 0.562192551 UNK
TRBV12-1 0 0 0 0 0 0 0 0 UNK
TRBV10-2 0.162337662 0.065832785 0 0 0 0 5.169867061 0.070274069 UNK
TRBV12-2 0 0 0 0 0 0 0 0 UNK
TRBV7-4 0.040584416 0.164581962 0.047014575 0.073855244 0 0.129254632 0.59084195 0.070274069 UNK
C

TRBV5-4 1.176948052 1.481237656 0.470145745 1.698670606 1.665741255 1.206376562 0 0.421644413 UNK

TRBV7-5 0 0 0 0 0 0 0 0 UNK
TRBV6-7 0 0 0 0 0 0 0 0 UNK
AC

TRBV7-6 1.866883117 3.324555629 1.833568406 0.664697194 1.665741255 4.825506247 2.215657312 2.951510892 UNK
TRBV5-8 1.176948052 1.547070441 0.564174894 2.363367799 1.776790672 1.292546316 0 0.421644413 UNK
TRBV6-8 0 0 0 0 0 0 0 0 UNK
TRBV7-8 5.438311688 4.805793285 3.94922426 3.249630724 7.828983898 3.920723826 2.067946824 4.427266339 UNK
TRBV7-7 1.785714286 3.159973667 1.692524683 0.664697194 1.554691838 4.739336493 2.215657312 2.670414617 UNK
TRBV5-7 0.568181818 1.11915734 2.209685002 0.295420975 1.998889506 0.603188281 0.147710487 0.281096275 UNK
TRBV7-9 3.571428571 5.892034233 5.970850964 1.846381093 4.664075514 1.206376562 0 4.567814476 UNK
TRBV11-3 1.948051948 1.316655695 3.667136812 1.550960118 1.166018878 0.430848772 0.147710487 0.562192551 UNK
TRBV12-5 0.324675325 0.032916392 0.047014575 0.443131462 0 0 0 0.210822207 UNK
TRBV15 1.339285714 1.11915734 1.175364363 0.443131462 0.610771793 0.086169754 1.033973412 2.178496135 UNK
TRBV16 0.081168831 0 0 0 0 0 0 0.140548138 UNK
TRBV17 0 0 0 0 0 0 0 0 UNK
TRBV21-1 0 0 0 0 0 0 0 0 UNK
TRBV22-1 0 0 0 0 0 0 0 0 UNK
TRBV23-1 0 0 0 0 0 0 0 0 UNK
TRBV24-1 0.324675325 0.098749177 0.282087447 0.443131462 0 0.387763895 3.397341211 0 UNK
 ACCEPTED MANUSCRIPT

TRBV26 0 0 0 0 0 0 0 0 UNK

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

Table S3. T cell receptor (TCR) Vβ usage in expanded microbiota-specific T cells using multiplex PCR assay and deep sequencing

Donor 3
S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vββ
TRBV9 1.762230405 0.883297645 2.598540146 0.599340725 0.821074239 0.882028666 0.393700787 0.198150594 ['1']

PT
TRBV20-1 12.62493425 11.88436831 6.540145985 8.210967935 5.371193979 10.17089305 0 10.03963012 ['2']
TRBV28 0.315623356 0.294432548 0.087591241 0 0.20526856 0.551267916 11.41732283 0 ['3']
TRBV29-1 4.497632825 3.533190578 3.649635037 7.222055739 3.592199795 9.481808159 0.131233596 4.227212682 ['4']

RI
TRBV5-1 6.785902157 6.959314775 5.372262774 2.96673659 0.547382826 7.552370452 0.787401575 4.623513871 ['5.1']
TRBV5-6 0.341925302 1.070663812 0.175182482 0.179802218 0.581594252 0.303197354 0 0.066050198 ['5.2']

SC
TRBV5-5 0.341925302 1.070663812 0.175182482 0.179802218 0.581594252 0.303197354 0 0.066050198 ['5.3']
TRBV4-1 0.263019463 1.177730193 0.291970803 0.059934073 1.060554225 2.287761852 0.131233596 2.113606341 ['7.1']
TRBV4-2 6.049447659 3.881156317 5.98540146 4.764758765 6.534382484 3.665931643 0.656167979 4.821664465 ['7.1']
TRBV4-3 6.049447659 3.961456103 6.01459854 4.764758765 6.534382484 3.638368247 0.656167979 4.953764861 ['7.1', '7.2']

U
TRBV4-3 6.049447659 3.961456103 6.01459854 4.764758765 6.534382484 3.638368247 0.656167979 4.953764861 ['7.1', '7.2']

AN
TRBV12-3 1.604418727 3.211991435 2.452554745 2.48726401 0.034211427 0.082690187 0.131233596 0.198150594 ['8']
TRBV12-4 1.604418727 3.211991435 2.452554745 2.48726401 0.034211427 0.082690187 0.131233596 0.198150594 ['8']
TRBV3-1 0.078905839 0.107066381 0.058394161 0.149835181 0.034211427 0.192943771 0 0 ['9']
TRBV25-1 0.026301946 0.026766595 0.087591241 0 0 0 0 0 ['11']

M
TRBV10-3 0 0.053533191 0 0 0 0 0.131233596 0 ['12']
TRBV6-1 0.420831142 1.097430407 3.416058394 2.187593647 3.660622648 0.110253583 0.656167979 0.396301189 ['13.1']

D
TRBV6-5 2.603892688 3.586723769 5.518248175 2.517231046 8.484433801 2.287761852 1.968503937 4.095112285 ['13.1']
TRBV6-6 2.603892688 3.479657388 5.343065693 2.367395865 8.176530961 2.177508269 1.968503937 4.029062087 ['13.1', '13.6']
TRBV6-9
TRBV6-2
0
0.815360337
0
1.070663812
0.02919708
2.335766423
TE 0
1.018879233 1.060554225
0 0.027563396
1.295479603
0
9.973753281
0
0.660501982
['13.1']
['13.2']
TRBV6-6 2.603892688 3.479657388 5.343065693 2.367395865 8.176530961 2.177508269 1.968503937 4.029062087 ['13.1', '13.6']
TRBV27 0.052603893 0.187366167 0.116788321 0.359604435 0.068422853 0.027563396 4.724409449 0 ['14']
EP

TRBV14 0.710152551 1.498929336 0.058394161 2.667066227 0 0.633958104 0 1.585204756 ['16']

TRBV19 0.078905839 0.080299786 0.145985401 0.029967036 0.376325693 0 1.312335958 0 ['17']
TRBV18 0.157811678 0.347965739 0.175182482 0.419538508 0.068422853 0.055126792 0 0 ['18']
C

TRBV30 5.812730142 3.185224839 3.766423358 7.521726101 4.721176873 5.760749724 32.67716535 7.661822985 ['20']
AC

TRBV11-2 0.920568122 1.472162741 1.664233577 5.154330237 0.273691413 0.771775083 0.524934383 0.264200793 ['21.3']
TRBV2 0.263019463 1.177730193 0.087591241 0.149835181 0.068422853 0.137816979 0.131233596 0 ['22']
TRBV13 0 0.107066381 0.058394161 0 0.034211427 0.110253583 0 0 ['23']
TRBV1 0 0 0 0 0 0 0 0 UNK
TRBV7-1 0 0 0 0 0 0 0 0 UNK
TRBV7-2 12.49342451 10.84047109 12.49635037 7.641594246 5.200136846 20.47960309 2.362204724 19.81505945 UNK
TRBV8-1 0 0 0 0 0 0 0 0 UNK
TRBV5-2 0 0 0 0 0 0 0 0 UNK
TRBV6-4 0.026301946 0.428265525 0 0 0 0 0.262467192 0 UNK
 ACCEPTED MANUSCRIPT

TRBV6-3 0.815360337 1.070663812 2.335766423 1.018879233 1.060554225 1.295479603 9.973753281 0.660501982 UNK
TRBV7-3 4.234613361 3.800856531 4.02919708 4.704824693 5.918576805 2.783902977 2.230971129 3.896961691 UNK
TRBV8-2 0 0 0 0 0 0 0 0 UNK
TRBV5-3 0.394529195 0.080299786 0.058394161 0 0 0.027563396 0.131233596 0.132100396 UNK
TRBV10-1 0 0 0 0 0 0 0 0 UNK
TRBV11-1 0.867964229 1.579229122 1.664233577 5.1243632 0.273691413 0.744211687 0.524934383 0.264200793 UNK

PT
TRBV12-1 0 0 0 0 0 0 0 0 UNK
TRBV10-2 0.052603893 0.053533191 0.058394161 0 0 0 3.018372703 0.066050198 UNK
TRBV12-2 0 0 0 0 0 0 0 0 UNK

RI
TRBV7-4 0.052603893 0.160599572 0.02919708 0.059934073 0.068422853 0.027563396 0.393700787 0.066050198 UNK
TRBV5-4 0.683850605 0.91006424 0.437956204 1.738088103 2.22374273 0.248070562 0.393700787 0 UNK
TRBV7-5 0 0 0 0 0 0 0 0 UNK

SC
TRBV6-7 0 0 0 0 0 0 0 0 UNK
TRBV7-6 1.525512888 2.248394004 0.204379562 0.329637399 2.22374273 2.425578831 1.181102362 3.038309115 UNK
TRBV5-8 0.683850605 0.91006424 0.437956204 1.708121067 2.22374273 0.33076075 0.393700787 0 UNK

U
TRBV6-8 0 0 0 0 0 0 0 0 UNK
TRBV7-8 3.340347186 2.221627409 1.284671533 1.528318849 6.87649675 3.169790518 1.837270341 5.085865258 UNK

AN
TRBV7-7 1.394003156 1.953961456 0.175182482 0.329637399 2.086897024 2.37045204 1.181102362 2.575957728 UNK
TRBV5-7 0.341925302 1.070663812 0.145985401 0.179802218 0.410537119 0.303197354 0 0.066050198 UNK
TRBV7-9 3.419253025 3.211991435 4.642335766 3.626011387 1.950051317 4.217199559 0 3.698811096 UNK

M
TRBV11-3 0.920568122 1.605995717 1.693430657 5.154330237 0.273691413 0.771775083 0.524934383 0.330250991 UNK
TRBV12-5 0.131509732 0.588865096 0.233576642 0 0 0 0 0 UNK
TRBV15 2.998421883 0.936830835 0.058394161 1.018879233 1.094765652 2.398015436 0 1.122853369 UNK

D
TRBV16 0.052603893 0.053533191 0 0 0 0 0 0 UNK
TRBV17 0 0 0 TE 0 0 0 0 0 UNK
TRBV21-1 0 0 0 0 0 0 0 0 UNK
TRBV22-1 0 0 0 0 0 0 0 0 UNK
TRBV23-1 0 0 0 0 0 0 0 0 UNK
EP

TRBV24-1 0.131509732 0.214132762 0 0.23973629 0.478959973 0 4.461942257 0 UNK

TRBV26 0 0 0 0 0 0 0 0 UNK
C
AC
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAGACCAAGGGCCGCGGATGGGATGCCGCAGTATTTT 22554 CAWRPRAADGMPQYF
TGTGCCTGGGAGCCAGTGGAGGCACTCACAGATACGCAGTATTTT 7027 CAWEPVEALTDTQYF
TGTGCCAGCAGCTTCGGGGACAGTATCCAAGAGACCCAGTACTTC 5842 CASSFGDSIQETQYF
TGTGCCTGGGACACTAGCGGGCAGGGCAATGAGCAGTTCTTC 5596 CAWDTSGQGNEQFF
TGTGCCTGGGGGGACGGAAGCAATCAGCCCCAGCATTTT 5367 CAWGDGSNQPQHF
TGTGCCAGCAGCTTCCTTCGGGGGAACACTGAAGCTTTCTTT 5210 CASSFLRGNTEAFF

PT
TGCGCCAGCAGCCAAGAGACGGGCCACGAGAGGAACATTCAGTACTTC 4524 CASSQETGHERNIQYF
TGTGCCAGCAGCTTTGAGGCAGGGCGGATCACTGAAGCTTTCTTT 4454 CASSFEAGRITEAFF
TGCAGTGCTAGCGCTGGCCTGGGGTACAATGAGCAGTTCTTC 4435 CSASAGLGYNEQFF
TGTGCCAGCAGCTTGGATAGCGGCTACAATGAGCAGTTCTTC 4025 CASSLDSGYNEQFF

RI
TGCGCCAGCAGCCAAGAGGTCGGGCCGTATGGCTACACCTTC 4016 CASSQEVGPYGYTF
TGTGCCACCTCTTCACCGGGCTGGGCAGATACGCAGTATTTT 3751 CATSSPGWADTQYF
TGTGCCTGGGACCCAGCGATCTCAGATACGCAGTATTTT 3581 CAWDPAISDTQYF

SC
TGTGCCGGCCAGACTAGCGGAGGTGTGGGTGAGCAGTTCTTC 3399 CAGQTSGGVGEQFF
TGTGCCAGCAGCCAAGCCACTATCCCTCCCAATGAGCAGTTCTTC 3276 CASSQATIPPNEQFF
TGTGCCACCAGCAGCTGGACAGGGTCTTCGGATGAGCAGTTCTTC 3081 CATSSWTGSSDEQFF
TGTGCCAGCAGCCCCTTTACGGACGAGCAGTACTTC 3001 CASSPFTDEQYF

U
TGCAGCGTTACGGGACTAGCGGGAGGCTCCTACAATGAGCAGTTCTTC 2750 CSVTGLAGGSYNEQFF
TGTGCCAGCAGTCCGGGACTAGCGGTACAAGAGACCCAGTACTTC 2596 CASSPGLAVQETQYF
AN
TGCGCCAGCAGCCAAGATACAGGGGACCGCGGGGGCTACACCTTC
TGTGCCAGCAGCTACTCTAGCGGGAGCGGTGGAGAGACCCAGTACTTC
2425
2416
CASSQDTGDRGGYTF
CASSYSSGSGGETQYF
TGTGCCAGCAGCCATACGACTAGTAGCGGGTTCTACGAGCAGTACTTC 2271 CASSHTTSSGFYEQYF
M
TGTGCCAGCTCCGGGGAGGCGCAGTATTTT 2263 CASSGEAQYF
TGTGCCAGCAGCTTAGCATCCCCCACTGAAGCTTTCTTT 2239 CASSLASPTEAFF
TGTGCCAGCAGCCCGTTACGGGGTGGAGATACGCAGTATTTT 2162 CASSPLRGGDTQYF
TGTGCCAGCAGCTTAAAGGCAGCGACCGACCTGGCTGAAGCTTTCTTT 2014 CASSLKAATDLAEAFF
D

TGTGCCTGGAGTGTGGGGGACTATGGCTACACCTTC 1868 CAWSVGDYGYTF

TGTGCCAGCAGTCCTTGGGGGGGAGGGGGAGATACGCAGTATTTT 1822 CASSPWGGGGDTQYF
TE

TGTGCCAGCAGCTTGGCTAGCCAAGAGACCCAGTACTTC 1757 CASSLASQETQYF

TGTGCCAGCAGCTTAAGCCTCATCCTCGCGGGCAATGAGCAGTTCTTC 1724 CASSLSLILAGNEQFF
TGTGCCAGCAGCTTATATGTCCGAGAGACCCAGTACTTC 1716 CASSLYVRETQYF
TGTGCCAGCAGCGCAGACTATGGCAATCAGCCCCAGCATTTT 1679 CASSADYGNQPQHF
EP

TGTGCCTGGAGTATCGGGACAGCTTACGAGCAGTACTTC 1636 CAWSIGTAYEQYF

TGTGCCAGCAGCTTAGGCGGGGCGAGGACTGAAGCTTTCTTT 1573 CASSLGGARTEAFF
TGTGCCTGGAGTGCGGGTCTACTGAGCTACGAGCAGTACTTC 1539 CAWSAGLLSYEQYF
C

TGCAGCGTTGGAACAGCGCCCACTGAAGCTTTCTTT 1526 CSVGTAPTEAFF

TGTGCCAGCAGCGGGACTAGCGGGAGTGAGCAGTTCTTC 1524 CASSGTSGSEQFF
AC

TGTGCCAGCTCGGGAGATACGCAGTATTTT 1433 CASSGDTQYF

TGTGCCAGCAGCATGGGGTGGAGGACTGAAGCTTTCTTT 1430 CASSMGWRTEAFF
TGTGCCAGCAGCCCGACCGGGACGTCGAGCTCCTACGAGCAGTACTTC 1410 CASSPTGTSSSYEQYF
TGTGCCAGCGCTACAGGGGGGGATCAGCCCCAGCATTTT 1402 CASATGGDQPQHF
TGTGCCAGCAGCTTATTGACAGGGGGGGCTTTCTTT 1402 CASSLLTGGAFF
TGTGCCAGCAGCTTAGATTGGCAGACCGCGGATGAGCAGTTCTTC 1396 CASSLDWQTADEQFF
TGTGCTAGCAGCCCATCCGGAGGTGCCGGCACAGATACGCAGTATTTT 1360 CASSPSGGAGTDTQYF
TGCAGTGCTAGATCCGGGACAGGGGTCGGGAGCAATCAGCCCCAGCATTTT 1337 CSARSGTGVGSNQPQHF
TGTGCCAGCAGCCCTTCGCGGTACAATGAGCAGTTCTTC 1312 CASSPSRYNEQFF
TGCGCCAGCAGCCTTTATTTAGCGGGAGCGTCGACCGGGGAGCTGTTTTTT 1296 CASSLYLAGASTGELFF
TGTGCCAGCAGCTTAGGGCTTAATCCCTCCTACAATGAGCAGTTCTTC 1204 CASSLGLNPSYNEQFF
TGTGCCTGGAGCCTACAGGGATCCTATGGCTACACCTTC 1199 CAWSLQGSYGYTF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCTTCGGGGACAGTATCCAAGAGACCCAGTACTTC 16040 CASSFGDSIQETQYF
TGTGCCAGCAGCCCCCAGACTGAAGCTTTCTTT 7638 CASSPQTEAFF
TGTGCCAGCGGGGTCAATGAGCAGTTCTTC 6655 CASGVNEQFF
TGTGCCTGGAGTGGGACAGGGGTTCGACAGCCCCAGCATTTT 6051 CAWSGTGVRQPQHF
TGTGCCAGCAGCCCGGGGGGGCTGAACACTGAAGCTTTCTTT 5779 CASSPGGLNTEAFF
TGCGCCAGCAGCCTCCAGGGCACTGAAGCTTTCTTT 5616 CASSLQGTEAFF

PT
TGTGCCAGCAGCTTTCTACAGGGGGCTCGCACTGAAGCTTTCTTT 4841 CASSFLQGARTEAFF
TGTGCCAGCAGCCCGGGACTAGCATCGTACATTCAGTACTTC 4396 CASSPGLASYIQYF
TGTGCCTGGAACACGGGCCGCCATGAGCAGTTCTTC 4380 CAWNTGRHEQFF

RI
TGTGCCACCAGCAGAGAAGGCGGCAGGGTCATGAACACTGAAGCTTTCTTT 4044 CATSREGGRVMNTEAFF
TGTGCCAGTCGACAGGGGGCGGATGGCTACACCTTC 3608 CASRQGADGYTF
TGTGCCTGGAGGGCGGAATACACTGAAGCTTTCTTT 3490 CAWRAEYTEAFF

SC
TGTGCCAGCAGCCCGGGGGGACTAGCGGGCGTCAATGAGCAGTTCTTC 3353 CASSPGGLAGVNEQFF
TGTGCCAGCAGCTGGGGGACAGGGGACACAGATACGCAGTATTTT 3226 CASSWGTGDTDTQYF
TGTGCCAGCAGCCCAGCAAGCGGGTCCTACGAGCAGTACTTC 3169 CASSPASGSYEQYF
TGTGCCAGCAGCCAACACCGGACAGCTGGGGCCAACGTCCTGACTTTC 2850 CASSQHRTAGANVLTF

U
TGCGCCAGCAGGAGAGGGGGGGTGGATTCACCCCTCCACTTT 2580 CASRRGGVDSPLHF
TGTGCCTGGACTAGCGCCGCCCGAGAGACCCAGTACTTC 2563 CAWTSAARETQYF
TGTGCCTGGAGTCGGGGGGGCCGAGCTTTCTTT AN
TGTGCCAGCAGCTCGACACCGACAGGGGGAGGCTACACCTTC
2498
2476
CAWSRGGRAFF
CASSSTPTGGGYTF
TGTGCCACCAGCAGGTACGGAGAACACTCTGAAGCTTTCTTT 2453 CATSRYGEHSEAFF
TGTGCCAGCATGGCGTCGGCAGGCTACGAGCAGTACTTC 2369 CASMASAGYEQYF
M
TGTGCCTGGACCCGGACTATCCAAGAGACCCAGTACTTC 2333 CAWTRTIQETQYF
TGCAGTGCTAGATCCGGGACAGGGGTCGGGAGCAATCAGCCCCAGCATTTT 2271 CSARSGTGVGSNQPQHF
TGTGCCTGGAACGAACAGGATTTAGCCGGGGAGCTGTTTTTT 2232 CAWNEQDLAGELFF
D

TGTGCCAGCAGCCCGGGGGGCGGGAGCTCCTACAATGAGCAGTTCTTC 2209 CASSPGGGSSYNEQFF

TGCGCCAGCAGCCAAGACTGGCTAGCGGGAGTCGAGCAGTACTTC 2173 CASSQDWLAGVEQYF
TE

TGTGCCAGCAGCTTAGGGAGGGGCACTGAAGCTTTCTTT 2172 CASSLGRGTEAFF

TGTGCCAGCAGCCGTTGGGACGGACAAGAGACCCAGTACTTC 2161 CASSRWDGQETQYF
TGTGCCAGCAGCGAACAGGGAGGGCAGCCCCAGCATTTT 2107 CASSEQGGQPQHF
EP

TGTGCCAGCAGCTTTTACATCGAAGAGACCCAGTACTTC 2053 CASSFYIEETQYF

TGTGCCAGCAGTCCGGGACTAGCGGTACAAGAGACCCAGTACTTC 2032 CASSPGLAVQETQYF
TGCGCCAGCAGCCGGGACCTAGCAACAGATACGCAGTATTTT 2025 CASSRDLATDTQYF
TGTGCCAGCAGCTTGGATAGCGGCTACAATGAGCAGTTCTTC 1975 CASSLDSGYNEQFF
C

TGTGCCAGCAGCTTCGTGGGGGGCACTGAAGCTTTCTTT 1941 CASSFVGGTEAFF

TGTGCCAGCAGCCGCATGAGGGGGGAGGAGACCCAGTACTTC 1930 CASSRMRGEETQYF
AC

TGTGCCTGGACTGGCGACAGGTCGTACTACGAGCAGTACTTC 1892 CAWTGDRSYYEQYF

TGTGCCTGGAGTGTCGGGTCGGATGGCTACACCTTC 1879 CAWSVGSDGYTF
TGTGCCAGCAGCCTTCCCGTGAACACTGAAGCTTTCTTT 1871 CASSLPVNTEAFF
TGTGCCAGCAGCGCAGAATGGAATCAGCCCCAGCATTTT 1801 CASSAEWNQPQHF
TGTGCCAGCAGCCCGACGGGGGTAGGTGAAGCTTTCTTT 1765 CASSPTGVGEAFF
TGTGCCTGGAACCGACTGGGAATGGATACGCAGTATTTT 1742 CAWNRLGMDTQYF
TGTGCCAGCAGCTTAACGCACGCTAGCGGGGAGCAGTACTTC 1739 CASSLTHASGEQYF
TGTGCCTGGAGAGATAACCTTGCGGAAAAACTGTTTTTT 1733 CAWRDNLAEKLFF
TGTGCCAGCAGCTTGGTCTCTAGCGGGAGCGATACGCAGTATTTT 1675 CASSLVSSGSDTQYF
TGTGCCTGGAGTGTACTCGGGCCGAGTCCCCAGTACGTC 1605 CAWSVLGPSPQYV
TGCAGTGTTAATAGTCCGGCTTTAACGGGACAGCCCCAGCATTTT 1563 CSVNSPALTGQPQHF
TGCGCCAGCAGCTCCCGGGACGGGACGAATTCACCCCTCCACTTT 1556 CASSSRDGTNSPLHF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCGCTTCCGGGACAGGGTCCTACGAGCAGTACTTC 33028 CASSASGTGSYEQYF
TGCGCCAGCAGCCAAGCCCCCTCTGGGGTTAGGCCCGGGGAGCTGTTTTTT 32838 CASSQAPSGVRPGELFF
TGTGCCTGGACGGGTGAAGGGGCCGCGGGGTTGCGCAATCAGCCCCAGCATTTT 22766 CAWTGEGAAGLRNQPQHF
TGTGCCAGCAGCCAAGTCGGAAGTCTAATTGAAAAACTGTTTTTT 21458 CASSQVGSLIEKLFF
TGTGCCTGGAACCTCTACCCGGGACTAGCGGGGACAGATACGCAGTATTTT 19128 CAWNLYPGLAGTDTQYF
TGCAGCGTTCCAGGGGTCTGGTCCTACAATGAGCAGTTCTTC 16761 CSVPGVWSYNEQFF

PT
TGTGCCAGCAGCTTGGGGGGGACCAGACTGAAAAATGAAAAACTGTTTTTT 15048 CASSLGGTRLKNEKLFF
TGTGCCAGCAGCTTAGTCGCGGGAGAGGGGGAGTTCTTC 11894 CASSLVAGEGEFF
TGTGCCAGCAGCGTAGACGTCGGGAGGTCCTACGAGCAGTACTTC 9692 CASSVDVGRSYEQYF

RI
TGCGCCAGCAGCTTGGGCTTGGATGTTGAGACCCAGTACTTC 8359 CASSLGLDVETQYF
TGTGCCAGCAGCTTGTGGGGGCAACAGACCCAGTACTTC 6894 CASSLWGQQTQYF
TGTGCCACCTCGTTTCCAGCGCGCCACAATGAGCAGTTCTTC 6173 CATSFPARHNEQFF

SC
TGTGCCAGCAGCTTTGATTCGGGACAAGGACGGGAAAAGTACTTC 5746 CASSFDSGQGREKYF
TGTGCCAGCAGCCCAAACTCACTAGCGGGAGTCAATGAGCAGTTCTTC 5624 CASSPNSLAGVNEQFF
TGTGCCAGCAGCTTGGCGAGCGGGACCGCCGGCGAGCAGTTCTTC 3550 CASSLASGTAGEQFF

U
TGTGCCAGCAGCTTCCTCCTCAACCCCCGGGATACGCAGTATTTT 3501 CASSFLLNPRDTQYF
TGTGCCAGCAGCCACACCGGGACTAGCGGGACCCCGCAGTACTTC 3410 CASSHTGTSGTPQYF
AN
TGCAGTGCTAGTTTTGATTCTGGAAACACCATATATTTT
TGTGCCAGCACCCATCTTAGCGGGACCTACAATGAGCAGTTCTTC
3320
3173
CSASFDSGNTIYF
CASTHLSGTYNEQFF
TGTGCCAGCAGCAGGACCGTCGGGAATGGCTACACCTTC 2899 CASSRTVGNGYTF
TGTGCCAGCAGTTACACCGGACAGGGGGTCTATGGCTACACCTTC 2664 CASSYTGQGVYGYTF
M
TGTGCCAGCAGCTTCGGAGCCACCATTAACTATGGCTACACCTTC 2417 CASSFGATINYGYTF
TGTGCCTGACAGGGTGGAATGGCTACACCTTC 2278 CA*QG_NGYTF
TGTGCCAGCAGCTTAGTTGGAAACTCTGGAAACACCATATATTTT 2235 CASSLVGNSGNTIYF
D

TGTGCCAGCAGCTTAGAGATCGGGAATGAGCAGTTCTTC 2208 CASSLEIGNEQFF

TGTGCCAGCAGCTTAAGACACACCACCGGGGAGCTGTTTTTT 2169 CASSLRHTTGELFF
TE

TGTGCCAGCAGCCTAGGACTAGCGGGAAACGTCCGGCATTACAATGAGCAGTTCTTC 2146 CASSLGLAGNVRHYNEQFF

TGTGCCAGCAGCTCAGCGGGAGAGGTCAAGGAGCAGTTCTTC 2135 CASSSAGEVKEQFF
TGTGCCAGCAGCTTCGACAGGGGGAATTCACCCCTCCACTTT 1987 CASSFDRGNSPLHF
EP

TGCAGCGTTGTCCCGGCCAGCCGGAACACCGGGGAGCTGTTTTTT 1796 CSVVPASRNTGELFF

TGTGCCAGCAGTATTTCGAGCTCCTACAATGAGCAGTTCTTC 1749 CASSISSSYNEQFF
TGCAGTGCCGGGGGACTAGCGATTAGCACAGATACGCAGTATTTT 1690 CSAGGLAISTDTQYF
C

TGTGCCAGCGCATCAGGAGGGGCCAACGTCCTGACTTTC 1588 CASASGGANVLTF

TGTGCCAGCAGCATCGGGAACTACAATGAGCAGTTCTTC 1500 CASSIGNYNEQFF
AC

TGTGCCTGGAGTGCTCTGGGGATGGGGGAGACCCAGTACTTC 1463 CAWSALGMGETQYF

TGTGCCTGGAGTGGGGGGGTCGGCTATAATTCACCCCTCCACTTT 1455 CAWSGGVGYNSPLHF
TGTGCCAGCAGCCCGCGCTCGGACAGGGGAGATTCACCCCTCCACTTT 1455 CASSPRSDRGDSPLHF
TGTGCCTGGAGAGGGGCTAGCACAGATACGCAGTATTTT 1444 CAWRGASTDTQYF
TGTGCCAGCAGCTTAAGGACTAGCGGGACGGAGACCCAGTACTTC 1442 CASSLRTSGTETQYF
TGTGCCAGCAGCTTAGCCCGATGGGCAACTAATGAAAAACTGTTTTTT 1438 CASSLARWATNEKLFF
TGCAGTGCTGGGGTAGGGAGGGACCACGGGGAGCTGTTTTTT 1436 CSAGVGRDHGELFF
TGCGCCAGCAGCGGGGGACTTAGCTCCTACGAGCAGTACTTC 1415 CASSGGLSSYEQYF
TGCAGTGCTGGGGGGGGAAGAGATACGCAGTATTTT 1372 CSAGGGRDTQYF
TGTGCCAGCAGCTTGCCCAGGGGGCATCCCGGGGAGCTGTTTTTT 1237 CASSLPRGHPGELFF
TGTGCCAGCCGACAGGGAACGAACACCGGGGAGCTGTTTTTT 1205 CASRQGTNTGELFF
TGTGCCAGCAGTTACTTCGGGAACACTGAAGCTTTCTTT 1075 CASSYFGNTEAFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGTCGACTAGCGGAGGGGAGGTACGAGCAGTACGTC 30769 CASSRLAEGRYEQYV
TGCAGTGCTTATTACACCTACCAAGAGACCCAGTACTTC 25592 CSAYYTYQETQYF
TGTGCCTGGAGTTTGGACACCCAGTGGAGCGGCTACACCTTC 25497 CAWSLDTQWSGYTF
TGCAGCGTTGCCCGAGACCCAAATGCCGGGGAGCTGTTTTTT 22391 CSVARDPNAGELFF
TGTGCCAGCAGCTTGTACGTTGGGAAAGCTTTCTTT 17185 CASSLYVGKAFF
TGTGCCAGCAGCTTAGTTGGAAACTCTGGAAACACCATATATTTT 16483 CASSLVGNSGNTIYF
TGTGCCTGGAGTGTCTTTCCAGCGACACAAGAGACCCAGTACTTC 16483 CAWSVFPATQETQYF

PT
TGTGCCTGGAGCAGGGGTGAGACCCAGTACTTC 13440 CAWSRGETQYF
TGTGCCAGCTCCCGGGGTACCTACGAGCAGTACGTC 11904 CASSRGTYEQYV
TGTGCCAGCAGCCAAGATTGGCGGAACACTGAAGCTTTCTTT 11250 CASSQDWRNTEAFF

RI
TGCAGCGTTGAAGGGACTAGCGGGAGTTACTCCTACAATGAGCAGTTCTTC 10468 CSVEGTSGSYSYNEQFF
TGTGCCAGCAGCCCTCTAGCCCAAGACAGACGACCTTCCCTCCACTTT 9927 CASSPLAQDRRPSLHF
TGTGCCAGCAGCTTAGGGTGGCAGGGGAGAGAGACCCAGTACTTC 9559 CASSLGWQGRETQYF
TGTGCCAGCACTCCTGTCAGGGTCGGCACTGAAGCTTTCTTT 9529 CASTPVRVGTEAFF

SC
TGTGCCTGGAGCCAGCCGGGGTCGGAGACCCAGTACTTC 8373 CAWSQPGSETQYF
TGTGCCAGCAGCTCCCTAGTGGTCTATGAGCAGTTCTTC 7806 CASSSLVVYEQFF
TGTGCCTGGAGGGATGAGACCGATTATGGCTACACCTTC 7682 CAWRDETDYGYTF
TGTGCCAGCAGCACAGACCGGGACGCCGTGAATTTCTTT 7380 CASSTDRDAVNFF

U
TGTGCCTGGGGTGACAGGGGTGACACTGAAGCTTTCTTT 7085 CAWGDRGDTEAFF
TGTGCCACCAGCAGAGTTAAGGGACGGGGAACTGAAGCTTTCTTT 6503 CATSRVKGRGTEAFF

TGTGCCAGCAGCTGGAGGGGGGCAGATACGCAGTATTTT
AN
TGTGCCAGCAGTTACGTGGGAAATCTTAACTATGGCTACACCTTC 5636
5460
CASSYVGNLNYGYTF
CASSWRGADTQYF
TGCGCCAGCAGGTCCAGTCCTGATGAGACCCAGTACTTC 5291 CASRSSPDETQYF
TGTGCCAGCAGCGGAATTCCGGGCGCGAAGCAGTTCTTC 4367 CASSGIPGAKQFF
M
TGCGCCAGCAGCCAAGGGACTAGCAGCGAGCAGTACTTC 4216 CASSQGTSSEQYF
TGTGCCAGCAGCTTAAGCAACACCGGGGAGCTGTTTTTT 4008 CASSLSNTGELFF
TGTGCCAGCAGCTTATCTTTAGGGGCTGGGGCCAACGTCCTGACTTTC 3770 CASSLSLGAGANVLTF
D

TGCGCCAGCAGCCAAAGGGGGAGCTCCTACAATGAGCAGTTCTTC 3631 CASSQRGSSYNEQFF

TGTGCCAGCAGCTGGGTTGGGGATACGCAGTATTTT 2651 CASSWVGDTQYF
TE

TGTGCCAGCAGTTCAGGACAGGGTTCCTACAATGAGCAGTTCTTC 2391 CASSSGQGSYNEQFF

TGTGCCAGCAGCACAGGGGTTGAAGCTTTCTTT 2099 CASSTGVEAFF
TGTGCCAGCAGCCATCGACAGGGGGCTGCAACTAATGAAAAACTGTTTTTT 2025 CASSHRQGAATNEKLFF
TGTGCCTGGAGTCCATCTAATCAGCCCCAGCATTTT 1964 CAWSPSNQPQHF
EP

TGTGCCAGCAGCTTTAGGGGCAGGGATACTGAAGCTTTCTTT 1955 CASSFRGRDTEAFF

TGTGCCAGCAGGGGTGGACTAGCGGGAGTTGTTGAGCAGTTCTTC 1858 CASRGGLAGVVEQFF
TGTGCCAGCAGTGAAGGACAGGGGACGCAGTATTTT 1731 CASSEGQGTQYF
TGTGCTAGCAGCGCACGGACTAGCGGGAACTACAATGAGCAGTTCTTC 1713 CASSARTSGNYNEQFF
C

TGCAGCGTTGACGGTTTAGGGCCCTACGAGCAGTACTTC 1429 CSVDGLGPYEQYF

TGTGCCAGCAGCTTAAACTCGGGAGATACGCAGTATTTT 1423 CASSLNSGDTQYF
AC

TGTGCCAGCAGCCAGTGGGACACCGGGGAGCTGTTTTTT 1329 CASSQWDTGELFF

TGTGCCTGCCCGGAGCTGGGACAGGGGTGTTGAGGGCTACACCTTC 1319 CACPELGQ_GVEGYTF
TGCAGTGGGACGTGGAATGAGCAGTTCTTC 1083 CSGTWNEQFF
TGCAGTGCTAGAGATCATCAACGTGGACAGTTCGGTGAGCAGTTCTTC 1078 CSARDHQRGQFGEQFF
TGCGCCAGCAGCTTCGAGGGGTTCGAATATGGCTACACCTTC 1059 CASSFEGFEYGYTF
TGTGCCAGCAGCTCGCGAGTAATACGACAGGGAACCAATGGCTACACCTTC 1017 CASSSRVIRQGTNGYTF
TGTGCCAGCAGCTTGGTAGAGAACACTGAAGCTTTCTTT 973 CASSLVENTEAFF
TGTGCCAGCAGCTTTGGGGGAGGCTATGGCTACACCTTC 961 CASSFGGGYGYTF
TGTGCCAGCAGCTTAAATGCAGCGGGAGTGGGCGAGACCCAGTACTTC 961 CASSLNAAGVGETQYF
TGTGCCAGCAGCCTAACACCGGGGCTCACAGATACGCAGTATTTT 904 CASSLTPGLTDTQYF
TGTGCCAGCAGCCAGGGTCGTGGCTACACCTTC 796 CASSQGRGYTF
TGTGCCACCAGTGAAGCAACGGGACTTTACAGTGGGCAGTTCTTC 774 CATSEATGLYSGQFF
TGTGCCAGCAGCTTGGAACGAACAGATACGCAGTATTTT 763 CASSLERTDTQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAAGGATCTAGCAAGGCCGGCAGATACGCAGTATTTT 50115 CAWKDLARPADTQYF
TGTGCCAGCAGCTTCGGGGTAACTAGCACAGATACGCAGTATTTT 37834 CASSFGVTSTDTQYF
TGCGCCAGCAGCCAAGAGGGGGGCCGGGGGAGCACAGATACGCAGTATTTT 36188 CASSQEGGRGSTDTQYF
TGTGCCAGCAGTTACGGACCGACAGGGGGTTATGGCTACACCTTC 30465 CASSYGPTGGYGYTF
TGCAGCGTTGATCCACGAGGAGCGGGAGCGGGGTACAATGAGCAGTTCTTC 26893 CSVDPRGAGAGYNEQFF
TGTGCCAGCAGCGCGGACAGGGCGGCTAACTATGGCTACACCTTC 24874 CASSADRAANYGYTF

PT
TGTGCCAGCAGTTGGGGGTTTCGGACTGGCAATGAGCAGTTCTTC 11409 CASSWGFRTGNEQFF
TGTGCCGGCGAGGGACCTAGAGCCGGGACAGGGTTCGATGAGCAGTTCTTC 9488 CAGEGPRAGTGFDEQFF
TGTGCCAGCAGCCATACCGGGACTAGCTACACAGATACGCAGTATTTT 8664 CASSHTGTSYTDTQYF

RI
TGCGCCAGCAGCCTGGGGGAGGGAGGGCCCTGGGAGACCCAGTACTTC 8244 CASSLGEGGPWETQYF
TGTGCCAGCAGCTCGTCTAGCGGGTCTTCCTACAATGAGCAGTTCTTC 6509 CASSSSSGSSYNEQFF
TGTGCCAGCAGTGCCCGGGACAACTCTGGAAACACCATATATTTT 5638 CASSARDNSGNTIYF

SC
TGTGCCAGCAGTTCCTTTAGCGGGGGGTCCTACAATGAGCAGTTCTTC 5384 CASSSFSGGSYNEQFF
TGTGCCAGCAGCCCCAGGAGGACAGGGGGGATCAACTATGGCTACACCTTC 4594 CASSPRRTGGINYGYTF
TGTGCCAGCAGCGCGACAGGGATGGTGGCAGATACGCAGTATTTT 4318 CASSATGMVADTQYF

U
TGTGCCAGCAGCTTATTAGGCGGAGAGACCCAGTACTTC 3500 CASSLLGGETQYF
TGTGCCAGCAGCTTAGTGCCAGGGGTGGTTGAAAAACTGTTTTTT 3310 CASSLVPGVVEKLFF

TGCAGTGCTGGGGGGGGAAGAGATACGCAGTATTTT
AN
TGCAGTGCTAGAGATCGCAGGGGATGGTCCTACACCTTC 3123
3015
CSARDRRGWSYTF
CSAGGGRDTQYF
TGCGCCAGCAGCCAAGATGGCGCAGATACGCAGTATTTT 2748 CASSQDGADTQYF
TGTGCCACCAGCTCCGGGACAGGGTTTTGGGAGCAGTACTTC 2518 CATSSGTGFWEQYF
M
TGTGCCTGGAGTCTCCGCGGACTAGCGGGATATGGGGCCAACGTCCTGACTTTC 2396 CAWSLRGLAGYGANVLTF
TGCAGCAGTTCAGTCCAGGGGGAGCAGTACTTC 1797 CSSSVQGEQYF
TGTGCCAGCAGCTTCTATGGACAGGGGGCGTCCAGTGGAGAGACCCAGTACTTC 1794 CASSFYGQGASSGETQYF
D

TGCAGTGCTAGATCGGGGGACAGGGGTGAAAAACTGTTTTTT 1556 CSARSGDRGEKLFF

TGTGCCAGCATTCCGGGGGCGAGAAACACCATATATTTT 1538 CASIPGARNTIYF
TE

TGTGCCTGGAGTGTCGCCAGGGAGATCGGCTACACCTTC 1536 CAWSVAREIGYTF

TGTGCCAGCAGCCAGGGACAGACTGAAGAGACCCAGTACTTC 1386 CASSQGQTEETQYF
TGCAGTGCGCGAGAGCAAGGTAGCGGGACCTACGAGCAGTACGTC 1255 CSAREQGSGTYEQYV
EP

TGTGCCACCAGTGATTTAACGAAGCGGGCCCGACGGGAGACCCAGTACTTC 1165 CATSDLTKRARRETQYF

TGTGCCAGCAGCGGCGGACAGGGGGTAGGAGGCTACACCTTC 1117 CASSGGQGVGGYTF
TGTGCCAGCAGTTACTCTGGGGACACCATATATTTT 1097 CASSYSGDTIYF
C

TGTGCCAGCAGACGGACACTGGGGCCCAATGAGCAGTTCTTC 1095 CASRRTLGPNEQFF

TGTGCCAGCAGCTTAGTTGGAAACTCTGGAAACACCATATATTTT 1013 CASSLVGNSGNTIYF
AC

TGTGCCAGCAGCCATCGACAGGGGGCTGCAACTAATGAAAAACTGTTTTTT 925 CASSHRQGAATNEKLFF

TGTGCCAGCAGCTTAATGGGCAGTAAAGAGACCCAGTACTTC 794 CASSLMGSKETQYF
TGTGCCAGCAGTTACAGGAATCACAATGAGCAGTTCTTC 766 CASSYRNHNEQFF
TGTGCCTGGAGTGTAGACCGGTTTCAGCCCCAGCATTTT 745 CAWSVDRFQPQHF
TGCAGTGCTAGAGGAGGGGGGGATTCACCCCTCCACTTT 695 CSARGGGDSPLHF
TGTGCCAGCGGGCCCCCCCGTAGCGATACGCAGTATTTT 661 CASGPPRSDTQYF
TGTGCCAGCAGCACGGACGGGAATCAGCCCCAGCATTTT 650 CASSTDGNQPQHF
TGTGCCAGCAGCTTAGGGAAGTTTGCAGTGGACCAGCCCCAGCATTTT 591 CASSLGKFAVDQPQHF
TGCAGTGCTGCCAGTGACGAAAAACTGTTTTTT 554 CSAASDEKLFF
TGCAGTGGAGGGGTAGGCTACACCTTC 507 CSGGVGYTF
TGTGCCAGCAGTATAGGGCAGGGGCACACTGAAGCTTTCTTT 504 CASSIGQGHTEAFF
TGTGCCAGCAGCTTAGCGCCCCTGGTCAATTCACCCCTCCACTTT 502 CASSLAPLVNSPLHF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAGTGTACTGTGGCTTCACCAAGAAGAGACCCAGTACTTC 36495 CAWSVLWLHQEETQYF
TGTGCCCAGGAGAAGGGAAGGGGCGGCACAGATACGCAGTATTTT 33746 CAQEKGRGGTDTQYF
TGTGCTAGCAGCCCCGGGCCCAGGGGTGAGCAGTTCTTC 29258 CASSPGPRGEQFF
TGTGCCAGCAGCTCCCCGCGAAGGCAAGAGACCCAGTACTTC 25715 CASSSPRRQETQYF
TGTGCCACCAGCAGAGATCCAGGGAGCTCCTACGAGCAGTACTTC 17695 CATSRDPGSSYEQYF
TGTGCCAGCAGCGGAGGGACAGGGTCCAACTATGGCTACACCTTC 15472 CASSGGTGSNYGYTF

PT
TGCAGCGTTGGGACAGCCAATCAGCCCCAGCATTTT 15266 CSVGTANQPQHF
TGCAGTGCTAGACAACAGCTTAATCAGCCCCAGCATTTT 11870 CSARQQLNQPQHF
TGTGCCAGCAGCGATCACCCGGGACAGGGAATTTCCTACGAGCAGTACGTC 11755 CASSDHPGQGISYEQYV

RI
TGTGCCAGCAGCGCCGGGACAGGGGCGTTTCAGTACTTC 9062 CASSAGTGAFQYF
TGTGCCAGCAGCTTAACTGGGCGGGACACTGAAGCTTTCTTT 8965 CASSLTGRDTEAFF
TGTGCCAGCAGTCTAGGGGTAGGGAACTACGAGCAGTACTTC 8587 CASSLGVGNYEQYF

SC
TGCGCCAGCAGCCAGAGGCCGGGACCCTGGGGTCTCACCTTC 7695 CASSQRPGPWGLTF
TGTGCCAGCAGCCAAGTGGGCGATCAGCCCCAGCATTTT 7323 CASSQVGDQPQHF
TGTGCCTGGAGTAAGCGGCCGCACACAGATACGCAGTATTTT 7103 CAWSKRPHTDTQYF
TGTGCCAGCAGCCTCGCCCCCGGGACAGGGAATGAAAAACTGTTTTTT 6747 CASSLAPGTGNEKLFF

U
TGTGCCAGCAGCCGTGCTGGGAGGGGAAACACTGAAGCTTTCTTT 5783 CASSRAGRGNTEAFF
TGCAGCGTTGGCCAGGGGAGGACTGAAGCTTTCTTT 5265 CSVGQGRTEAFF
AN
TGCAGTGCTAGAGATATAGGGGACAGCGGAATTTATAGCAATCAGCCCCAGCATTTT
TGCGCCAGCAGCCAAGGGACTAGCGGGAGTTTAACCGGGGAGCTGTTTTTT
5121
4853
CSARDIGDSGIYSNQPQHF
CASSQGTSGSLTGELFF
TGTGCCAGCACCTTGGAGGGGGCGCAGGAGACCCAGTACTTC 4464 CASTLEGAQETQYF
M
TGTGCCAGCAGCTTAGGATCAGGGAGCTCTGGGGCCAACGTCCTGACTTTC 4456 CASSLGSGSSGANVLTF
TGTGCCAGCAGCTTAGTCGCCGTAGGTAGCACAGATACGCAGTATTTT 4346 CASSLVAVGSTDTQYF
TGCAGCGTTGTACTCTCGGGGAATGAAAAACTGTTTTTT 3906 CSVVLSGNEKLFF
D

TGTGCCAGCAGTGAGGGGAACACTGAAGCTTTCTTT 3867 CASSEGNTEAFF

TGTGCCAGCAGCTTCGGGGGGGACTATGGCTACACCTTC 3534 CASSFGGDYGYTF
TGTGCCAGCAGCATCCTGGCGGATACGCAGTATTTT 3377 CASSILADTQYF
TE

TGTGCCAGCAGCTATGCGGGACTAGCGAATGAGCAGTTCTTC 3312 CASSYAGLANEQFF

TGTGCCTGGAGTCGAGGGTTCCAAGAGACCCAGTACTTC 3103 CAWSRGFQETQYF
TGCGCCAGCAGCTTTGAGGGAGGCACAGATACGCAGTATTTT 3050 CASSFEGGTDTQYF
EP

TGCAGTGCTAGGGGACAGGCCCCGTACCAAGAGACCCAGTACTTC 2614 CSARGQAPYQETQYF

TGTGCCAGCGGGGATTTTGGAGGGCCAGATACGCAGTATTTT 2501 CASGDFGGPDTQYF
TGTGCCAGCAGCATCGGACTAGCGGACAATGAGCAGTTCTTC 2491 CASSIGLADNEQFF
TGCAGCGTTGTAGGGCTAGCCGGCGAGCAGTTCTTC 2376 CSVVGLAGEQFF
C

TGCGCCAGCAGCCAAGATCCCGGGAGCCCTCTCACAGATACGCAGTATTTT 2168 CASSQDPGSPLTDTQYF

TGTGCCAGCAGTTCCCATTGGGACCGCGGGCAGTTCTTC 2135 CASSSHWDRGQFF
AC

TGTGCCAGCAGCCCCGGGACTAGCGGGATCGGCAATGAGCAGTTCTTC 2129 CASSPGTSGIGNEQFF

TGTGCCAGCAGCTCCGTGGGACTAGCGGGAGCCGAGCAGTTCTTC 1986 CASSSVGLAGAEQFF
TGTGCCAGCAGCTTAAGTTCTAGCCGGCAGTATTTT 1835 CASSLSSSRQYF
TGTGCCAGCAGCGACATCGGCACTGAAGCTTTCTTT 1547 CASSDIGTEAFF
TGTGCCAGCAGCTTGAGATCCGGTTGGGGCCCCCTTAATTCACCCCTCCACTTT 1501 CASSLRSGWGPLNSPLHF
TGTGCTAGCAGCTTAGATATCCAGGGGGTAGGGGGTGAGCAGTTCTTC 1486 CASSLDIQGVGGEQFF
TGTGCCAGCAGCCAAGATCCCAGGGAGACCCAGTACTTC 1465 CASSQDPRETQYF
TGTGCCAGCAGCGAAGTAGCGGGAGAGGGCGAGACCCAGTACTTC 1449 CASSEVAGEGETQYF
TGTGCCAGCAGCTTAATCCCGGGACTAAGTGAGCAGTTCTTC 1449 CASSLIPGLSEQFF
TGTGCCAGCAGCCCCCAGCGGGACCTTGAGCCCCAGCATTTT 1443 CASSPQRDLEPQHF
TGTGCCTGGAGTGCCGGGACAGCTAATGAAAAACTGTTTTTT 1408 CAWSAGTANEKLFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAGTGGCCAAAACATTCAGTACTTC 5713 CAWSGQNIQYF
TGTGCCGGCAGAGAGACCCAGTACTTC 2426 CAGRETQYF
TGTGCCTGGAGTGTTCAGGGGGCGTATGGCTACACCTTC 2229 CAWSVQGAYGYTF
TGTGCCAGCAGCTTAATGGTATGGGGATATCTTCCCTCGTTGCTCTTC 2138 CASSLMVWGYLPSLLF
TGTGCCTGGAGTCGATCCGGGACTAGCGGCAATGAGCAGTTCTTC 1858 CAWSRSGTSGNEQFF
TGTGCCTGGGAGTCTGGAGGGAACACTGAAGCTTTCTTT 1827 CAWESGGNTEAFF

PT
TGTGCCTGGAGCGAGGGACTCCAGCTGAGCACAGATACGCAGTATTTT 1769 CAWSEGLQLSTDTQYF
TGTGCCAGCAGCCCAAATCGCCCCTACGAGCAGTACGTC 1666 CASSPNRPYEQYV
TGTGCCTGGAGTGTAGCCGTTAACTATGGCTACACCTTC 1636 CAWSVAVNYGYTF

RI
TGTGCCTGGAGTATCGGACAGGGAAACACTGAAGCTTTCTTT 1520 CAWSIGQGNTEAFF
TGTGCCTGGACGGCCCCCCAGGGTGGGGTGATCAATCAGCCCCAGCATTTT 1441 CAWTAPQGGVINQPQHF
TGTGCCTGGAGTGAAAGGGTTACTCAGCCCCAGCATTTT 1381 CAWSERVTQPQHF

SC
TGTGCCTGGAGTGTTCAACTGGACTACGAGCAGTACTTC 1316 CAWSVQLDYEQYF
TGTGCCTGGAGCCCCTCCAATACGCAGTATTTT 1255 CAWSPSNTQYF
TGTGCCTGGGCGAAGGGCGGACGCAGCACAGATACGCAGTATTTT 1229 CAWAKGGRSTDTQYF
TGTGCCGGGGGGCGGATGAACACTGAAGCTTTCTTT 1202 CAGGRMNTEAFF

U
TGTGCCAGCAGCCCCGGACTAGCGGGAGGGATATACGAGCAGTACGTC 1164 CASSPGLAGGIYEQYV
TGTGCCTGTATGACAGGGGGCTTGGCCACTGAAGCTTTCTTT 1146 CACMTGGLATEAFF
TGTGCCAGCAGCTTAGTGTCCTGGTCCGAGCAGTACTTCAN
TGTGCCAGCAGCTTGCCGGATTAGCAATCAGCCCCAGCATTTT
1120
1069
CASSLVSWSEQYF
CASSLPD_SNQPQHF
TGTGCCTGGAGTACGGGAGGGGGGCTTGGCTACACCTTC 1020 CAWSTGGGLGYTF
M
TGTGCCTGGAGTGTACTAGCGGGATCGAATGAGCAGTTCTTC 1011 CAWSVLAGSNEQFF
TGTGCCTGGAGTGAGGGGCAGGGCAGCGGCTACACCTTC 992 CAWSEGQGSGYTF
TGTGCCTGGCAGTCCGGGGTAGCGGGAGCAAACTCCTACAATGAGCAGTTCTTC 917 CAWQSGVAGANSYNEQFF
D

TGTGCCGGCGGGAGGGGTAATCAGCCCCAGCATTTT 900 CAGGRGNQPQHF

TGTGCCTGGAACAGGGGCGGGGATGGGGGCACAGATACGCAGTATTTT 856 CAWNRGGDGGTDTQYF
TGCGCCAGCAGCCAAGATTGGACTAGCGGGCCGTACTCCTACAATGAGCAGTTCTTC 851 CASSQDWTSGPYSYNEQFF
TE

TGTGCCTGGAGACCGACAGGGGGCCGAGATCAGCCCCAGCATTTT 807 CAWRPTGGRDQPQHF

TGTGCCTGGCGGACAGGGACCAACTATGGCTACACCTTC 790 CAWRTGTNYGYTF
TGTGCCTGGAGTCCCCGAAGCGAGTCCCAGTACTTC 776 CAWSPRSESQYF
EP

TGTGCCAGCCGGGGACAAACGAACACTGAAGCTTTCTTT 767 CASRGQTNTEAFF

TGTGCCTGGGGCAGTGCTTATGGCTACACCTTC 745 CAWGSAYGYTF
TGCAGCGTACGGGACAGGCGGAATTCACCCCTCCACTTT 721 CSVRDRRNSPLHF
TGTGCCTGGATGCGACAGACGAACACTGAAGCTTTCTTT 694 CAWMRQTNTEAFF
C

TGTGCCTGGAGTTCGGGACAGCACAATCAGCCCCAGCATTTT 686 CAWSSGQHNQPQHF

TGTGCCAGCAATCCGGGACTTGCCCCCTACGAGCAGTACTTC 662 CASNPGLAPYEQYF
AC

TGTGCCTGGACCACCCTGATGGAAAACACCATATATTTT 654 CAWTTLMENTIYF

TGTGCCACCAGGGAGGAAGTCGAAAAACTGTTTTTT 651 CATREEVEKLFF
TGTGCCTGGAGCAGCGGGAGGGAGAAACTGTTTTTT 611 CAWSSGREKLFF
TGTGCCAGCAGACGGGTGACAGGGAGGAGCAATGAGCAGTTCTTC 606 CASRRVTGRSNEQFF
TGTGCCTGGAACCGGGGCCGGCAAAACATTCAGTACTTC 604 CAWNRGRQNIQYF
TGTGCCTGGAGTGTGGGGGGCGTGGTGAACACTGAAGCTTTCTTT 560 CAWSVGGVVNTEAFF
TGTGCCTGGAGATCGGATAATAGCAATCAGCCCCAGCATTTT 549 CAWRSDNSNQPQHF
TGTGCCTGGAGTGTTGGACTGTCAGGGGAGCTGTTTTTT 542 CAWSVGLSGELFF
TGTGCCTGGAGTGCGGGGACTAGCACAGATACGCAGTATTTT 537 CAWSAGTSTDTQYF
TGTGCCTGGAGTGTACAGGGGGGTAATACCGGGGAGCTGTTTTTT 531 CAWSVQGGNTGELFF
TGTGCCAGCAGCTTAGCGGTGGCAGGACGGCCGCTACACCTTC 519 CASSLAV_QDGRYTF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGAGAGGGGTCTGGCACAGATACGCAGTATTTT 1975 CASREGSGTDTQYF
TGTGCCAGCAGCAAGGGGACAGCAAATCAGCCCCAGCATTTT 1670 CASSKGTANQPQHF
TGTGCCTGTGTGGGACAGATTTACAATGAGCAGTTCTTC 1380 CACVGQIYNEQFF
TGTGCCTGGAGTGCGGGGGCGAACACTGAAGCTTTCTTT 1157 CAWSAGANTEAFF
TGTGCCTGGAGTGCGGGTGCCAGAAGCACAGATACGCAGTATTTT 1027 CAWSAGARSTDTQYF
TGTGCCTGGGCGAAGGGCGGACGCAGCACAGATACGCAGTATTTT 982 CAWAKGGRSTDTQYF
TGTGCCCGGACAGGGGGCCCGGGGTCAACGGGCTACACCTTC 954 CARTGGPGSTGYTF

PT
TGTGCCTGGAGTGGAGACAGGGTGGAGACCCAGTACTTC 942 CAWSGDRVETQYF
TGTGCCTGGAGTGGCCAAAACATTCAGTACTTC 930 CAWSGQNIQYF
TGTGCCTGGAACGAGGCGGCAGGCAATCAGCCCCAGCATTTT 843 CAWNEAAGNQPQHF

RI
TGTGCCAGCAGCTTAGCGACCGACAGCGCTGAAGCTTTCTTT 822 CASSLATDSAEAFF
TGTGCCAGCAGCCTCGGTCCTAGCGGGAGTGTTGGGGAGCAGTTCTTC 818 CASSLGPSGSVGEQFF
TGTGCCGGGGTAGGGGGCACTGAAGCTTTCTTT 813 CAGVGGTEAFF

SC
TGTGCCAGCAGCCAAGGGGGGGGCCGCGAGCCCCAGCATTTT 809 CASSQGGGREPQHF
TGTGCCAGCAGCCCAGGAAGGGGGTCCGGCACAGATACGCAGTATTTT 795 CASSPGRGSGTDTQYF
TGTGCCTGTGATAGCGGGATCGCCTACAATGAGCAGTTCTTC 768 CACDSGIAYNEQFF
TGTGCCTGGAGTGTACGGTCCCCAGATACGCAGTATTTT 737 CAWSVRSPDTQYF

U
TGTGCCTGGAGTGTACAGCAGAACACTGAAGCTTTCTTT 713 CAWSVQQNTEAFF
TGTGCCAGCAGCTTCAGGACAGTCGCATCCTACGAGCAGTACTTC 643 CASSFRTVASYEQYF
TGTGCCTGGAAACTCCAGGGCTCCTACAATGAGCAGTTCTTC
AN
TGTGCCAGCAGCCGCCCGGGACAGATGAACTATGGCTACACCTTC
TGTGCCAGCAGCTTAGGGGGCAGGGAATCTGTCTACGAGCAGTACTTC
638
634
598
CAWKLQGSYNEQFF
CASSRPGQMNYGYTF
CASSLGGRESVYEQYF
TGTGCCTGGGACAAACCAGGGGGAAACTACAATGAGCAGTTCTTC 569 CAWDKPGGNYNEQFF
M
TGTGCGGCCGGGACTTACACCTACGAGCAGTACTTC 561 CAAGTYTYEQYF
TGTGCCAGCAGCCGGGACCTTTCCGGGGAGACCCAGTACTTC 561 CASSRDLSGETQYF
TGTGCCTGGACGGGTTTCGGTAACAATGAGCAGTTCTTC 544 CAWTGFGNNEQFF
TGTGCCTGGGACAGCGGCAGGTGGGAGCAGTACTTC 541 CAWDSGRWEQYF
D

TGTGCCAGCAGCTTGAGCGGAGGCCAAGAGACCCAGTACTTC 535 CASSLSGGQETQYF

TGTGCCTGGAGGGATCAGGGGGCTAACTATGGCTACACCTTC 519 CAWRDQGANYGYTF
TE

TGTGCCAGCAGCTTAGGGGCAGGGACTGAAGCTTTCTTT 516 CASSLGAGTEAFF

TGTGCCAGCAGCTTAAACGGGCAGCGGGAGACCCAGTACTTC 509 CASSLNGQRETQYF
TGTGCCTGGAGTGTAGGGACAGCGAACACTGAAGCTTTCTTT 505 CAWSVGTANTEAFF
TGTGCCAGCAGCCAAAAAGTGATCCTAGCTACTAGCACAGATACGCAGTATTTT 501 CASSQKVILATSTDTQYF
EP

TGCGCCAGCAGCTATTGGGGGGGCGAGACCCAGTACTTC 486 CASSYWGGETQYF

TGCGCCAGCAGCCAAGATTATGTGGAGACCCAGTACTTC 473 CASSQDYVETQYF
TGTGCCTGGGGGAGTGGGGATACGCAGTATTTT 466 CAWGSGDTQYF
TGTGCCTGGTCATGGGGTGGCTATGGCTACACCTTC 451 CAWSWGGYGYTF
C

TGTGCCAGCAGCTTCGCGGCAACGGCTACTGAAGCTTTCTTT 445 CASSFAATATEAFF

TGTGCCAGCAGCTTAGGGGCAGACACTGAAGCTTTCTTT 440 CASSLGADTEAFF
AC

TGTGCCAGCAGCTCACCGGGACAGGGCGACGAGCAGTACTTC 432 CASSSPGQGDEQYF

TGCGCCAGCAGCCCTTTTTTTGTGGGACTAGAGATGGAGACCCAGTACTTC 429 CASSPFFVGLEMETQYF
TGTGCCTGGAGTTCCTATCTGAATACGCAGTATTTT 413 CAWSSYLNTQYF
TGTGCCTGGAGTACCCGTGGGACTCTCCAAGAGACCCAGTACTTC 408 CAWSTRGTLQETQYF
TGTGCCAGCAGCCCGGTGACTTGGACCAATTCACCCCTCCACTTT 404 CASSPVTWTNSPLHF
TGTGCCAGCAGCCAACGCGCGGGAGCCGCCTACGAGCAGTACGTC 397 CASSQRAGAAYEQYV
TGTGCCTGGACCGAGGGGGGGCCCTCAGGAGCTTTCTTT 394 CAWTEGGPSGAFF
TGTGCCAGCAGGCCGGGACTAGCGTCCTGGGCCGGGGAGCTGTTTTTT 393 CASRPGLASWAGELFF
TGTGCCAGCAGCCAACTGGGAAGGGACACTGAAGCTTTCTTT 393 CASSQLGRDTEAFF
TGTGGGGGGGACACAGGAGTAGAGAACACTGAAGCTTTCTTT 392 CGGDTGVENTEAFF
TGTGCCTGGAGTACCGGGACAGTTTCCTATGGCTACACCTTC 385 CAWSTGTVSYGYTF
TGTGCTAGCAGCAAAGGGGCCGGGGGCACCGGGGAGCTGTTTTTT 385 CASSKGAGGTGELFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGTATAGGGGGAGGGCAGTCCGAGCAGTACTTC 19802 CASSIGGGQSEQYF
TGTGCCAGCAGCCCGGGCTCCTATAATTCACCCCTCCACTTT 6613 CASSPGSYNSPLHF
TGCAGTGCTGGCCCTCAGGCACTAGCGGGTAATGAGCAGTTCTTC 4686 CSAGPQALAGNEQFF
TGCGCCAGCAGCTCCTCCATAGCGGGAGATGGTGCGAATGAGCAGTTCTTC 4057 CASSSSIAGDGANEQFF
TGTGCCAGCAGCACAATGCACGGACTGCAAGAGACCCAGTACTTC 3238 CASSTMHGLQETQYF
TGTGCCTGGAGCGGGGATGATCAGCCCCAGCATTTT 2883 CAWSGDDQPQHF
TGTGCCAGCAGCCCCCCTAGCGGGAACTACGAGCAGTACTTC 2455 CASSPPSGNYEQYF

PT
TGTGCCAGCAGCCTAGCGGAGCCCGGCACAGATACGCAGTATTTT 2429 CASSLAEPGTDTQYF
TGTGCCTGGAGTCGGGAGTTCGAGCAGTACTTC 2383 CAWSREFEQYF
TGTGCCAGCAGCTTAGTTGGCGGTTCTAGCACAGATACGCAGTATTTT 2363 CASSLVGGSSTDTQYF
TGTGCCTGGAGTGTGGGGGCCTCAGAGACCCAGTACTTC 1823 CAWSVGASETQYF

RI
TGTGCCAGCACGCAAGCCGGGACTAGCGGGTCCCAATGGTACTTC 1801 CASTQAGTSGSQWYF
TGTGCCAGCAGCCAAGATCGGGGGACAGGGGCCAAGGCTTGGGGCTACACCTTC 1722 CASSQDRGTGAKAWGYTF
TGCGCCAGCAGCTTGGATGGGTATGGCTACACCTTC 1680 CASSLDGYGYTF

SC
TGTGCCACCAGCAGAGGCCGGGAAAACACTGAAGCTTTCTTT 1639 CATSRGRENTEAFF
TGTGCCAGCAGCCCGGGACTCAGCACCGGGGAGCTGTTTTTT 1591 CASSPGLSTGELFF
TGTGCCAGCAGCTTGGACGTCTACACTGAAGCTTTCTTT 1562 CASSLDVYTEAFF
TGTGCCTGGGGGATGGGGTTCTACGAGCAGTACTTC 1544 CAWGMGFYEQYF

U
TGTGCCAGCAGCAGGGGGCTGAGCTCCTACGAGCAGTACTTC 1494 CASSRGLSSYEQYF
TGTGCCAGCAGCTTAGCAGGCTCCTATAATTCACCCCTCCACTTT 1492 CASSLAGSYNSPLHF
TGTGCCAGCAGCTTCGAGGGGCGATGGGGCGAGCAGTACTTC
TGTGCCTGGAGTCTTGCTCTTAGCAATCAGCCCCAGCATTTT AN
TGTGCCAGCAGCAATTTCCCGGGACCGGACAATGAGCAGTTCTTC
1453
1441
1386
CASSFEGRWGEQYF
CAWSLALSNQPQHF
CASSNFPGPDNEQFF
TGTGCCAGCAGTACTAGCGGAGCTTTCACCGGGGAGCTGTTTTTT 1339 CASSTSGAFTGELFF
M
TGTGCCAGCAGCCCGGGGGGGGCGCTGGAGACCCAGTACTTC 1335 CASSPGGALETQYF
TGTGCCTGGAGTGTCGGACTAGCCACGGACGAGCAGTACTTC 1293 CAWSVGLATDEQYF
TGTGCCAGCAGCAAATATGAGGTGCAGTACTTC 1274 CASSKYEVQYF
TGTGCCAGCAGCTCAGGACAGGTGACCTACGAGCAGTACTTC 1265 CASSSGQVTYEQYF
D

TGCAGCGCAGAACGACAGAACACTGAAGCTTTCTTT 1230 CSAERQNTEAFF

TGTGCCAGCAGCGAGGGACAGGCGTCCTGGGAGCAGTACTTC 1211 CASSEGQASWEQYF
TE

TGTGCCAGCACCATCCCAAACAGTCCCCAGTACTTC 1109 CASTIPNSPQYF

TGTGCCAGCAGCCAATCTACGGGACTCATCACAGATACGCAGTATTTT 1100 CASSQSTGLITDTQYF
TGTGCCTGGCCGTGGGGGGAAAGGTACGAGCAGTACTTC 1070 CAWPWGERYEQYF
TGTGCCTGGAGCGGGAGCTCCCACGAGCAGTACTTC 1023 CAWSGSSHEQYF
EP

TGTGCCAGCAGCACCGGGGCGTCAGATACGCAGTATTTT 1023 CASSTGASDTQYF

TGTGCCAGCAGTTCCCAGGGACGCTACAATGAGCAGTTCTTC 983 CASSSQGRYNEQFF
TGTGCCAGCAGCTTAGGGGATACGCAGTATTTT 973 CASSLGDTQYF
TGTGCCTGGAATACAGGGGCGAGGGGCTACACCTTC 954 CAWNTGARGYTF
C

TGCGCCAGCAGCCAAGATGGGGGAGCTTCAGATACGCAGTATTTT 945 CASSQDGGASDTQYF

TGTGCCAGCAGCCGGGGGGGGGCATTGGAGACCCAGTACTTC 914 CASSRGGALETQYF
TGTGCCAGCAGCTTAGAGGGACTACCCCAGCATTTT 905 CASSLEGLPQHF
AC

TGTGCCTGGAGCGGAAACAGGGAATCAGCCCCAGCATTTT 898 CAWSGNR_NQPQHF

TGTGCCAGCAGCCCACCAGCTAGAAACGCCTACGAGCAGTACTTC 870 CASSPPARNAYEQYF
TGTGCCAGCAGTTACCATGGACTAGCCCAAGAGACCCAGTACTTC 853 CASSYHGLAQETQYF
TGTGCCTGGCGCCCCGACAGGGCCAGTTCACCCCTCCACTTT 847 CAWRPDRASSPLHF
TGCGCCAGCAGCTTGGACAGGGGGACTGAAGCTTTCTTT 832 CASSLDRGTEAFF
TGTGCCTGGGACAGGGGCCTGGAGACCCAGTACTTC 830 CAWDRGLETQYF
TGTGCCTGGAGTCCGGGACTAGCGGGACTCACAGATACGCAGTATTTT 823 CAWSPGLAGLTDTQYF
TGTGCCAGCAGCGAGACGACGGTGAACACTGAAGCTTTCTTT 817 CASSETTVNTEAFF
TGTGCCAGCAGCTCCCCCACTAGCTTACAAGAGACCCAGTACTTC 805 CASSSPTSLQETQYF
TGTGCCTGGAGTGTACCCCCCGGGACCGAGTATGCAGATACGCAGTATTTT 797 CAWSVPPGTEYADTQYF
TGTGCCAGCAGCCAAGAAGGGCAGAACTATGGCTACACCTTC 794 CASSQEGQNYGYTF
TGCGCCAGCAGCTTTGCGGGAGGCGGCACAGATACGCAGTATTTT 793 CASSFAGGGTDTQYF
TGTGCCAGCAGCTTAGAGACAGGGAAGCAGTACTTC 793 CASSLETGKQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCCCGGGCTCCTATAATTCACCCCTCCACTTT 4530 CASSPGSYNSPLHF
TGTGCCAGCAGCCCCCGCTTTCGCTACACCTTC 4500 CASSPRFRYTF
TGTGCCAGCAGCCCTGGTGGGACTTTATACGAGCAGTACTTC 3591 CASSPGGTLYEQYF
TGCAGTGCTTCCGGGACTAGCGGGCGCAGCTACGAGCAGTACTTC 3217 CSASGTSGRSYEQYF
TGTGCCAGCAGCTTAGAGCTGGACTATGGCTACACCTTC 3184 CASSLELDYGYTF
TGTGCCAGCAGCCGGGGGCCTTATAATTCACCCCTCCACTTT 3082 CASSRGPYNSPLHF

PT
TGTGCCAGCAGCTTAGGGCAGGGCCATGGCTACACCTTC 3015 CASSLGQGHGYTF
TGTGCCAGCAGCCGACAACAGCCTGGCACCGGGGAGCTGTTTTTT 2898 CASSRQQPGTGELFF
TGTGCCCGGGGGGTCTACCCGAACACCGGGGAGCTGTTTTTT 2779 CARGVYPNTGELFF

RI
TGTGCCAGCAGCGATACCGGGACTAGCGGGATGCCCTACGAGCAGTACTTC 2674 CASSDTGTSGMPYEQYF
TGTGCCAGCAGCCCCATGCGCACAGATACGCAGTATTTT 2601 CASSPMRTDTQYF
TGTGCCAGCAGGACGGCCTCCCAAGAGACCCAGTACTTC 2576 CASRTASQETQYF

SC
TGTGCCAGCAGCTTAGTTGGCGGTTCTAGCACAGATACGCAGTATTTT 2343 CASSLVGGSSTDTQYF
TGTGCCACCAGCAGAGGAATATACGACACTGAAGCTTTCTTT 2296 CATSRGIYDTEAFF
TGTGCCAGCAGCTACAACATGAACACTGAAGCTTTCTTT 2280 CASSYNMNTEAFF
TGTGCCAGCACTCGGGGGTTAAGGGCTGAAGCTTTCTTT

U
2143 CASTRGLRAEAFF
TGTGCCTGTCAGGGACAGAGCTACAATGAGCAGTTCTTC 2071 CACQGQSYNEQFF

AN
TGCGCCAGCAGCTTTGCGGGAGCTGGGACAGATACGCAGTATTTT
TGTGCCAGCAGCCCGACAGGCACTGGGGCCAACGTCCTGACTTTC
TGTGCCAGCAGCCCCCCCACTAGCGGGCCTCACAATGAGCAGTTCTTC
1980
1797
1761
CASSFAGAGTDTQYF
CASSPTGTGANVLTF
CASSPPTSGPHNEQFF
TGTGCCTGGTCGGAGAGGCCAACTGACAATGAGCAGTTCTTC 1733 CAWSERPTDNEQFF
M
TGTGCCAGCAGCTCCGGGACAGGGGGAGGAGAAGCTTTCTTT 1711 CASSSGTGGGEAFF
TGTGCCTGGGGAACGACAGGGTCCCATGGCTACACCTTC 1674 CAWGTTGSHGYTF
TGCAGCGTTGAAGGGCTAGCGGGAGGGCCTCGAGAGACCCAGTACTTC 1659 CSVEGLAGGPRETQYF
D

TGTGCCTGGTCTCCCTCCGGGTCCCCTTATGGCTACACCTTC 1650 CAWSPSGSPYGYTF

TGTGCCTGGAGCGGAGCCGGGCTCAATCAGCCCCAGCATTTT 1604 CAWSGAGLNQPQHF
TE

TGTGCCAGCAGCCGGACGGTCGCGGGAGGACCCACAGATACGCAGTATTTT 1483 CASSRTVAGGPTDTQYF

TGTGCCTGGAGCCCTACCCCCGGTGGCTACACCTTC 1453 CAWSPTPGGYTF
TGTGCCTGGAGTACTTTTACAGGGGGAAGCACTGAAGCTTTCTTT 1300 CAWSTFTGGSTEAFF
TGTGCCACCAGCAGAGATAGGAGACAGGGGGTAGAGCAGTTCTTC 1253 CATSRDRRQGVEQFF
EP

TGCGCCAGCAGCTTTGCGGGAGGCGGCACAGATACGCAGTATTTT 1166 CASSFAGGGTDTQYF

TGTGCCACCAGCAGAAATGTGGCAGAGACCCAGTACTTC 1118 CATSRNVAETQYF
TGTGCCAGCAGCCCCTACCAAGAGACCCAGTACTTC 1102 CASSPYQETQYF
C

TGTGCCTGGAGCTCCTCTGCGAACACAGATACGCAGTATTTT 1089 CAWSSSANTDTQYF

TGTGCCAGCAGTCCAACCCTTAAATCCTACGAGCAGTACTTC 1082 CASSPTLKSYEQYF
AC

TGTGCCAGCAGCCCGGGACTCAGCACCGGGGAGCTGTTTTTT 1005 CASSPGLSTGELFF

TGTGCCAGCAGCTGGGGTTGGACAGGCAATGAGCAGTTCTTC 987 CASSWGWTGNEQFF
TGTGCCAGCAGCAAATATGAGGTGCAGTACTTC 976 CASSKYEVQYF
TGTGCCAGCAGCTTATTCGCTAGCGGGAGCACAGATACGCAGTATTTT 941 CASSLFASGSTDTQYF
TGTGCCAGCAGCTCCCAAGGCGGGTCCAACACCGGGGAGCTGTTTTTT 941 CASSSQGGSNTGELFF
TGCAGCGTGGGTGAGGCCGCTAATCAGCCCCAGCATTTT 940 CSVGEAANQPQHF
TGTGCCAGCAGCTTATCGGCTAGCGGGCATACCGGGGAGCTGTTTTTT 917 CASSLSASGHTGELFF
TGTGCCAGCAGCACCGGACAGTGGGGCTACACCTTC 913 CASSTGQWGYTF
TGTGCCAGCAGCTTAGCAGGGCTGTACAATGAGCAGTTCTTC 875 CASSLAGLYNEQFF
TGTGCCTGGAGTCGACAGGGAAACGAGCAGTACTTC 837 CAWSRQGNEQYF
TGTGCCAGCAGCCAAGTTTGGGGACAGGGTCGGTTCTTC 835 CASSQVWGQGRFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCCGATCTTCGGCCCCAGGGGACAGCGAAAAACTGTTTTTT 33006 CASSRSSAPGDSEKLFF
TGCAGCGTTGGCAGCCCCATTACCTACGAGCAGTACTTC 16214 CSVGSPITYEQYF
TGTGCCAGCAGCTTCGACAGGGGTTCACCCCTCCACTTT 14121 CASSFDRGSPLHF
TGTGCCTGGAGAGTTGGGACACCGAACACTGAAGCTTTCTTT 11011 CAWRVGTPNTEAFF
TGCGCCAGCAGCTTGCAGGCCGGCTACACCGGGGAGCTGTTTTTT 9585 CASSLQAGYTGELFF
TGCAGTGGGAAACGGGAGTCCGACAATGAGCAGTTCTTC 6010 CSGKRESDNEQFF

PT
TGTGCCAGCAGCGATGAGCCAGGGGTCTATGGCTACACCTTC 5129 CASSDEPGVYGYTF
TGTGCCAGCAGCTCCCCTAGGGCTAACAGGAGGTCGGGCTACACCTTC 5061 CASSSPRANRRSGYTF
TGTGCCAGCAGCTTGTGGGGACAGGGGAGTCAGCCCCAGCATTTT 4748 CASSLWGQGSQPQHF

RI
TGTGCCAGCCAGCCCGGCTATGGCTACACCTTC 4288 CASQPGYGYTF
TGTGCCACCAGCAGAGAGGGACTAGCGGGAGGCACAGATACGCAGTATTTT 3584 CATSREGLAGGTDTQYF
TGTGCCAGCAGCTTAAGAGCGGACTCCTACAATGAGCAGTTCTTC 3247 CASSLRADSYNEQFF

SC
TGTGCCAGCAGCCGAACACGTCAGCATTCACCCCTCCACTTT 2931 CASSRTRQHSPLHF
TGTGCCAGCAGCTTAGGGCCCGCGGGAGCCAAAGAGACCCAGTACTTC 2865 CASSLGPAGAKETQYF
TGTGCCAGCAGCTTAGAGATCTTACTAGGGGGTATCGAGACCCAGTACTTC 2806 CASSLEILLGGIETQYF

U
TGTGCCAGCTGCCCCGGGACAGGGAGCTCACCCCTCCACTTT 2526 CASCPGTGSSPLHF
TGCGCCAGCAGCCGTACTAGCGGGAGATCCGATACGCAGTATTTT 2379 CASSRTSGRSDTQYF

TGTGCCAGCAGCTTCGGGGGCGGTGGCTACACCTTC
AN
TGTGCCAGCAGTTACTCCCCGCCAGCCAATGAGCAGTTCTTC 2235
2020
CASSYSPPANEQFF
CASSFGGGGYTF
TGTGCCAGCAGCCCACGGACTAGCCTTGACATTCAGTACTTC 1864 CASSPRTSLDIQYF
TGTGCCTGGAGGTCAGGGACACCCCCGGTAGAGCTGTTTTTT 1785 CAWRSGTPPVELFF
M
TGTGCCTGGAGGGTAGGGACACCGAACACTGAAGCTTTCTTT 1684 CAWRVGTPNTEAFF
TGTGCCAGCAGCCAAGGACAGCACCATGGCTACACCTTC 1602 CASSQGQHHGYTF
TGTGCCAGCAGCTCCCTGACAGGTAACACTGAAGCTTTCTTT 1465 CASSSLTGNTEAFF
D

TGTGCCACCAGCAGAGAGGGACTAGCGGGAGGGACAGATACGCAGTATTTT 1387 CATSREGLAGGTDTQYF

TGCAGTGCTGGGCGGGAGCAGGATAATGAGCAGTTCTTC 1355 CSAGREQDNEQFF
TE

TGTGCCAGCACCCTCCTAGACCGGGGTGAGCAGTTCTTC 1303 CASTLLDRGEQFF

TGTGCCAGCAGCTTCGGGTTCTTC 1298 CASSFGFF
TGTGCCAGCAGCTTAAGTGACAGGGGACACGAGCAGTACTTC 1261 CASSLSDRGHEQYF
EP

TGTGCCAGCAGCCTTGGTGGAGGCACTGAAGCTTTCTTT 1193 CASSLGGGTEAFF

TGTGCCAGCAGCTTAATTAGAGTCAGGGGATTGGAGACCCAGTACTTC 1054 CASSLIRVRGLETQYF
TGTGCCAGCAGTTTATATCGGGGAGGTGAAGCTTTCTTT 1004 CASSLYRGGEAFF
C

TGCGCCAGCAGATCGGGACTAGCGGGAGATGTGCATACGCAGTATTTT 984 CASRSGLAGDVHTQYF

TGCAGTGCTAGCCTTAGCGGGAGAGATGCGGGTGAGCAGTTCTTC 943 CSASLSGRDAGEQFF
AC

TGTGCCAGCAGAACCTCCGCCCACGGGGACGAGCAGTACTTC 930 CASRTSAHGDEQYF

TGCGCCAGCAGCCAAGCCAGGAACCCACGTCGGTTCTTC 904 CASSQARNPRRFF
TGTGCCTATGACCGGGGTCCTAATCAGCCCCAGCATTTT 840 CAYDRGPNQPQHF
TGTGCCAGCAGCTTTAGCAGGTTCGAGCAGTACTTC 801 CASSFSRFEQYF
TGTGCCAGCACCTTGTTGAGGAGCACAGATACGCAGTATTTT 775 CASTLLRSTDTQYF
TGCGCCAGCAGCTTGGCACCGAACCCTAACAATGAGCAGTTCTTC 744 CASSLAPNPNNEQFF
TGTGCCAGTGTCCGGGACAGGGGACGTTCTGGGGCCAACGTCCTGACTTTC 726 CASVRDRGRSGANVLTF
TGTGCCTGGAGGTTGGACAGGCGAAACTATGGCTACACCTTC 725 CAWRLDRRNYGYTF
TGTGCCAGCAGCTTAACGTCGGCGGGAGAGCAGTTCTTC 722 CASSLTSAGEQFF
TGCAGTGCTAGAAATATTCGGACAGGGGGCCTCCGAGCCTACGAGCAGTACTTC 704 CSARNIRTGGLRAYEQYF
TGTGCCAGCAGCTCCGGGACAGCCGACTATTTTCATTTT 691 CASSSGTADYFHF
TGTGCCAGCAGCTCCGGACTGAACACTGAAGCTTTCTTT 686 CASSSGLNTEAFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCCGTCAGTGGAACACTGAAGCTTTCTTT 28532 CASSRQWNTEAFF
TGCAGTGCTAGAGATTGGAGCGGAAGAACAGATACGCAGTATTTT 18973 CSARDWSGRTDTQYF
TGTGCCTGGAGTGTACTGGACACACATGGCTACACCTTC 9961 CAWSVLDTHGYTF
TGTGCCTGGAACCGGGCCTTCGACGAGCAGTACTTC 9880 CAWNRAFDEQYF
TGTGCCAGCAGCCAAGGACAGGGATACGGCTACACCTTC 7706 CASSQGQGYGYTF
TGTGCCAGCAGCTTGGACTTATTAGCCAAAAACATTCAGTACTTC 7146 CASSLDLLAKNIQYF

PT
TGCGCCAGCAGCACTCTGACTAGCGGGAATCAGGAGCAGTTCTTC 5909 CASSTLTSGNQEQFF
TGTGCCAGCAGTTACCGAGGCGGAGAAGCTTTCTTT 4891 CASSYRGGEAFF
TGTGCCAGCAGCGGACTAGCACCTAGCTCCTACAATGAGCAGTTCTTC 4848 CASSGLAPSSYNEQFF

RI
TGTGCCAGTATCCAAGCCGGACTAGTTCAAGAGACCCAGTACTTC 3685 CASIQAGLVQETQYF
TGCAGCGTTGGCAGCCCCATTACCTACGAGCAGTACTTC 2775 CSVGSPITYEQYF
TGTGCCAGCGGACAGCGTGGGAATGAAAAACTGTTTTTT 2417 CASGQRGNEKLFF

SC
TGTGCCAGCAGCGCCGACGGCGGGCGCATGACCTCCTACAATGAGCAGTTCTTC 2183 CASSADGGRMTSYNEQFF
TGCGCCAGCAGCTTGCGGACTAGTGGCAATACGCAGTATTTT 2061 CASSLRTSGNTQYF
TGTGCCAGCAGCACTTGGACAGATCAGCCCCAGCATTTT 1756 CASSTWTDQPQHF

U
TGTGCCTTGGGTTTTTC 1671 CAL_FF
TGTGCCAGCAGCTTTTCTAGCACAGATACGCAGTATTTT 1670 CASSFSSTDTQYF
AN
TGTGCCAGCAGCCAGGGCAGTACTGCTGGCCAGCCCCAGCATTTT
TGTGCCAGCACCCCGCGAAGGCGGAACACCGGGGAGCTGTTTTTT
1491
1398
CASSQGSTAGQPQHF
CASTPRRRNTGELFF
TGTGCCTGGAGTTTGGGGCGGAATCAGCCCCAGCATTTT 1258 CAWSLGRNQPQHF
TGTGCCAGCAGCGCCCGACAGGGGGAGAACACTGAAGCTTTCTTT 1200 CASSARQGENTEAFF
M
TGTGCCAGCTGCCCCGGGACAGGGAGCTCACCCCTCCACTTT 1171 CASCPGTGSSPLHF
TGTGCCAGCAGACTCACCAGACCTACGCCCTACGAGCAGTACTTC 1125 CASRLTRPTPYEQYF
TGTGCCAGCAGTTCTGACAGGGTTGCACCCCTCCACTTT 1122 CASSSDRVAPLHF
D

TGCAGTGCTAGAGGGACGCCGGCCTCCACAGATACGCAGTATTTT 1104 CSARGTPASTDTQYF

TGTGCCTGGGGGGGACTAGCGGGAGAGGGGACCCAGTACTTC 1079 CAWGGLAGEGTQYF
TE

TGCGCCAGCAGCTTTATTGTAACGGGAGGAGGGAATGAGCAGTTCTTC 1061 CASSFIVTGGGNEQFF

TGCAGCGTTGGTGAAGGGTGGGGTCGCTATGGCTACACCTTC 1003 CSVGEGWGRYGYTF
TGTGCCAGCCGTGGGGGGACAGGGTCAAACACTGAAGCTTTCTTT 1003 CASRGGTGSNTEAFF
EP

TGTGCCAGCAGCTATGGAGCGCCAGTGTCCTACGAGCAGTACTTC 995 CASSYGAPVSYEQYF

TGTGCCAGCAGCTTGACAGGGGGACTAACGCGCTCTGGGGCCAACGTCCTGACTTTC 940 CASSLTGGLTRSGANVLTF
TGTGCCTGGAGTGCAAACGGGAAATACAATGAGCAGTTCTTC 925 CAWSANGKYNEQFF
C

TGTGCCACCAGCAGCCGTCGGGGGGGCTGGGAATCGAACACTGAAGCTTTCTTT 913 CATSSRRGGWESNTEAFF

TGTGCCACCTCCCTGGGACAGGGACGTTACACCTTC 912 CATSLGQGRYTF
AC

TGTGCCAGCAGCGACCAACCGAACACTGAAGCTTTCTTT 888 CASSDQPNTEAFF

TGTGCCAGCAGCTCTCGCCTCGGCGAGCAGTACTTC 866 CASSSRLGEQYF
TGTGCCTGGAGGCCTCGGGGGTTCGGGTACGAGCAGTACTTC 850 CAWRPRGFGYEQYF
TGTGCCAGCAGCGTAGCCCAGACGGGTGAGCAGTTCTTC 823 CASSVAQTGEQFF
TGTGCCAGCAGTTTACACGGGAATGAAAAACTGTTTTTT 809 CASSLHGNEKLFF
TGTGCCAGCAGCTTAGATATCCGATCCTACGAGCAGTACTTC 805 CASSLDIRSYEQYF
TGTGCCAGCATTCCTAGCGGGAGGTCCTACGAGCAGTACTTC 800 CASIPSGRSYEQYF
TGTGCCAGCGGTTCCGCTGGCAATCAGCCCCAGCATTTT 792 CASGSAGNQPQHF
TGCAGTGCTAGTGTCCTAGCGCACATATCTGTGCAGTTCTTC 785 CSASVLAHISVQFF
TGTGCCAGCAGCTTCGCATATGGCTACACCTTC 772 CASSFAYGYTF
TGTGCCAGCAGCTTATCCGGGGCGGGATCTTTCGACACGCAGTATTTT 740 CASSLSGAGSFDTQYF
TGTGCCAGCAGCCCTCCGGGGGCGAGGCGAAACGAGCAGTACTTC 726 CASSPPGARRNEQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGCGCCAGCAGCCGGGACAGAACTAATGGCTACACCTTC 12343 CASSRDRTNGYTF
TGTGCCAGCAGCTTAGTAAGCGAGCAGTACTTC 11145 CASSLVSEQYF
TGTGCCAGCAGCTTCTCCCGGACTAGCGGGAGTAAACGAGATACGCAGTATTTT 5901 CASSFSRTSGSKRDTQYF
TGTGCCTGGAACCCGGACGGAGATGGCTACACCTTC 5516 CAWNPDGDGYTF
TGTGCCTGGAGTGGTGGGATCTCTGGAAACACCATATATTTT 4140 CAWSGGISGNTIYF
TGTGCCAGCAGGCAGACCGGGACAGGGACCGGGGAGCTGTTTTTT 4103 CASRQTGTGTGELFF

PT
TGTGCCAGCAGCCGGCCGGGACTGGCGCAGTATTTT 3850 CASSRPGLAQYF
TGTGCCAGCAGCTTATTTCCGCGGGGGACCCAGTACTTC 3410 CASSLFPRGTQYF
TGTGCCAGCAGGACGGGACAGGGGGGCACAGATACGCAGTATTTT 3123 CASRTGQGGTDTQYF

RI
TGTGCCAGCAGCTTTGGGGAGGGGTACAATGAGCAGTTCTTC 2965 CASSFGEGYNEQFF
TGTGCCAGCAGCTTAGAGATCTTACTAGGGGGTATCGAGACCCAGTACTTC 2892 CASSLEILLGGIETQYF
TGTGCCAGCAGCTTCAGCGGGAGCAGCCCAGATACGCAGTATTTT 2087 CASSFSGSSPDTQYF

SC
TGTGCCTGGAGTGAAGGTAGCGGGAGCGTTGATACGCAGTATTTT 2072 CAWSEGSGSVDTQYF
TGTGCCAGCAGCTTAGCAAGGGGTCGGGGAGCTTTCTTT 2068 CASSLARGRGAFF
TGTGCCAGCAGCTTAGCAACCTTCACCGGGGAGCTGTTTTTT 2013 CASSLATFTGELFF

U
TGTGCCAGCTCGACTAGCGGGAGTTACGAGCAGTACTTC 1838 CASSTSGSYEQYF
TGTGCCAGCAGCTTAGGTGTGGCTCGCAATCAGCCCCAGCATTTT 1814 CASSLGVARNQPQHF
AN
TGTGCCAGCAGCCAAGAGCTCAGCTTCGATACGCAGTATTTT
TGTGCCAGCAGCTTAGGCCCCAATCAGCCCCAGCATTTT
1749
1638
CASSQELSFDTQYF
CASSLGPNQPQHF
TGTGCCAGCAGCTTAGGCGGACAGGGATTTAAGCCCCAGCATTTT 1553 CASSLGGQGFKPQHF
TGTGCCAGCAGCTTAGGAAGCACAGATACGCAGTATTTT 1495 CASSLGSTDTQYF
M
TGTGCCAGCAGCCAAGACCGCCGAGCGGGGTTAGACGAGCAGTACTTC 1471 CASSQDRRAGLDEQYF
TGCGCCAGCAGCCAAGGACAGGGGCCGGATTCACCCCTCCACTTT 1235 CASSQGQGPDSPLHF
TGTGCCAGCACCTCCCCCCGGACAGGGGGTAACGAGCAGTACTTC 1210 CASTSPRTGGNEQYF
D

TGTGCCAGCAGCTTGAACGGGGGAAAAGGGAATCAGCCCCAGCATTTT 1185 CASSLNGGKGNQPQHF

TGTGCCAACCTTGACTAATGGTTTCCAGATACGCAGTATTTT 1161 CANLD*WFPDTQYF
TE

TGCAGTGCTAGTTTGGGGTTTTCCAGCTATGGCTACACCTTC 1068 CSASLGFSSYGYTF

TGCAGTGCTAGGGCAGGGATCGGGAGCAATCAGCCCCAGCATTTT 1054 CSARAGIGSNQPQHF
TGTGCCAGCAGCTTAACGGGACAGGGGCCCCAGTTCTTC 1052 CASSLTGQGPQFF
EP

TGCGCCAGCAGCCACCTACTAGCGGGCTACGAGCAGTACTTC 992 CASSHLLAGYEQYF

TGTGCCAGCAGCGCCGGACAGGGCCCCTACGAGCAGTACTTC 958 CASSAGQGPYEQYF
TGCAGCGTTGGTGGCAAGGAGGGACGGTGGACTGAAGCTTTCTTT 937 CSVGGKEGRWTEAFF
C

TGTGCCAGCAGCGATCGAGGGGGCGGGTTCTTC 867 CASSDRGGGFF

TGTGCCAGCAGCTCCTCTACCGGGGTACAGGTAGCTTTCTTT 845 CASSSSTGVQVAFF
AC

TGTGCCAGCAGCCTACGGACAGGGATCCTCCCGGAGACCCAGTACTTC 838 CASSLRTGILPETQYF

TGCGCCAGCAGCCATAGTCCTAGCGAGAGGGAGACCCAGTACTTC 835 CASSHSPSERETQYF
TGTGCCAGCAGTTACGGACAGGGAGGAACAGATACGCAGTATTTT 811 CASSYGQGGTDTQYF
TGCAGTGCTTTGGACAGGGGGCTTCGAGAGACCCAGTACTTC 700 CSALDRGLRETQYF
TGTGCCAGCAGTTACAGACAGGGAGACTACGAGCAGTACTTC 700 CASSYRQGDYEQYF
TGTGCCAGAGGGACGGCTCCCACAGATACGCAGTATTTT 681 CARGTAPTDTQYF
TGTGCCAGCAGTTCCCAGGACCGAGCGGGGGTTCGGGGTGAGCAGTTCTTC 653 CASSSQDRAGVRGEQFF
TGTGCCAGCAGCTTAAATCCTAGCGGGACATACACAGATACGCAGTATTTT 624 CASSLNPSGTYTDTQYF
TGCACCAGCAGCCGGGACAGAACTAATGGCTACACCTTC 604 CTSSRDRTNGYTF
TGCAGTGCTAGAGGGACAGTGTTTAACACTGAAGCTTTCTTT 601 CSARGTVFNTEAFF
TGTGCCTGGAGTGGTGAGAGGAGTGCTGAAGCTTTCTTT 571 CAWSGERSAEAFF
TGTGCCAGCAGTCCAGTCTTGGGGTGGCCCAATGAGCAGTTCTTC 569 CASSPVLGWPNEQFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCTAGCAGCTTAGATGGGGGCCGGAACAATGAGCAGTTCTTC 61638 CASSLDGGRNNEQFF
TGTGCCAGCAGCTCCCCCCCGACAGGGGTCTACGAGCAGTACTTC 25157 CASSSPPTGVYEQYF
TGTGCCTGGAGGATCGGTTCTGGGGCCAACGTCCTGACTTTC 13979 CAWRIGSGANVLTF
TGTGCCAGCAGCCAAGAGACTAGCGGGAGAAGAGATACGCAGTATTTT 12753 CASSQETSGRRDTQYF
TGTGCCAGCAGCCTTCGACAGGGGCGGAATGAAAAACTGTTTTTT 9797 CASSLRQGRNEKLFF
TGTGCCAGCAGCCCTAGCAACTCCTACGAGCAGTACTTC 8664 CASSPSNSYEQYF

PT
TGTGCCTGGAGTACAGGGAGTCTCAACGAGCAGTACTTC 5454 CAWSTGSLNEQYF
TGCGCCAGCAGCTTGACGGAGGGGATGAACACTGAAGCTTTCTTT 4830 CASSLTEGMNTEAFF
TGCAGTGCCCTAGGCAATGAGCAGTTCTTC 4359 CSALGNEQFF

RI
TGTGCCTGGAGTTTTTCGACGGCGGGAGGCTACACCTTC 3789 CAWSFSTAGGYTF
TGTGCCTGGAGGTCGGGACGAATAATCACACCCCTCCACTTT 3344 CAWRSGRIITPLHF
TGTGCCAGCAGCTTAGGGGGAAGATACGAGCAGTACTTC 3327 CASSLGGRYEQYF

SC
TGTGCCTGGAGTGAAGGGACCCACTACGAGCAGTACTTC 3103 CAWSEGTHYEQYF
TGTGCCAGCAGCTTAGCCTACAGGATAAACTCTGGGGCCAACGTCCTGACTTTC 2728 CASSLAYRINSGANVLTF
TGTGCCTGGAGTTCCCAGTGGACCGGGCAGTTCTTC 2710 CAWSSQWTGQFF

U
TGTGCCAGCAGCCTTGACCGCTCGAGGGAGCAGTACTTC 2620 CASSLDRSREQYF
TGTGCCTGGAGTTCCGAAACGACAGGGAGGGGTGAAAAACTGTTTTTT 2592 CAWSSETTGRGEKLFF
AN
TGTGCCAGCAGCACAACAGCACCCGGCGAGCAGTACTTC
TGTGCCTGGAGGAGACTAGCCGCCACCGTTACGCAGTATTTT
2301
2159
CASSTTAPGEQYF
CAWRRLAATVTQYF
TGTGCCAGCAGCTTGGGACAGGGAGGAGGCTACACCTTC 2097 CASSLGQGGGYTF
TGCGCCAGCAGCCAAAGTTACTCCGAGAAAGCGTACTTC 1915 CASSQSYSEKAYF
M
TGTGCCAGCAGTTTAGCGGGGGGCTATGGCTACACCTTC 1816 CASSLAGGYGYTF
TGTGCCTGGAGTGTAGGGCTGATCTCCTACGAGCAGTACTTC 1414 CAWSVGLISYEQYF
TGTGCCTGCCGACAGGGTTGGGCAGATACGCAGTATTTT 1383 CACRQGWADTQYF
D

TGTGCCAGCAGCTTAGTGGCGTCTACCTACGAGCAGTACTTC 1321 CASSLVASTYEQYF

TGTGCCTGGAGTGGCCCCACAGGGAGCGGTGGCTACACCTTC 1030 CAWSGPTGSGGYTF
TE

TGTGCCAGCAGCCTAGACTCAGGGAAGCAGTATTTT 956 CASSLDSGKQYF

TGTGCCAGCAGTTCGGGGGGCTCCTACAATGAGCAGTTCTTC 855 CASSSGGSYNEQFF
TGTGCCAGCAGCTTTTTGCCACAAGAGACCCAGTACTTC 841 CASSFLPQETQYF
EP

TGCGCCAGCAGCCAAGCCCCGGGGAATGAGCAGTTCTTC 830 CASSQAPGNEQFF

TGTGCCAGCAGCTTAGGAGGGAGCTCAGATACGCAGTATTTT 763 CASSLGGSSDTQYF
TGTGCCAGCAGTTATACAGGCTATAATTCACCCCTCCACTTT 762 CASSYTGYNSPLHF
C

TGTGCCAGCAGTTACGGCGGTTCTAACTATGGCTACACCTTC 727 CASSYGGSNYGYTF

TGTGCCTGCTCGACTTTTAACACCGGGGAGCTGTTTTTT 702 CACSTFNTGELFF
AC

TGTGCCAGCAGCTTATATGACAGGGGAGGCGAGCAGTTCTTC 676 CASSLYDRGGEQFF

TGTGCCAGCAGAACCTCCGCCCACGGGGACGAGCAGTACTTC 622 CASRTSAHGDEQYF
TGTGCCAGCAGCTTGCCCACGGATCCTGAAAAACTGTTTTTT 528 CASSLPTDPEKLFF
TGTGCCAGCAGTGACCCCCAAGAGCCCCAGTTCTTC 511 CASSDPQEPQFF
TGTGCCAGCAGTACTAGCGGCTTTTACGAGCAGTACTTC 506 CASSTSGFYEQYF
TGTGCCTGGAGTCCTTTAGCGGCCAACGTCCTGACTTTC 455 CAWSPLAANVLTF
TGTGCCAGCAGCCAAGATGAGGGCGGCGGGAGTAGACAAGATACGCAGTATTTT 449 CASSQDEGGGSRQDTQYF
TGTGCCAGCGGCCGCGGGGGGAGCTCCTACAATGAGCAGTTCTTC 406 CASGRGGSSYNEQFF
TGTGCCAGCAGCTTAGTCGGTGGCACCGGGGAGCTGTTTTTT 385 CASSLVGGTGELFF
TGTGCCAGCAGCTCCCCTCGACGTTACAATGAGCAGTTCTTC 368 CASSSPRRYNEQFF
TGCGCCAGCAGCTTGCGGACTAGTGGCAATACGCAGTATTTT 365 CASSLRTSGNTQYF
TGTGCCAGCAGCTTAACGGTTACCTACGAGCAGTACTTC 348 CASSLTVTYEQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAGAAGGCTCAGGGGGAACACTGAAGCTTTCTTT 2797 CAWRRLRGNTEAFF
TGTGCCTGGAGCGTCCGGCGAGCGGCGGATGAGTTCTTC 2135 CAWSVRRAADEFF
TGTGCCTGGAGCGCCGGGCCCGCTGGAAAACTGTTTTTT 1934 CAWSAGPAGKLFF
TGTGCCTGGAGTGTACTGGCGGGGGGTCAGCCCCAGCATTTT 1839 CAWSVLAGGQPQHF
TGTGCCAGCAGCTTAGCATCGGGACAGATGAACACTGAAGCTTTCTTT 1800 CASSLASGQMNTEAFF
TGTGCCTGGAGTGTTGGGTTAGGCCCCTATAGCAATCAGCCCCAGCATTTT 1623 CAWSVGLGPYSNQPQHF

PT
TGTGCCCTTATAAGGGAGGGCCGTGAGACCCAGTACTTC 1526 CALIREGRETQYF
TGTGCCTGGAGTGGCCGGACAGTCTATGGCTACACCTTC 1503 CAWSGRTVYGYTF
TGTGCCTGGCAAACTGGGGGCAGGTTTCAGCCCCAGCATTTT 1257 CAWQTGGRFQPQHF

RI
TGTGCCTGGACGAGCGGCAACTCTGGAAACACCATATATTTT 1008 CAWTSGNSGNTIYF
TGTGCCTGGAGAAACGCCGGGACTAGCGGGGACAATGAGCAGTTCTTC 992 CAWRNAGTSGDNEQFF
TGTGCCTGGAGTGTAAGAAGCACAGATACGCAGTATTTT 988 CAWSVRSTDTQYF

SC
TGTGCCTTACGTCGGGGAGGCGGCGGCTACACCTTC 975 CALRRGGGGYTF
TGTGCCTGGAGTCCGCGGAGGGCCGATACGCAGTATTTT 940 CAWSPRRADTQYF
TGTGCCTGGAGTTACGGTTACGAGCAGTACTTC 933 CAWSYGYEQYF

U
TGTGCCTGGAGTAAAGATCGCTCCTACGAGCAGTACTTC 906 CAWSKDRSYEQYF
TGTGCCTGGAGTGAGGGGCTTACCTACGAGCAGTACTTC 877 CAWSEGLTYEQYF
TGTGCCTGGAGTGCGGGGTATGGCTACACCTTC AN
TGTGCCAGCAGCCCTAAGGGGCGGGAGGGCCAGCGCCTACAATGAGCAGTTCTTC
862
855
CAWSAGYGYTF
CASSPKGRE_ASAYNEQFF
TGTGCCTGGAGTGTTCGGACAGGGACAGGTCAGCCCCAGCATTTT 855 CAWSVRTGTGQPQHF
TGTGCCTGGAGTCGGAACAGGGGGACCTCAGGCTACACCTTC 832 CAWSRNRGTSGYTF
M
TGTGCCTGGAGGGGGGACAGTGGGGCCTACGAGCAGTACTTC 824 CAWRGDSGAYEQYF
TGTGCCAGCAGCCCCCGGAAATGGTGACGTTCAAGGTGGCCACCCCTCCACTTT 823 CASSPRKW*RSRWPPLHF
TGTGCCAGCAGCCCCTCCTGGACCCCAGAAAAAACACTGAAGCTTTCTTT 794 CASSPSWT_RKNTEAFF
D

TGTGCCTGGACCGGGACAGGGGTAGGCTACACCTTC 776 CAWTGTGVGYTF

TGTGCCTGGAGTTGGCAGCTGGGCGGCTACACCTTC 765 CAWSWQLGGYTF
TE

TGTGCCTGCACACGAGCGGAATCCTATAATTCACCCCTCCACTTT 751 CACTRAESYNSPLHF

TGTGCCTGGAGTGAAGCGATTAACTATGGCTACACCTTC 740 CAWSEAINYGYTF
TGTGCCTGGAGTTCGGCTATGGATACGCAGTATTTT 735 CAWSSAMDTQYF
EP

TGTGCCTGGAGGGAACTAGCGGGAAGAAGCTATAATGAGCAGTTCTTC 723 CAWRELAGRSYNEQFF

TGTGCCGGCGGAGGGGATTGCACTGAAGCTTTCTTT 717 CAGGGDCTEAFF
TGTGCCTGGAGCAGGGGGACAGCGAAGAATTCACCCCTCCACTTT 705 CAWSRGTAKNSPLHF
C

TGTGCCTGGAATCCCGACTACCGCTATGGCTACACCTTC 677 CAWNPDYRYGYTF

TGCGCCAGCAGCCAACTCGCCAGTGAGACCCAGTACTTC 661 CASSQLASETQYF
AC

TGTGCCTGGAGTGATGGGCGCCCCAATCAGCCCCAGCATTTT 643 CAWSDGRPNQPQHF

TGTGCCAGCCGGGACAGGGTATATGGCTACACCTTC 642 CASRDRVYGYTF
TGTGCCTGGAGTTTGGGAGGGACCTATAATTCACCCCTCCACTTT 638 CAWSLGGTYNSPLHF
TGTGCCTGGAGTAGGTCAGATTCACTGTTGACTGAAGCTTTCTTT 627 CAWSRSDSLLTEAFF
TGTGCCTGGAGTACCGACTGGGTGCAGTACTTC 622 CAWSTDWVQYF
TGTGCCTACGTGCGGACAGGGGTCTACGAGCAGTACTTC 621 CAYVRTGVYEQYF
TGTGCCTGGGAACTACAGAGGAACACTGAAGCTTTCTTT 620 CAWELQRNTEAFF
TGTGCCCGGAAACCAGACAGGGCAGGGTGCAATCAGCCCCAGCATTTT 607 CARKPDRAGCNQPQHF
TGTGCCTGGAGTGTCCCAGGGATTGGGAACTATGGCTACACCTTC 600 CAWSVPGIGNYGYTF
TGTGCCGGAGGGGGCAGACTCCAAGAGACCCAGTACTTC 592 CAGGGRLQETQYF
TGTGCCTGGTCGGGTGAGGCCAACGTCCTGACTTTC 580 CAWSGEANVLTF
TGTGCCAGAAGGCCTCCAGCCGCAGGGGAAGGTCAGCCCCAGCATTTT 570 CARRPPAAGEGQPQHF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGTTATTATCAGGGGGCGACTGAAGCTTTCTTT 7459 CASYYQGATEAFF
TGTGCCAGCAGTTTGAGCGGGGGAGCCGGGGAGCTGTTTTTT 1003 CASSLSGGAGELFF
TGTGCCTTAATTCCGGGACAGGGGGATGAAAAACTGTTTTTT 797 CALIPGQGDEKLFF
TGTGCCTGGAGTGTGTATGCGGCAGAGACCCAGTACTTC 656 CAWSVYAAETQYF
TGTGCCAGCAGCAACGGCTGGGAGCAGTTCTTC 635 CASSNGWEQFF
TGTGCCAGCAGCCCAAGAAACAATGAGCAGTTCTTC 531 CASSPRNNEQFF

PT
TGTGCCAGCAGCAAACTAGCGGGAGGGGTAGGCAATGAGCAGTTCTTC 508 CASSKLAGGVGNEQFF
TGTGCCAGCAGCTTAGGACAGGGCTCTGGGGCCAACGTCCTGACTTTC 495 CASSLGQGSGANVLTF
TGTGCCTGGAGTGTACGGCTCAGGAATGAGCAGTTCTTC 456 CAWSVRLRNEQFF

RI
TGTGCCAGCAGCTTCGGACAGTCAGGTGGCTACACCTTC 424 CASSFGQSGGYTF
TGTGCCTGGATCGGACAGAACTATGGCTACACCTTC 405 CAWIGQNYGYTF
TGTGCCACCAGCAGCCGGGACACGGAACGGGGCTACACCTTC 401 CATSSRDTERGYTF

SC
TGTGCCAGCCACTTCAGGAACACTGAAGCTTTCTTT 400 CASHFRNTEAFF
TGTGCCAGCAGCTTAGGGCTGAGCAATCAGCCCCAGCATTTT 386 CASSLGLSNQPQHF
TGTGCCTGGAGTTGGGGTAGTAAGGATCAGCCCCAGCATTTT 385 CAWSWGSKDQPQHF
TGTGCCAGCAGCTTACCCTCGGTGGGTAACTATGGCTACACCTTC 382 CASSLPSVGNYGYTF

U
TGTGCCAGCAGCGACGGGGGCCACAGGGGGCGTCAGCCCCAGCATTTT 379 CASSDGGHRGRQPQHF
TGTGCCAGCAGCCAAGATCCCAATCAGCCCCAGCATTTT 376 CASSQDPNQPQHF
AN
TGTGCCAGCAGCCCGCGGACAGGGAATCAGCCCCAGCATTTT
TGTGCCAGCAGCTCGACCGGGACTAGCGGGAGGAGGGACCAGTACTTC
372
363
CASSPRTGNQPQHF
CASSSTGTSGRRDQYF
TGTGCCAGCAGCTTAAAAAAGACCCAGTACTTC 356 CASSLKKTQYF
M
TGTGCCAGCAGCTCTCAGCAGGGGTACAATGAGCAGTTCTTC 355 CASSSQQGYNEQFF
TGCGCCAGCAGCCAAGATCGGTCTAGCGGGAGGGTCGATGAGCAGTTCTTC 352 CASSQDRSSGRVDEQFF
TGTGCCAGCGCCTCGGATAGGGTAGGCACTGAAGCTTTCTTT 351 CASASDRVGTEAFF
D

TGTGCCTGGAGTTACGCGCGGGGGATCATGAACACTGAAGCTTTCTTT 346 CAWSYARGIMNTEAFF

TGTGCCAGCAGCTTAGGGCTGGTGGCTGAAGCTTTCTTT 329 CASSLGLVAEAFF
TE

TGTGCCTGGAGACTACAGGGACCCGGGGAGCTGTTTTTT 326 CAWRLQGPGELFF

TGTGCCAGCAGCTGGCGGGAGGACGCATCCAATCAGCCCCAGCATTTT 321 CASSWREDASNQPQHF
TGTGCCAGCAGCTTGGGACAGGGGTCGACCGGGGAGCTGTTTTTT 309 CASSLGQGSTGELFF
TGTGCCTGGACACAAGGCTCCTACTATGGCTACACCTTC 299 CAWTQGSYYGYTF
EP

TGTGCCAGCAGCGCAGACAGTGCCGTTAGCTATGGCTACACCTTC 297 CASSADSAVSYGYTF

TGTGCCAGCAGGCTACAGGGGGCCACTGAAGCTTTCTTT 293 CASRLQGATEAFF
TGTGCCAGCAGCTTGGGGACAGGGTATTCACCCCTCCACTTT 290 CASSLGTGYSPLHF
C

TGTGCCAGCAGCTCTTGGAGCTCGAGGGGCAGGGACGAGCAGTACTTC 289 CASSSWSSRGRDEQYF

TGTGCTGGGGGAGTAGCGACGGGTGAGCAGTTCTTC 289 CAGGVATGEQFF
AC

TGTGCCTGGACCGACGGAGGGACTGAAGCTTTCTTT 284 CAWTDGGTEAFF

TGTGCCAGCAGCTTAGGTCTCAACACTGAAGCTTTCTTT 283 CASSLGLNTEAFF
TGTGCCTGGAGTGTCCGGGAAAGAGATACGCAGTATTTT 278 CAWSVRERDTQYF
TGTGCTAGCAGTCTAACAACTAGCGGGCCCAACACCGGGGAGCTGTTTTTT 277 CASSLTTSGPNTGELFF
TGTGCCAGCAGCTTAGACGGCTCTGGGGCCAACGTCCTGACTTTC 275 CASSLDGSGANVLTF
TGTGCCAGCAGCCAATCTTCCCCCTACGAGCAGTACTTC 272 CASSQSSPYEQYF
TGTGCCAGCAGCTTAGGGGCAGGGAGGCATTCACCCCTCCACTTT 266 CASSLGAGRHSPLHF
TGTGCCAGCAGCTTGACAGGGGGACTAACGCGCTCTGGGGCCAACGTCCTGACTTTC 264 CASSLTGGLTRSGANVLTF
TGTGCCACCAGCAGAGATTACGGTTCGGACCAGGTGGGCGAGCAGTACTTC 262 CATSRDYGSDQVGEQYF
TGTGCTAGCAGCTTTCAGGGGCAAGAGACCCAGTACTTC 257 CASSFQGQETQYF
TGTGCCAGCAGCTTAACGGCCGAGGGGGAAGCTTTCTTT 257 CASSLTAEGEAFF
TGTGCCAGCAGCTTAGGGGGGGCAGGAGAAGGCTACACCTTC 256 CASSLGGAGEGYTF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCCCCCTAGCGGGAGTAGGTTACAATGAGCAGTTCTTC 23724 CASSPLAGVGYNEQFF
TGTGCCAGCAGCTTGCGGCGGCAGCGAGGAGATACGCAGTATTTT 19583 CASSLRRQRGDTQYF
TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 12924 CSVLRDPNSYEQYF
TGTGCCTGGAGCGCCGGGCCCTACGAGCAGTACTTC 10982 CAWSAGPYEQYF
TGTGCCAGCAGCACGTTTGGGGGAGGCTACACCTTC 7151 CASSTFGGGYTF
TGTGCCAGCAGGCGAAGCGGGCGGGGATACACCGGGGAGCTGTTTTTT 5211 CASRRSGRGYTGELFF

PT
TGTGCCAGCAGCTTAGGCAGATTCAATCAGCCCCAGCATTTT 4553 CASSLGRFNQPQHF
TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 3477 CSVHRDPNSHEQFF
TGTGCCAGCAGCCAGGGTGGGGGAGTGCTTGATACGCAGTATTTT 3476 CASSQGGGVLDTQYF

RI
TGTGCCAGCAGCTTAACGGGGGAGCCCGCCTACGAGCAGTACTTC 3181 CASSLTGEPAYEQYF
TGTGCCAGCAGCTTAAGGGACGCCGGGTATGGCTACACCTTC 3074 CASSLRDAGYGYTF
TGCGCCAGCAGCCTAATTAGCGGGAGGGCCGGAAGAACCGGGGAGCTGTTTTTT 2845 CASSLISGRAGRTGELFF

SC
TGTGCCAGCAGCTTACAAAGCCCCTCAATGAACACTGAAGCTTTCTTT 2719 CASSLQSPSMNTEAFF
TGTGCCAGCAGTTACTCGCGACAGGGGGCTTTTGAAGCTTTCTTT 2662 CASSYSRQGAFEAFF
TGTGCCTGGAGTCTCGGAGACTATCAGCCCCAGCATTTT 2474 CAWSLGDYQPQHF

U
TGTGCCAGCAGCCTTAGGGGGGGACGGCAGAACACTGAAGCTTTCTTT 2228 CASSLRGGRQNTEAFF
TGTGCCAGCAGCCTTCTATTGGCCCAGTACTTC 1895 CASSLLLAQYF
AN
TGTGCCAGCAGCCGACCCCGAGGTGACTCCTCCTACGAGCAGTACTTC
TGTGCCCTGGAGGGGGGGGCAGTAGGGACCCAGTACTTC
1605
1604
CASSRPRGDSSYEQYF
CALEGGAVGTQYF
TGCGCCAGCAGGAGGACAGGGGAAGGCAATCAGCCCCAGCATTTT 1587 CASRRTGEGNQPQHF
TGCGCCAGCAGCCTGACGTCCGGGACCTCCTACGAGCAGTACTTC 1412 CASSLTSGTSYEQYF
M
TGTGCCCGCCAGGGGGAAGGCCCAGATACGCAGTATTTT 1307 CARQGEGPDTQYF
TGCGCCAGCAGCCAAGATTGGGCAGGGAGAGGTCATGGCTACACCTTC 1305 CASSQDWAGRGHGYTF
TGCGCCAGCAGCTCGACAGGGACGAACACTGAAGCTTTCTTT 1269 CASSSTGTNTEAFF
D

TGTGCCAGCAGCTTAGCACAGGGTCCCCACGAGCAGTACTTC 1260 CASSLAQGPHEQYF

TGCGCCAGCAGCTTGACAGGCACTGGAGGGGCCAACGTCCTGACTTTC 1257 CASSLTGTGGANVLTF
TE

TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 1178 CASSFTGAMNTEAFF

TGTGCCAGCAGCTTAGAGGCGGGGGGTCCCCAGTACTTC 1163 CASSLEAGGPQYF
TGTGCCAGCAGCTTAATCGGGTACGAGCAGTACTTC 1127 CASSLIGYEQYF
EP

TGCAGTGCCGCCCCCGGGTCGGGAGGAGGGGAGACCCAGTACTTC 1074 CSAAPGSGGGETQYF

TGTGCCAGCAGCACCCACCTGTGGGAGACCCAGTACTTC 1026 CASSTHLWETQYF
TGTGCCAGCAGCTTAGCTGTCGGACGACAGGGGAATGAGCAGTTCTTC 1026 CASSLAVGRQGNEQFF
C

TGTGCCACCACACCGGGACAGGGGGCGCGCGGCTACACCTTC 1007 CATTPGQGARGYTF

TGTGCCAGCAGCTTAGGGCTGAGGAGCGGGAGGGACAATGAGCAGTTCTTC 1004 CASSLGLRSGRDNEQFF
AC

TGTGCCAGCAGCCAACGTCCTCAGGGAGCTAATGAAAAACTGTTTTTT 977 CASSQRPQGANEKLFF

TGTGCCAGCAGCCAAAGATCAGCGGATTACACCGGGGAGCTGTTTTTT 932 CASSQRSADYTGELFF
TGTGCCAGCAGCTTAGTTGCGGGAAAAGAGACCCAGTACTTC 915 CASSLVAGKETQYF
TGTGCCAGCAGCCTAGGGGGGAACACTGAAGCTTTCTTT 883 CASSLGGNTEAFF
TGTGCCTGGAGTCCCGGGTGGAACATTCAGTACTTC 873 CAWSPGWNIQYF
TGCAGTGCTACACCCACCGGGGTCGCTGGTAAACTGTTTTTT 870 CSATPTGVAGKLFF
TGCAGCGTTTCGACAGGGGGCCAGCCCCAGCATTTT 806 CSVSTGGQPQHF
TGCAGTGCTAGAGAAGGGTATAATGAAAAACTGTTTTTT 741 CSAREGYNEKLFF
TGCGCCAGCAGCCAAGATAGAGTGGGCTCCTACAATGAGCAGTTCTTC 735 CASSQDRVGSYNEQFF
TGTGCCAGCAGCCAAACATCTAGCGGTTCTACAGATACGCAGTATTTT 712 CASSQTSSGSTDTQYF
TGTGCCACCAGCAGTGAGCCGTTGGGCGAGCAGTACTTC 697 CATSSEPLGEQYF
TGTGCCAGCACCTTGACCGGGACGGCGAACACTGAAGCTTTCTTT 656 CASTLTGTANTEAFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGTTACTCGCGACAGGGGGCTTTTGAAGCTTTCTTT 33215 CASSYSRQGAFEAFF
TGTGCCAGCAGCTTAAAGGGGCGACTAGCGGGAGGGCGGGAGCAGTTCTTC 9603 CASSLKGRLAGGREQFF
TGCGCCAGCAGGGCCCAGGGGCAGGGGAATGAGCAGTTCTTC 7217 CASRAQGQGNEQFF
TGTGCCAGCAGCCTGGGGACAGGCCAAGAGACCCAGTACTTC 4168 CASSLGTGQETQYF
TGTGCCTCCGAGAGGGGTCAGGGAAACATTCAGTACTTC 4025 CASERGQGNIQYF
TGTGCCAGCAGCTTAGCTTACAATCAGCCCCAGCATTTT 3651 CASSLAYNQPQHF

PT
TGTGCCAGCAGCGTGGGACAGGGGGAAGGCTACACCTTC 3272 CASSVGQGEGYTF
TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 3244 CSVLRDPNSYEQYF
TGTGCCTCTGCGACTAGCGGGTTCCCAGATACGCAGTATTTT 2871 CASATSGFPDTQYF

RI
TGTGCCCGCCAGGGGGAAGGCCCAGATACGCAGTATTTT 2801 CARQGEGPDTQYF
TGCGCCAGCAGCCAAGGTACCGCGAGCTCCTACAATGAGCAGTTCTTC 2693 CASSQGTASSYNEQFF
TGTGCCAGCAGCTTAGTCGGAGTTCAGTTTAATGAAAAACTGTTTTTT 2506 CASSLVGVQFNEKLFF

SC
TGTGCCAGCAGCTTCCGAATAACCGGGGAGCTGTTTTTT 2426 CASSFRITGELFF
TGTGCCTGGAGTGTACTCGACGGGGGGAGAAATGGCTACACCTTC 2302 CAWSVLDGGRNGYTF
TGTGCCAGCAGCTTATTTGGGACAGGGGACAATCAGCCCCAGCATTTT 2011 CASSLFGTGDNQPQHF
TGTGCCTGGAGTGTACGCCAGGGGGACGAGCAGTACTTC 2004 CAWSVRQGDEQYF

U
TGTGCCAGCAGCTTTTCGGGACAGGGACCCTATGGCTACACCTTC 1815 CASSFSGQGPYGYTF
TGTGCCTGGAGTGTACGCGTTAGAGAGACCCAGTACTTC 1772 CAWSVRVRETQYF
TGTGCCTGGAGTCGGCAGGGAGATGGCTACACCTTC AN
TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC
1723
1637
CAWSRQGDGYTF
CSVHRDPNSHEQFF
TGTGCCAGCAGCTTAGTGGGGGACAGGGCTTACGAGCAGTACTTC 1600 CASSLVGDRAYEQYF
M
TGTGCCAGCAGCTTCACCGCCACGGGGTCCCAGTACTTC 1490 CASSFTATGSQYF
TGTGCCAGCAGCTACCCGGGACTTTACACCGGGGAGCTGTTTTTT 1462 CASSYPGLYTGELFF
TGTGCCTGGAGCGCCGGGCCCTACGAGCAGTACTTC 1453 CAWSAGPYEQYF
D

TGTGCCAGCAGCCTGTGGTCCCACCAAGAGACCCAGTACTTC 1442 CASSLWSHQETQYF

TGTGCCAGCAGCTTAACTGGACAGGGACATAGTGGCTACACCTTC 1346 CASSLTGQGHSGYTF
TGTGCCAGCTCACCGGGACTAGCGGGAGGTGGAGAGACCCAGTACTTC 1344 CASSPGLAGGGETQYF
TE

TGTGCCGTCGGCGGTTTAGCCTCCTACAATGAGCAGTTCTTC 1326 CAVGGLASYNEQFF

TGTGCCAGCAGCTTAGAGGCGGGACGCACAGATACGCAGTATTTT 1247 CASSLEAGRTDTQYF
TGCGCCAGCAGCCGGGACCGACAGGGAGAGACGGATGAGCAGTTCTTC 1240 CASSRDRQGETDEQFF
EP

TGTGCCAGCAGCTTAGTTGCGGGAAAAGAGACCCAGTACTTC 1192 CASSLVAGKETQYF

TGTGCCTGGAGTGCCCAAATTTCGGGTCAGCCCCAGCATTTT 1171 CAWSAQISGQPQHF
TGTGCCAGCAGCCCCCAGACAGGGACGAGCAATCAGCCCCAGCATTTT 1059 CASSPQTGTSNQPQHF
TGTGCCACCAGCAGAGCCCCGGACGGACCCTACGAGCAGTACTTC 1037 CATSRAPDGPYEQYF
C

TGCAGCGTAGCCGGGACAGGGGCCGAGACCCAGTACTTC 990 CSVAGTGAETQYF

TGCGCCAGCAGCCAAGGGGGGTCCACTGCTTTCTTT 979 CASSQGGSTAFF
AC

TGTGCCAGCAGCCCATACAGGATTCGGACGAACACCGGGGAGCTGTTTTTT 890 CASSPYRIRTNTGELFF

TGTGCCTGGAGTGTAGCGGGAGAGACCCAGTACTTC 853 CAWSVAGETQYF
TGTGCCAGCAGCCGCCAGACAGGGCGGAACACTGAAGCTTTCTTT 799 CASSRQTGRNTEAFF
TGTGCCAGCAGCTTAGTCCGATCTGGAAACACCATATATTTT 766 CASSLVRSGNTIYF
TGCAGCTCAGCTCGGGACACAGATACGCAGTATTTT 759 CSSARDTDTQYF
TGCAGCCCCCTGGCTAAAGAGACCCAGTACTTC 755 CSPLAKETQYF
TGTGCCAGCAGCTTAACCCCGTTAGGGGGAGGCACAGATACGCAGTATTTT 730 CASSLTPLGGGTDTQYF
TGTGCCAGCAGCCGACCCCGAGGTGACTCCTCCTACGAGCAGTACTTC 713 CASSRPRGDSSYEQYF
TGTGCCAGCAGCCCCGGGGGAGCGGGATTTCGGGAGACCCAGTACTTC 707 CASSPGGAGFRETQYF
TGCAGTGCTAGGGACAACATGAACACTGAAGCTTTCTTT 707 CSARDNMNTEAFF
TGTGCCAGCAGCTTAGTACCTAGCGGGAACTCCTACGAGCAGTACTTC 705 CASSLVPSGNSYEQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGGGGGGACTAGCGGGAGAGGGGACCCAGTACTTC 22581 CAWGGLAGEGTQYF
TGCAGCGCTAGCGGGAGGCGCTACAATGAGCAGTTCTTC 18305 CSASGRRYNEQFF
TGTGCCAGCAGCGCCGGACAGGGCCCCTACGAGCAGTACTTC 14208 CASSAGQGPYEQYF
TGTGCCAGCAGCTCTCGCCTCGGCGAGCAGTACTTC 12246 CASSSRLGEQYF
TGCGCCAGCACCTTAACGGCCGCGGGAGGAGGCACAGATACGCAGTATTTT 9508 CASTLTAAGGGTDTQYF
TGTGCCAGCAGCCCCTTGACAGGGGGGATGACTGAAGCTTTCTTT 6217 CASSPLTGGMTEAFF

PT
TGCAGTGCCCCGACTAGCAGCACAGATACGCAGTATTTT 5813 CSAPTSSTDTQYF
TGCGCCAGCAGCCAAGATCTCAACCCAACTAATGAAAAACTGTTTTTT 5129 CASSQDLNPTNEKLFF
TGCAGTGCTAGTTTGGATAATCTAAAATATGGCTACACCTTC 4446 CSASLDNLKYGYTF

RI
TGTGCCAGCAGACCTTCGTCCGCAGGGGGATCTGGAAACACCATATATTTT 4055 CASRPSSAGGSGNTIYF
TGTGCCTGGAACGCAGGGGGCATAAACACTGAAGCTTTCTTT 3904 CAWNAGGINTEAFF
TGCGCCAGCAGCTCTCCGTCTAGCGGGATAACGGATACGCAGTATTTT 3171 CASSSPSSGITDTQYF

SC
TGTGCCAGCAGCTCCTATAGCGGGAAGAACACCGGGGAGCTGTTTTTT 2912 CASSSYSGKNTGELFF
TGTGCCAGCAGCTTAGACCCCGGGACAGGAGGCTATGGCTACACCTTC 2741 CASSLDPGTGGYGYTF
TGTGCCAGCACTTGGGGAGGAAACACAGATACGCAGTATTTT 2710 CASTWGGNTDTQYF

U
TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 2503 CSVHRDPNSHEQFF
TGTGCCAGCAGTGGCGGACAGGTATCGCGCCACGAGCAGTACTTC 2341 CASSGGQVSRHEQYF
AN
TGCAGTGCTAGAGTTCCCCCGAACACTGAAGCTTTCTTT
CGTGCCAGCAGCTTAGGACTAGCGGACAATGAGCAGTTCTTC
2006
1791
CSARVPPNTEAFF
RASSLGLADNEQFF
TGTGCCAGCAGCCTTGGGACAGGGGCGTTCTATGGCTACACCTTC 1658 CASSLGTGAFYGYTF
TGCAGTGCTACCACCCACAGTAGACAGGGTTACACTGAAGCTTTCTTT 1554 CSATTHSRQGYTEAFF
M
TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 1512 CSVLRDPNSYEQYF
TGTGCCAGCAGCTTATCGGGGGAGACCCAGTACTTC 1430 CASSLSGETQYF
TGTGCCAGCAGCTCCCGGACAGGGCTTTTACAGACCCAGTACTTC 1258 CASSSRTGLLQTQYF
D

TGTGCCTGGAGTGTAACGTCGTCCCCTCCTTTTAACTATGGCTACACCTTC 1247 CAWSVTSSPPFNYGYTF

TGCGCCAGCAGCCAAGATCGGGGAGGCTCCTACAATGAGCAGTTCTTC 1206 CASSQDRGGSYNEQFF
TE

TGTGCCAGCAGCGGGGGGACTAGCGGGAGTTTGGGTGAGCAGTTCTTC 1168 CASSGGTSGSLGEQFF

TGTGCCAGCAGCTCATGGGGGACCTCTGGAAACACCATATATTTT 1092 CASSSWGTSGNTIYF
TGTGCCGGCAGGCCAGGGGGTTACTTGAGCACTGAAGCTTTCTTT 846 CAGRPGGYLSTEAFF
EP

TGTGCCAGCAGCCACCGGAGCGGGACCGGTTACGAGCAGTACTTC 759 CASSHRSGTGYEQYF

TGCAGTGCTAGGGGGGGGTCCACTGAAGCTTTCTTT 725 CSARGGSTEAFF
TGTGCCAGCAGGGGGGCCTACGAGCAGTACTTC 619 CASRGAYEQYF
C

TGTGCCAGCAGCTTAACACAGGGGTCACAGCAGTACTTC 611 CASSLTQGSQQYF

TGTGCCAGCAGCTTAGTGGGGGCACTGAACACTGAAGCTTTCTTT 578 CASSLVGALNTEAFF
AC

TGTGCCAGCAGCTTGACAGTTAACTATGGCTACACCTTC 555 CASSLTVNYGYTF

TGTGCCAGCAGCCCTTTACGGGAGAAAGGCGGGGAGCTGTTTTTT 511 CASSPLREKGGELFF
TGTGCCAGCAGCCCATCGGGACAGGGGTCCTACGAGCAGTACTTC 438 CASSPSGQGSYEQYF
TGTGCCAGCAGTTGGAAGAAAGAGTCAATTGAAGAGACCCAGTACTTC 432 CASSWKKESIEETQYF
TGTGCCAGCAGCGACAGGGAAAGGGCGCCCCAGCATTTT 397 CASSDRERAPQHF
TGTGCCAGCAGCTCGGGACAGGGGAGGCCCCAGCATTTT 394 CASSSGQGRPQHF
TGTGCCAGCAGCTTAGGAGTGGGATTCACAAATGAGCAGTTCTTC 389 CASSLGVGFTNEQFF
TGTGCCAGCAGTCCAGACAGGGAATGGGAGCAGTACTTC 338 CASSPDREWEQYF
TGTGCCTGCCCCTCCGGGTCGCAAGGCTATGGCTACACCTTC 333 CACPSGSQGYGYTF
TGTGCCAGCAGCCCCCGCCTGGGCACCTACGAGCAGTACTTC 327 CASSPRLGTYEQYF
TGCAGCGTTGGAGGTCCTGAACAGCCCCAGCATTTT 309 CSVGGPEQPQHF
TGTGCCTGGAGTGTAGGGACTGCCCTTGGAGAGCAGTACTTC 292 CAWSVGTALGEQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAATCCTGGTGTAGTTACAGATACGCAGTATTTT 28173 CAWNPGVVTDTQYF
TGCAGTGCTAGAAGGGGCGACACTGAAGCTTTCTTT 27297 CSARRGDTEAFF
TGTGCCAGCAGCTTAGTGGGGGCACTGAACACTGAAGCTTTCTTT 17916 CASSLVGALNTEAFF
TGTGCCAGCACTTTATACGGGACAGGGGGCTATGGCTACACCTTC 9190 CASTLYGTGGYGYTF
TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 8937 CSVHRDPNSHEQFF
TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 7799 CASSFTGAMNTEAFF

PT
TGTGCCAGCACCAGGACTAGCGATACGCAGTATTTT 7085 CASTRTSDTQYF
TGTGCCAGCAGCTTAGACCCCGGGACAGGAGGCTATGGCTACACCTTC 6352 CASSLDPGTGGYGYTF
TGTGCCAGCAGTTACAGGGGGCCGGTTAGCGAGACCCAGTACTTC 4778 CASSYRGPVSETQYF

RI
TGTGCCAGCAGCCCTAGTGGAGGCACTGAAGCTTTCTTT 4232 CASSPSGGTEAFF
TGTGCCAGCAGTTCAGGACAGGGGGTTACCGGGGAGCTGTTTTTT 3976 CASSSGQGVTGELFF
TGCAGTGCTAGAGCCCCGTGGGGGGGAGATACGCAGTATTTT 3044 CSARAPWGGDTQYF

SC
TGCGCCAGCACCTTAACGGCCGCGGGAGGAGGCACAGATACGCAGTATTTT 2092 CASTLTAAGGGTDTQYF
TGTGCCTGGAGTGTACGATACGAGCAGTACTTC 1540 CAWSVRYEQYF
TGTGCCAGCAGCTCCCGGGTGGGACAGGGCAGTCAGCCCCAGCATTTT 1278 CASSSRVGQGSQPQHF
TGCAGTGCTACGACAGGGGGCTCGCAGGTTAATGGCTACACCTTC

U
1264 CSATTGGSQVNGYTF
TGTGCCAGCAGCGCTAGCGTAGCGGGGGTATACGAGCAGTACTTC 1106 CASSASVAGVYEQYF
TGTGCCAGCACCGCTCGAGGGAACTATGGCTACACCTTC
AN
TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC
TGTGCCAGCAGCTTAGGAGGGCAGAGACCCAGTACTTC
897 CASTARGNYGYTF
736 CSVLRDPNSYEQYF
718 CASSLG_AETQYF
TGCAGCGTTTTAAAGGACCCAAACTACAATGAGCAGTTCTTC 614 CSVLKDPNYNEQFF
M
TGTGCCTGGAGTGGATCCAATCAGCCCCAGCATTTT 531 CAWSGSNQPQHF
TGTGCCAGCAGTCGGGACAGGGGGGATCAGCCCCAGCATTTT 443 CASSRDRGDQPQHF
TGTGCCAGCAGTGACTGGTCCTACACCTTC 261 CASSDWSYTF
D

TGTGCCTGCCAAGTCGCGGGAGGTTTAGGTCATGATGAGCAGTTCTTC 204 CACQVAGGLGHDEQFF

TGTGCCAGCAGCAATCTCGGGGGCCCCAGAAGCGAGCAGTTCTTC 201 CASSNLGGPRSEQFF
TE

TGTGCCAGCAGCCTATACAGGAACACCGGGGAGCTGTTTTTT 167 CASSLYRNTGELFF

TGTGCCAGCGGGAGCAGGGGAGTTGCAACTAATGAAAAACTGTTTTTT 144 CASGSRGVATNEKLFF
TGCAGTGCTAGAGATGGGGGGACAGGGTTTTCACCCCTCCACTTT 144 CSARDGGTGFSPLHF
TGCAGTGCTAGAGTGGGCGGAGCTTGGGGTGGCTACACCTTC 132 CSARVGGAWGGYTF
EP

TGTGCCAGCAGCCCCAGAACCGGGACTAGCGGGAGAAGAATGGAAGAGACCCAGTACTTC 112 CASSPRTGTSGRRMEETQYF

TGTGCCGCGAGGGGGAGTGATACGCAGTATTTT 111 CAARGSDTQYF
TGTGCCAGCAGTTTAACGGCCCATGCTAGCAATCAGCCCCAGCATTTT 104 CASSLTAHASNQPQHF
C

TGCAGCGTTGGAAGGGACCCGAACACCGGGGAGCTGTTTTTT 93 CSVGRDPNTGELFF
TGCGCCAGCAGCCAGCCTTGCCAGTGCAGTACTTC 88 CASSQP_PVQYF
AC

TGTGCCAGCAGCGCGTTAGGTGCGGGAGCTTCAGATACGCAGTATTTT 84 CASSALGAGASDTQYF
TGTGCCAGCAGATGGGCTAGCACAGATACGCAGTATTTT 50 CASRWASTDTQYF
TGTGCCAGCAGCCAAGATGGTCGGCAGGGGGGCACTGAAGCTTTCTTT 42 CASSQDGRQGGTEAFF
TGTGCCAGCAGTTCCCCTAGCGGGAGTGGATACAATGAGCAGTTCTTC 41 CASSSPSGSGYNEQFF
TGCAGTGCTAGTTCATACCGCGAGCAGTACTTC 35 CSASSYREQYF
TGCAGTGCTCCGGGACAGGGAAATCAGCCCCAGCATTTT 30 CSAPGQGNQPQHF
TGTGCCAGCAGCCTAGGACTAGCGGGAGATTACGAGCAGTACTTC 30 CASSLGLAGDYEQYF
TGTGCCAGCAGCTTAGCTCGCACCGGACTAGGAAACAATACAATGAGCAGTTCTTC 30 CASSLARTG_RKQYNEQFF
TGTGCCAGCAGCTTAGTTCTCCAAGAGGCCCCTTATTTT 29 CASSLVLQEAPYF
TGCAGTGCTAGAGAGAGCTTATCCTACGAGCAGTACTTC 28 CSARESLSYEQYF
TGTGCCAGCATTGCCCACCAAGGCGGAGGGCAGTACTTC 28 CASIAHQGGGQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGCAGTGCTAGAGGCACCGGTGGCGCCAGGGATTACACTGAAGCTTTCTTT 16754 CSARGTGGARDYTEAFF
TGTGCCAGCAGCCCCCAGGGGGCTATCGAGCAGTACTTC 13946 CASSPQGAIEQYF
TGTGCCAGCAGCCAAAGGCGGGAGGGGATCTACGAGCAGTACTTC 12405 CASSQRREGIYEQYF
TGTGCCTGGAGTGTACAGGGGAACTATGGCTACACCTTC 11075 CAWSVQGNYGYTF
TGTGCCTGGAGGGACCGGACAGGGGGCCGTAGCAATCAGCCCCAGCATTTT 9891 CAWRDRTGGRSNQPQHF
TGTGCCAGCAGCTCCTATAGCGGGAAGAACACCGGGGAGCTGTTTTTT 9331 CASSSYSGKNTGELFF

PT
TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 6818 CSVHRDPNSHEQFF
TGTGCCAGCAGCCCAAAAGGGGGTTCCGGGGCCAACGTCCTGACTTTC 5603 CASSPKGGSGANVLTF
TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 5304 CSVLRDPNSYEQYF

RI
TGTGCCAGCAGCGGGCCCGGGACCGGGGAGACCCAGTACTTC 4166 CASSGPGTGETQYF
TGTGCCTGGAGTGGGGGGTGGTACGAGCAGTACTTC 3082 CAWSGGWYEQYF
TGCAGCGGGGGACAGGGGAACTATGGCTACACCTTC 2902 CSGGQGNYGYTF

SC
TGTGCCAGCAAATCACAGGGAGGGGAGACCCAGTACTTC 2838 CASKSQGGETQYF
TGTGCCAGCAGCTCATCGCCCAACGAGGGGTATGGCTACACCTTC 2275 CASSSSPNEGYGYTF
TGCGCCAGCAGCCAAGACTCCAGGGAAAAACTGTTTTTT 2197 CASSQDSREKLFF

U
TGCAGCGTTGGACGGGACCCGAACACCGGGGAGCTGTTTTTT 1948 CSVGRDPNTGELFF
TGCGCCAGCAGCCAAGAACGGGGGCTGCCCTACGAGCAGTACTTC 1892 CASSQERGLPYEQYF
AN
TGTGCCAGCAGCTTAGCGCTTAGCGGGAAGGGTCAAGAGACCCAGTACTTC
TGTGCCAGCAGCTTAAGGGACGCCGGGTATGGCTACACCTTC
1832
1822
CASSLALSGKGQETQYF
CASSLRDAGYGYTF
TGTGCCAGCAGATTCTCTGGGGCCAACGTCCTGACTTTC 1739 CASRFSGANVLTF
TGTGCCAGCACCCGAGGCCCTGAAGCTTTCTTT 1649 CASTRGPEAFF
M
TGTGCCAGCCAAAATCAGGCGGCGAAGGTTGAGACCCAGTACTTC 1539 CASQNQAAKVETQYF
TGCAGTGCTGGGGGGGACAGGGGGTTTAGAGGTGGCACTGAAGCTTTCTTT 1425 CSAGGDRGFRGGTEAFF
TGTGCCAGCAGCCTAGACTCAAACACCGGGGAGCTGTTTTTT 1312 CASSLDSNTGELFF
D

TGTGCCTGGGCGGCGGGGACAGCTTTGTACGAGCAGTACTTC 1248 CAWAAGTALYEQYF

TGCGCCAGCAGCCAAGATCTTCCCGGGACAGGGTATTACGCTGAGCAGTTCTTC 1238 CASSQDLPGTGYYAEQFF
TE

TGTGCCAGCAGTCGGACTAGCGGGAACGGCGAGCAGTACTTC 1156 CASSRTSGNGEQYF

TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 1095 CASSFTGAMNTEAFF
TGTGCCAGCAGGGGAGGTGGGGCCAACGTCCTGACTTTC 1019 CASRGGGANVLTF
EP

TGTGCCAGCAGCTTAGGACAGGGAGGTCAGCCCCAGCATTTT 995 CASSLGQGGQPQHF

TGTGCCAGCAGCCCAAGTAGCGGGAGAGTGAACACCGGGGAGCTGTTTTTT 959 CASSPSSGRVNTGELFF
TGCGCCAGCAGTTCGGACCCTTCCTCCTCAGATACGCAGTATTTT 835 CASSSDPSSSDTQYF
C

TGTGCCAGCAGCCACACGGGTCCGGGGGAGCAGTACTTC 769 CASSHTGPGEQYF

TGTGCCAGCAGCTTCCTAGCGGGTACCGTTAGGGGCGAGCAGTACTTC 749 CASSFLAGTVRGEQYF
AC

TGTGCCAGCAGCTTAGGACAGGGACTAGCCAAAAACATTCAGTACTTC 730 CASSLGQGLAKNIQYF

TGTGCCAGCAGCCTCTCGAGGCTAGCGGGAGGGAGTAACACCGGGGAGCTGTTTTTT 576 CASSLSRLAGGSNTGELFF
TGTGCCAGCAGCTTAGACCCCGGGACAGGAGGCTATGGCTACACCTTC 570 CASSLDPGTGGYGYTF
TGTGCCTGGAGTCGGCAGGGGGCGAGTAAGAAACTGTTTTTT 552 CAWSRQGASKKLFF
TGTGCCAGCAGGGATGCGGGGGGTGGGGCTTACAATGAGCAGTTCTTC 547 CASRDAGGGAYNEQFF
TGTGCCAGCACCCGGGACAGGGGGCGGTGGGAGCAGTACTTC 531 CASTRDRGRWEQYF
TGCGCCAGCAGCCAAGGGAGCGGTGGATTTTATGGCTACACCTTC 529 CASSQGSGGFYGYTF
TGTGCCAGCAGCTACTCCAATACAGGGAGCTACGAGCAGTACTTC 487 CASSYSNTGSYEQYF
TGTGCCAGCAGCTTAGCAGGTGAAGCTTTCTTT 441 CASSLAGEAFF
TGTGCCAGCAGCTTATTCACAGATACGCAGTATTTT 402 CASSLFTDTQYF
TGTGCCAGCAGCTTATTTCAGGACACCGGGGAGCTGTTTTTT 392 CASSLFQDTGELFF
TGTGCCAGCAGCGTAGGTGTCAGGGGGGGCGAGCAGTACTTC 359 CASSVGVRGGEQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCTTGATGTGGGAGCAGTACTTC 27446 CASSLMWEQYF
TGTGCCTGGAGTGGAACAGGGAGGAGTGAGCAGTTCTTC 22505 CAWSGTGRSEQFF
TGTGCCAGCAGCTTGACTAGCGGGGGCTCCTACGAGCAGTACTTC 9382 CASSLTSGGSYEQYF
TGTGCCAGCAGTTGGACAGGGGGCGTTTACGAGCAGTACTTC 8999 CASSWTGGVYEQYF
TGTGCCAGCAGCTGGGGACAGGGGCCCTATGGCTACACCTTC 8588 CASSWGQGPYGYTF
TGTGCCAGCCTAAACAGGGGGCGCGGCACTGAAGCTTTCTTT 8536 CASLNRGRGTEAFF

PT
TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 8032 CSVLRDPNSYEQYF
TGCGCCAGCAGCCAAGGCAGGAACACCGGGGAGCTGTTTTTT 7069 CASSQGRNTGELFF
TGTGCCAGCAGCTTTTCATCATCATACTCTGGAAACACCATATATTTT 6826 CASSFSSSYSGNTIYF
TGCGCCAGCAGCCAAGAGGGCGGCGAGCGCTTCAATGAGCAGTTCTTC 4006 CASSQEGGERFNEQFF

RI
TGCGCCAGCAGCTTGGCCGGGACAGGGAACACTGAAGCTTTCTTT 3059 CASSLAGTGNTEAFF
TGTGCCTGGAAACTAGCGGGAGTCGAGCAGTACTTC 3050 CAWKLAGVEQYF
TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 2305 CSVHRDPNSHEQFF

SC
TGTGCCAGCAGCTTAGGACCTTACACCGGGGAGCTGTTTTTT 2144 CASSLGPYTGELFF
TGTGCCTGGAGTTCAGGGACAGGGCTTCGCGGCTACACCTTC 1942 CAWSSGTGLRGYTF
TGTGCCAGCAGCTTTTACACAGATACGCAGTATTTT 1884 CASSFYTDTQYF
TGTGCCTGGAACCTCAGGTTGGCAGAGACCCAGTACTTC 1830 CAWNLRLAETQYF

U
TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 1758 CASSFTGAMNTEAFF
TGTGCCAGCAGCTTAGAGGGGTCTAGCGGGAGGCAGACCCAGTACTTC 1656 CASSLEGSSGRQTQYF
TGCAGTGCTAGAAGGGGCGACACTGAAGCTTTCTTT AN
TGTGCCAGCAGCCGGGCATATCGGGGGTATGGCTACACCTTC
1606 CSARRGDTEAFF
1432 CASSRAYRGYGYTF
TGTGCCAGCAGCGAGAGACAGGGTCTCTTCAGTGGCTACACCTTC 1257 CASSERQGLFSGYTF
TGTGCCAGCCAAAATCAGGCGGCGAAGGTTGAGACCCAGTACTTC 1121 CASQNQAAKVETQYF
M
TGTGCCTGGAGCCCAGGGGGGGCTATCAGTCAGCCCCAGCATTTT 1120 CAWSPGGAISQPQHF
TGCGCCAGCAGCCAAGAACGTGGGACTAGTGGCTCTTGGCAGTTCTTC 1091 CASSQERGTSGSWQFF
TGTGCCAGCACCCCCCAAGGGACGGGAGAAAAAGCTTTCTTT 985 CASTPQGTGEKAFF
D

TGTGCCAGCAGCTTGGACTACCAAGAGACCCAGTACTTC 952 CASSLDYQETQYF

TGTGCCTGGAGCCCTCCAGGGCCAGATACGCAGTATTTT 940 CAWSPPGPDTQYF
TE

TGTGCCAGCAGCAGACAGGGGGCCGTTCACGAGCAGTACTTC 940 CASSRQGAVHEQYF

TGTGCCTGGAGTGTTCGGCGCACAGATACGCAGTATTTT 925 CAWSVRRTDTQYF
TGTGCCAGCAGCTCACAGGGGGCGATGGAGACCCAGTACTTC 883 CASSSQGAMETQYF
TGCGCCAGCAGCTTGGAGACAGGGCAATGGGGTTCACCCCTCCACTTT 838 CASSLETGQWGSPLHF
EP

TGTGCCAGCAGCTCCTCTGGAAACACCATATATTTT 764 CASSSSGNTIYF

TGCGCCAGCCGAAGACAGAATATGAACACTGAAGCTTTCTTT 737 CASRRQNMNTEAFF
TGCAGTGCTAGTGGACAGGCCGGCGAGCAGTACTTC 712 CSASGQAGEQYF
C

TGTGCCAGCAGCTTGAATTCGCGGGAGAGGGAAGAGACCCAGTACTTC 710 CASSLNSREREETQYF

TGTGCCAGCAGCGATACAGGGGCCTATAATTCACCCCTCCACTTT 668 CASSDTGAYNSPLHF
TGTGCCAGCAGCCTGACTAGCGGGTCCTACAATGAGCAGTTCTTC 639 CASSLTSGSYNEQFF
AC

TGCAGTCTAGCGGGAGAGGTGTGGGAGACCCAGTACTTC 606 CSLAGEVWETQYF

TGTGCCACCAGCAGAGAAGGACAGGTGTATGGCTACACCTTC 587 CATSREGQVYGYTF
TGCAGTGCTAGAAAGGAGGCGTTTAGCTCCTACAATGAGCAGTTCTTC 572 CSARKEAFSSYNEQFF
TGTGCCAGCAGCAATGGACTAGCCAGCACGAATGAGCAGTTCTTC 559 CASSNGLASTNEQFF
TGTGCCAGCGCTCCCCCCCGAGCTTCGGCTAATCAGCCCCAGCATTTT 526 CASAPPRASANQPQHF
TGCAGCGTTGCTGGACAGGGGGTGGGCTACACCTTC 505 CSVAGQGVGYTF
TGCGCCAGCAGACCAGGGACTATGAACACTGAAGCTTTCTTT 473 CASRPGTMNTEAFF
TGCGCCAGCAGCCAAGCCCTAACCGGTGAGCAGTTCTTC 425 CASSQALTGEQFF
TGCGCCAGCAGCCGCTTCCCGGGACTAGCGGGAGAGCCTAAGTCTAGCACAGATACGCAGTATTTT 424 CASSRFPGLAGEPKSSTDTQYF
TGTGCCAGCAGGCGACAGGACAATCAGCCCCAGCATTTT 421 CASRRQDNQPQHF
TGTGCCAGCAGCTTAAAGGGGCGACTAGCGGGAGGGCGGGAGCAGTTCTTC 350 CASSLKGRLAGGREQFF
TGCAGCGTTTTAAAGGACCCAAACTACAATGAGCAGTTCTTC 348 CSVLKDPNYNEQFF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCTGGAGTGTTGGGTCAAACAGCAATCAGCCCCAGCATTTT 1690 CAWSVGSNSNQPQHF
TGTGCCTGGAGTGTACGGGAGGCGTCCCAACCCAGTTTT 1396 CAWSVREASQPSF
TGTGCCTGGAGTCTGCCGGACACTGAAGCTTTCTTT 1157 CAWSLPDTEAFF
TGTGCCTGGAGTGTCAGAAGCCAAGATACGCAGTATTTT 896 CAWSVRSQDTQYF
TGTGCCTGGAGTGTGAGGGGCAGGGGATGGACTGAAGCTTTCTTT 867 CAWSVRGRGWTEAFF
TGTGCCAGCAGCCATGACACGAAGCTTTCTTT 820 CASSH_HEAFF

PT
TGTGCCTGGAGCAAAACAGGGGGCAGTGAGCAGTTCTTC 806 CAWSKTGGSEQFF
TGTGCCTGGACCCAGGCTAGGAGGCAGCGGGAGACCCAGTACTTC 728 CAWTQARRQRETQYF
TGTGCCAGCAGCTTAGCGAAGAGCGGGAGGGTTAACGAGCAGTACTTC 707 CASSLAKSGRVNEQYF
TGTGCCTCCTCCCACGGCGGAGATCAGCCCCAGCATTTT 659 CASSHGGDQPQHF

RI
TGTGCCTGGAGTGTACAACAGGGGATGGAGTACTTC 650 CAWSVQQGMEYF
TGTGCCAGCAGTTTCAAGGGGTTTTTT 618 CASSFKGFF
TGTGCCTCGACTTCCGGGACAGGGATTGGAAACACCGGGGAGCTGTTTTTT 614 CASTSGTGIGNTGELFF

SC
TGTGCCTGGAGCGGGGGCAGCCTCGGAGGGAATCAGCCCCAGCATTTT 608 CAWSGGSLGGNQPQHF
TGTGCCTGGGGCAGGGGCAGCTATGGCTACACCTTC 600 CAWGRGSYGYTF
TGTGCCTGGAGTACTAGCGGGCACGCGAACACCGGGGAGCTGTTTTTT 597 CAWSTSGHANTGELFF
CGTGCCAGCAGCCCCCGCCGGGCTAGCGGGGCCAGCTCCTACAATGAGCAGTTCTTC 592 RASSPRRASGASSYNEQFF

U
TGTGCCTGGAGTGTGGGAGGCTACACCTCTGGAAACACCATATATTTT 590 CAWSVGGYTSGNTIYF
TGTGCCTGGAGTGTATCGGGTTTGGGGTCTACGGAGACCCAGTACTTC 578 CAWSVSGLGSTETQYF
TGTGCCTGGAGTGTCCGGGAGGCAGATACGCAGTATTTT
TGTGCCTGGAGGGGGGCGGTACAAGAGACCCAGTACTTC
AN
TGTGCCTGGAGTGTGGGGAGGGCGTGGGAGACCCAGTACTTC
576
559
551
CAWSVREADTQYF
CAWRGAVQETQYF
CAWSVGRAWETQYF
TGTGCCAGCAGCTTAGGGGTCGGGACAGGGTTCAGTCAGCCCCAGCATTTT 548 CASSLGVGTGFSQPQHF
M
TGTGCCAGCAGCTTAGGCGGCTTCCGGGATCACTGAAGCTTTCTTT 543 CASSLGGF_GITEAFF
TGTGCCAGCACCCCGGGACTAGCGGGAGGAACCGGGGAGCTGTTTTTT 528 CASTPGLAGGTGELFF
TGTGCCTGGTCAACGGGAGCGGTCGAGCAGTACTTC 510 CAWSTGAVEQYF
D

TGTGCCTGGAAACCACCGCCCCAGCGGTACTACGAGCAGTACTTC 507 CAWKPPPQRYYEQYF

TGTGCCTGGAGTGTACGGGACAGGGTAGGCTATGGCTACACCTTC 506 CAWSVRDRVGYGYTF
TE

TGTGCCTGGAGTGTACGTCAGCGGGGACTCCCCTACGAGCAGTACTTC 500 CAWSVRQRGLPYEQYF

TGTGCCTGGAGTGTACGGGCAACAGGGAGGGCCTATGGCTACACCTTC 500 CAWSVRATGRAYGYTF
TGTGCCAGCGCGCCCCCCTCGTCAGCCCCAGCATTTT 496 CASAPP_RQPQHF
TGTGCCTGGAGAGCGGCAGGGGCTCACTACGAGCAGTACTTC 491 CAWRAAGAHYEQYF
EP

CGTGCCAGCAGCTTAACCTGGGCGAGCACAGATACGCAGTATTTT 489 RASSLTWASTDTQYF

TGTGCCTGGCGCAGGGAGGCTGAAGCTTTCTTT 485 CAWRREAEAFF
TGTGCCTGCCCGGTGGGGGACCTAGCTGGGGTGAACACTGAAGCTTTCTTT 476 CACPVGDLAGVNTEAFF
TGTGCCTGGAGTGATCCCCTAGCGGGAGGCCACGAGTACTTC 466 CAWSDPLAGGHEYF
C

TGTGCCACCAGCAGATTAGGGACGGCAGTATATGGCTACACCTTC 465 CATSRLGTAVYGYTF

TGTGCCTGGTGGCAGGGTGAATCCTACGAGCAGTACTTC 451 CAWWQGESYEQYF
AC

TGTGCCTGGAGTGGTCCAGGGACCAACGTCCTGACTTTC 443 CAWSGPGTNVLTF

TGTGCCTGGAGCCCCGGGACCTCACCCCTCCACTTT 441 CAWSPGTSPLHF
TGTGCCTGCTCAAGTCGCTACGAGCAGTACTTC 435 CACSSRYEQYF
TGTGCCAGCAGCTACTCCAATACAGGGAGCTACGAGCAGTACTTC 434 CASSYSNTGSYEQYF
TGTGCCTGGACCACGGGACAGGCCTCCTACGAGCAGTACTTC 431 CAWTTGQASYEQYF
TGTGCCTGGAGTGTCTTGGGGACCCAGTACTTC 427 CAWSVLGTQYF
TGTGCCTGGAGTGTTTTACAGGGATTCGCGAACACCGGGGAGCTGTTTTTT 409 CAWSVLQGFANTGELFF
TGTGCCTGGAGTCAGAGAAGGGCCACTGAAGCTTTCTTT 400 CAWSQRRATEAFF
TGTGCCTGGGGCCTCGGGGACAGACCCTCCTACGAGCAGTACTTC 384 CAWGLGDRPSYEQYF
TGTGCCTGGACACCAGGAGCGCTTATTGAAGCTTTCTTT 380 CAWTPGALIEAFF
TGTGCCTGTCTTCGGGACAGGGCACCCAACTATGGCTACACCTTC 378 CACLRDRAPNYGYTF
TGTGCCTGGAGGCCCGGACAGGACCCTACCTACGAGCAGTACTTC 365 CAWRPGQDPTYEQYF
 ACCEPTED MANUSCRIPT

Clone count CDR3 AA Seq

CDR3 DNA Seq
TGTGCCAGCAGCTTTCGGACTAGCGGCGAGCAGTACTTC 2677 CASSFRTSGEQYF
TGTGCCAGCAGCCCCCCCCGCGGGGTCCGGGAGCTGTTTTTT 1392 CASSPPRGVRELFF
TGTGCCTGGAGTGTTTGGGGGACCCGGAACACTGAAGCTTTCTTT 979 CAWSVWGTRNTEAFF
TGTGCCTGGCAGAAACAGGACAACTATGGCTACACCTTC 885 CAWQKQDNYGYTF
TGTGCCAGCAGCCAAGAGCCAGGGCACAATCAGCCCCAGCATTTT 840 CASSQEPGHNQPQHF
TGTGCCTGGAGTGTGGACAGTGGCAATCAGCCCCAGCATTTT 769 CAWSVDSGNQPQHF

PT
TGTGCCTGGAGTGTACTGGCTGCAGGCCAAGAGACCCAGTACTTC 706 CAWSVLAAGQETQYF
TGTGCCAGCAACAAAGCGGCAAACCAAGAGACCCAGTACTTC 694 CASNKAANQETQYF
TGTGCCTGGAGTGTACGTGACAGGGAGGTCATTGAAGCTTTCTTT 647 CAWSVRDREVIEAFF
TGTGCCTGGAGCAAAACAGGGGGCAGTGAGCAGTTCTTC 635 CAWSKTGGSEQFF

RI
TGCGCCAGCAGCCAGTTAGGGCAGTACACCGGGGAGCTGTTTTTT 558 CASSQLGQYTGELFF
TGTGCCAGCAGCTTAGGAACAGAACCATCCTACGAGCAGTACTTC 478 CASSLGTEPSYEQYF
TGTGCCTGGAGTACCCGGACCTACCAGGGAGAATTC 442 CAWSTRTYQGEF

SC
TGTGCCAGCAGCTTAGCGAGCCTAGCGGGAGAGCAGTACTTC 440 CASSLASLAGEQYF
TGTGCCAGCAGCCCCGGGACAGTCACAGATACGCAGTATTTT 412 CASSPGTVTDTQYF
TGTGCCTGGAGCGTCGGGCCCGGCCTGAACACTGAAGCTTTCTTT 408 CAWSVGPGLNTEAFF

U
TGTGCCTGGACAAGACTCGGAAACACCATATATTTT 406 CAWTRLGNTIYF
TGTGCCTGGAGACCTAGGACCAGCGGGGAGCTGTTTTTT 404 CAWRPRTSGELFF
TGTGCCTGGGCGACAGGGGAAAATCAGCCCCAGCATTTT
TGTGCCTGGAGTGTCCGCGGAGAGACCCAGTACTTC
AN
TGTGCCAGCAGCTCAGGGGCTAGCCCTCAAGAGACCCAGTACTTC
403
391
372
CAWATGENQPQHF
CAWSVRGETQYF
CASSSGASPQETQYF
TGTGCCTGGAGTGTCGTTCTAGCGGGATTAGAGACCCAGTACTTC 355 CAWSVVLAGLETQYF
M
TGTGCCAGCAGTCCCTTACTGAACACTGAAGCTTTCTTT 343 CASSPLLNTEAFF
TGTGCCAGCAGCTCCTCGAATGAAAAACTGTTTTTT 331 CASSSSNEKLFF
TGCGCCAGCAGCCAAATTGGGGAGACCCAGTACTTC 325 CASSQIGETQYF
D

TGTGCCAGCAGCCGGCCAGCAACGAACACTGAAGCTTTCTTT 311 CASSRPATNTEAFF

TGTGCCTGCCGTGGCGGACAGGGGAACTATGGCTACACCTTC 307 CACRGGQGNYGYTF
TGTGCCTGGATAGCGGCGGGGAGAGAGACCCAGTACTTC 306 CAWIAAGRETQYF
TE

TGTGCCTGGAGTGGGGTTTCCAACACTGAAGCTTTCTTT 300 CAWSGVSNTEAFF

TGTGCCAGCAGCCCTCGTCCCCGTGAGCAGTTCTTC 299 CASSPRPREQFF
TGTGCCAGCAGCGAGGGGACAGGGAACTATGGCTACACCTTC 299 CASSEGTGNYGYTF
EP

TGTGCCTGGAGGGTAGCGGGAGGCCTTCTAGAGCAGTACTTC 295 CAWRVAGGLLEQYF

TGTGCCAGCAGCCCCCCGGACAGCGGCTTACCCAATCAGCCCCAGCATTTT 288 CASSPPDSGLPNQPQHF
TGTGCCTGGAGTGTCCTACAAGAGACCCAGTACTTC 288 CAWSVLQETQYF
TGTGCCAGCAGCTTAAACAGGGGCTCCTACGAGCAGTACTTC 282 CASSLNRGSYEQYF
C

TGTGCCTGGAGTAGCGGGCTAGCGGGCGGGGAGCTGTTTTTT 275 CAWSSGLAGGELFF

TGCAGCGTTGAAAGGGGGTCCAATGAGCAGTTCTTC 267 CSVERGSNEQFF
AC

TGTGCCTGGCGTGGGGACAGATACTACGAGCAGTACTTC 267 CAWRGDRYYEQYF

TGTGCCTGGAGTGTAGGCGGGGGAGGAGATACGCAGTATTTT 262 CAWSVGGGGDTQYF
TGTGCCAGCAGCTCCCGACTAGCCTCCGAGCAGTACTTC 257 CASSSRLASEQYF
TGTGCCAGCGGGACTAGCGGAGGCACAGATACGCAGTATTTT 255 CASGTSGGTDTQYF
TGTGCCTGGAGACCTACCGGTGCGCGAGATACGCAGTATTTT 254 CAWRPTGARDTQYF
TGTGCCTGGATGGTGTCCTATGGCTACACCTTC 254 CAWMVSYGYTF
TGTGCCAGCAGCCAAGCGGAAAGCAATCAGCCCCAGCATTTT 254 CASSQAESNQPQHF
TGTGCCAGCAGCGCCCGACTAGCGGAAGAGACCCAGTACTTC 248 CASSARLAEETQYF
TGTGCCTGGAGAGACTACCGGGACAGGGGGAACACCTTC 247 CAWRDYRDRGNTF
TGTGCCAGCAGCTTCGGGGTGTGGAATTCACCCCTCCACTTT 242 CASSFGVWNSPLHF
TGTGCCAGCAGCTTGGGGCTAGCGGAAGATACGCAGTATTTT 241 CASSLGLAEDTQYF
TGTGCCAGCAGCTTAGCGGGGGGACTAACCTACGAGCAGTACTTC 240 CASSLAGGLTYEQYF
 ACCEPTED MANUSCRIPT
Supplementary Table 5. Clinical characteristics of Oxford cohort patients
assessed in this study for gene expression analysis.

Characteristic Control UC CD
(n=13) (n=31) (n=27)
Male/female 3/9 13/17 11/16
Median (IQR) age at sampling (years) 58 48 37
(41–65) (35–62) (24–61)

PT
Median (IQR) age at diagnosis (years) n/a 31 25
(24–39) (18–46)
Median (IQR) disease duration (years) n/a 9 9
(3–23) (6–13)

RI
Median (IQR) C-reactive protein (mg/l) n/a 6.0 11.1
(1.1–14.9) (3.8–46.3)
Median (IQR) peripheral blood leukocytes 8.8 8.3 8.0

SC
(10^9/l) (8.3–9.0) (7.0–9.7) (6.5–10.7)
Current medication at sampling
5-Aminosalicylates 21 1
Corticosteroids 8 1

U
Azathioprine/6-mercaptopurine 7 10
Infliximab/adalimumab 1 9
AN
Unknown 4 7
Demographic and clinical characteristics of IBD patients analysed in Figure 5E and 7D
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Supplementary Table 6. Clinical characteristics of Oxford cohort patients
assessed in this study for the analysis of microbiota-specific CD4 T cells.

Characteristic All patients UC CD

(n=44) (n=20) (n=24)
Male/female 24/20 10/10 14/10
Median (IQR) age at sampling (years) 41 42 ( 38.5

PT
(31.5-54.75) 34-55.75) (31-52)
Median (IQR) age at diagnosis (years) 31 39 26.5
(21-47) (25-55) (19.5-37.75)
Median (IQR) disease duration (years) 7 4 12

RI
(2-15) (1-12) (6-15.75)
Median (IQR) C-reactive protein (mg/l) 2.1 1.3 2.6
(0.9-4.425) (0.85-3.56) (0.92-4.65)

SC
Median (IQR) peripheral blood 7.065 7.1 6.9
leukocytes (10^9/l) (5.4-8.11) (5.35-9.11) (5.33-8)
Current medication at sampling
5-Aminosalicylates 10 2

U
Corticosteroids 4 2
Azathioprine/6-mercaptopurine 3 12
AN
Infliximab/adalimumab 2 10
Unknown 3 1
Demographic and clinical characteristics of IBD patients analysed in Figure 7A, B and
Supplementary 6A-H
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Supplementary Table 7. Clinical characteristics of Oxford cohort patients
assessed in this study for cell accumulation in the mucosa.

Characteristic Control UC CD
(n=13) (n=8) (n=9)
Male/female 6/7 6/2 4/5
Median (IQR) age at sampling (years) 32.5 29.5 24

PT
(25–45.5) (25.75–39.5) (18.5–29.5)
Median (IQR) age at diagnosis (years) 25 25 23.5
(22-46) (17.5–28.5) (19.5–33.75)
Median (IQR) disease duration (years) n/a 4 1

RI
(1–10.5) (0–6)
Median (IQR) C-reactive protein (mg/l) 1.75 0.9 20.7
(0.15-8.3) (0.4–6.3) (6.3–43.2)

SC
Median (IQR) peripheral blood 7.02 6.73 6.12
leukocytes (10^9/l) (5.8–8.3) (6.018–8.54) (5.35–11.68)
Current medication at sampling
5-Aminosalicylates 4 2

U
Corticosteroids 1 0
Azathioprine/6-mercaptopurine 1 4
AN
Infliximab/adalimumab 0 1
Unknown or no treatment 2 4
Demographic and clinical characteristics of IBD patients analysed in Figure 3B, C and
Figure 7C
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
A Firmicutes Actinobacteria B
Clostridiaceae Bifidobacteriaceae **** *
10
Clostridium difficile Bifidobacterium animalis

CD154+ of CD4+ cells (%)

Lactobacillaceae Mycobacteriaceae
Lactobacillus acidophilus 1
Mycobacterium tuberculosis
Lachnospiraceae
0.1
Roseburia intestinalis Bacteroidetes ns
Ruminococcus obeum Bacteroidaceae
Bacteroides vulgatus 0.01
Ruminococcaceae
Faecalibacterium prausnitzii
Fungi 0.001
Staphylococcaceae
Staphylococcus aureus Saccharomycetaceae
Candida albicans 0.0001
Proteobacteria

PT
Enterobacteriaceae 0.00001
Obligate aerobe
Escherichia coli

.d m
ile
be
im PS
Facultative anaerobe

C riu
ic
Salmonella typhimurium

ro
L

iff
Obligate anaerobe

u
ic
m

ph
no

ty
RI
C

S.
gated on live, CD3+
E. coli L. acidophilus B. vulgatus S. aureus no microbe
10
5

4
0.123 10 5 0.0146 10
5 2.63e-3 10
5 0.118 10
5 0
D CD154+ CD4+ T cells
CD154-

SC
10 10 4 10 4 10 4 10
4

100
10 3 10
3
10 3 10 3 10
3

2
80
10 10 2 10
2
10
2
10
2

0 0 0 0 0
60

0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10
3
10
4
10
5
0 10
3
10
4
10
5
0 10
3
10
4
10
5

S. typhimurium F. prausnitzii R. intestinalis C. albicans LPS

40

U
10
5 0.1 10
5 4.08e-3 10
5 4.17e-3 10 5 0.0752 10
5 4.84e-4
20

% of maximum
4
10 10 4 10 4 10 4 10 4
CD154

0
AN
2 3 4 5
10 3
0 10 10 10 10
10 3 10 3 10 3 10 3

10 2 2 2 2 2
CD69
10 10 10 10

100
0 0 0 0 0

80
0 10 3 10 4 10 5 0 10
3
10
4
10
5
0 10
3
10
4
10
5
0 10 3 10 4 10 5 0 10
3
10
4
10
5

B. animalis C. difficile R. obeum M. tuberculosis SEB 60

M

10
5 0.0241 10
5 0.024 10
5 3.92e-3 10
5 0.0113 10
5 4.78

4 4 4 4 4 40
10 10 10 10 10

3 3 3 3 3
10 10 10 10 10 20

10 2 10
2
10
2
10
2
10
2
0
10 2 10 3 10 4 10 5
D

0 0 0 0 0 0

3 4 5 3 4 5 3 4 5 3 4 5 3 4 5
ICOS
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

CD4
TE

E S. typhimurium B. animalis L. acidophilus

100 ** 100
*** 100
**
G S. typhimurium C. difficile
0.08 *** 0.03 **

F
Reduction in CD154+ CD4+ T cells (%)

80 80 80 0.06
10 5 0.02
EP

60 60 60
**** * 0.04
CD154+ of CD4+ cells (%)
Microbiota-reactive cells

40 40 40 10 4 0.01
microbe 0.02
/106 CD4+ cells

20 20 20
Adult blood
microbe 10 3
0 0 0 + α-MHCII 0 0.00 Cord blood
C

C. difficile F. prausnitzii S. aureus C. albicans B. vulgatus R. intestinalis R. obeum

** *** ** **** 10 2 0.015 * 0.008 * 0.008 **
100 100 100 100

80 80 80 0.006 0.006
AC

80
10 1 0.010
60 60 60 60
0.004 0.004
40 40 40 40 10 0 0.005
0.002 0.002
.d m
ile

20 20 20 20
C uriu
ic
iff
im

0 0 0 0 0.000 0.000 0.000

ph
ty
S.

Supplementary Figure S1
A E. coli
(3.84x)
S. typhimurium
(6.6x)
ACCEPTED
B. animalis
(2.5x)
MANUSCRIPT
L. acidophilus
(3.7x)

B
1 1 1 1
**** **** **** ****
**** ****
0.1 0.1 0.1 0.1 100
90
0.01 0.01 0.01 0.01
80
70

Memory (%)
0.001 0.001 0.001 0.001
60
0.0001 0.0001 0.0001 0.0001 50
F. prausnitzii C. difficile B. vulgatus R. intestinalis 40
(3.5x) (6.2x)
1
**** 1
**** 1
(3.2x) 1 (5.8x) 30
**** ****
3.5x
20
CD154+ (%)

0.1 0.1 0.1 0.1

10
0

PT
0.01 0.01 0.01 0.01

lls
ta di m
4 e
ce
D icil
To C. riu

T
l C ff
0.001 0.001 0.001 0.001

u
im
ph
ty
0.0001 0.0001 0.0001 0.0001

S.

RI
R. obeum S. aureus C. albicans M. tuberculosis SEB
1 (4.3x) 1 (10x) 1 (6.2x) 1 (32.5x) 100 (0.6x)
**** **** **** **** ****
10
0.1 0.1 0.1 0.1
****

SC
1
0.01 0.01 0.01 0.01
0.1
0.001 0.001 0.001 0.001
0.01

0.0001 0.0001 0.0001 0.0001 0.001

U
CD154+ of CD154+ of
naive T cells (CD45RA+) memory T cells (CD45RA-)
AN
Memory CD4 T cells
C 100 (live CD3+ CD4+ CD154+ CD45RA-)
D CD4+ CD154+ CD45RA- T cells
CD4+ CD154- CD45RA- T cells B. animalis stimulation
90 10
5 93.6
62
2.6
2.7
95.1
55
1.3
1.1
51.8
33.4
44.7
31.6
80
M

10 4

70 3
TNF-α (%)

10

60 0

50 34.7 0.5 43.2 0.6 32 3.0

3.7 0.1 3.5 0.1 3.1 0.28
40
D

0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 3 10 4 10 5

30 Integrin β7 CCR9 CCR2

20 76.6 6.0 90.3
20 10 5
20.8 44.4 4.0 60.8
96.1
54.5
0.4
2.6
10
TE

10 4
CCR6

0 10 3

80 0

9.6 25.1 2.1 33.1 39.6 3.2

70 1.6 1.6 0.07 3.6 3.4 0.06
10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
60
0 0 10 2 10 3 10 4 10 5

CCR4 CCR7 CCR10

EP

50 87.6 9.1 83.7 10 17.4 79.3

IL-2 (%)

5
10

55.6 9.0 56.6 7.6 16.5 48.7

40 10
4

30 10
3

20
C

28.2 6.1 31.6 4.1 28.4 6.3

2.25 0.9 3.5 2.8 1.6 1.5
10 0 10
3
10
4
10
5
0 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5

0 CXCR3 CXCR5 CD161

AC
lb sis
be lis
B. E. um

C sn s

R stin s
. i lg ile

um

s
R vu ffic i

C rcu us
pr ph s

i i
au ilu

e u
B. . d itz

an
ac nim li
F. ido ali

.o a

. a lo
L. a co

nt at

e e
i
ur

ic
ub ur
m

.t a
i
ph

M S.
ty
S.

Supplementary Figure S2
 ACCEPTED MANUSCRIPT
A 10

10
5

4
37.4 50.6 10 5

10 4
82.3 1.13 10

10 4
5
55.6 3.11 10

10 4
5
87.1 3.09 10

10
5

4
12.1 0.16

10 3 10 3 10
3 10 3 10
3
LPMCs
10
2 10
2
10
2
CD4+ T cells

Integrin β7
0 0
0
0
0

CD127
CCR7
CD69

CD69
2.89 9.12 14.5 2.05 41.2 0.06 9.62 0.16 84.7 3.02
0 10 2 10 3 10 4 10 5 0 10
3
10
4
10
5
0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5

10 5
0.1 0.9 10 5
0.26 0.42 10 5
32.2 61.6 10 5
32.6 60.7 10 5
3.5 24.4
4 4 4 4 4
10 10 10 10 10

10 3
3
10 3
PBMCs
CD4+ T cells
10 3 10 3 10

10 2 10 2
10 2
0 0
0
0
0

11.6 87.3 37.7 61.6 5.79 0.39 5.09 1.61 34.5 37.6
2 3 4 5 3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

CCR7 CD45RA

PT
B D S. typhimurium C. difficile
LPMCs
6000 1600
Live dead (EF780)

****
250K 250K

cells/106 CD4+ cells

80.3
5
10 10
5 10 5

Microbiota-reactive
10 4
200K 200K
96.9 10 4 79.2 10 4 5000 1400
56.5
1200
SSC-A

FSC-H

RI
CD3

CD4
150K 150K

4000
3
10 10
3
10 3
85.4
10 2
100K 100K
2
1000
10
0 50K 50K 0
0
3000 800
0 50K 100K 150K 200K 250K
0
0 50K 100K 150K 200K 250K
0
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 2000 600
FSC-A FSC-A FSC-A FSC-A FSC-A 400
1000

SC
200
10 5 8.44 4.38 10 5 40.3 9.06 0 0
Control mucosa
E. coli LPS
4
10 10 4

Peripheral blood
IFN-γ
IL-22

10 3
10
3 CD154+ 10 5
0.36 10 5
0.007
TNF-α+
TNF-α

TNF-α

10 4 10 4
0
0

U
3 3
10 10
70.9 16.2 39.1 11.6
3 4 5 3 4 5
0 10 10 10 0 10 10 10
10 2 10 2

Control mucosa
E
IL-17A
0 0

2.24 0.731
5
0 0 5
0 0 Peripheral blood
10 10
AN
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5

10
4 4 CD154 CD154
IL-17A
10

CD154+ IFN-γ
IFN-γ

15 IL-22
IL-22

10
3
10 3
60 100

Cytokine+ of TNF-α+ CD154+

TNF-α-

****
ns 90

***
0
0

50

*
100
3 4
0
5
100
3 4
0
5
80

CD4+ cells (%)

0 10 10 10 0 10 10 10

IL-17A 40 70 10 ns ns
M

60
gated on Live, 30 50
C PBMCs
10 5 1.57 0 10 5 48.8 7.09
lymphocyte, singlets,
CD3+, CD4+ 20
40
30 5
E. coli LPS
D

10 4 10
4

10 20
CD154+
IFN-γ
IL-22

10 5 10 5

10
3
10
3
10
TNF-α+
4 4
10 10
TNF-α

TNF-α

10 2
0
0
10 3
0.028 10 3
0.0008 0 0 0
TE

81.1 17.3 33.9 10.2

.d m
ile

. d um

ile
.d m
ile
2 2
10 10
C uriu

C uriu
ic

ic
ic
0 10 3 10 4 10 5 0 10 3 10 4 10 5
C uri
0 0
iff

iff
IL-17A 0.10 0.02 iff
im

im

im
2 3 4 5 2 3 4 5
ph

0 10 10 10 10 0 10 10 10 10
0 0 0.204 0
ph

ph
10 5 10 5

CD154 CD154
ty

ty

10 4
10
4 ty
CD154+
S.

S.

S.
IFN-γ
IL-22

3 3
10 10

TNF-α-
EP

10 2
0
0

99.8 0.204 99.6 0.204

0 10 3 10 4 10 5 0 10 3 10 4 10 5

IL-17A

F
C

IL-2 GMCSF IL-4

100 60 20
****

Control mucosa
AC
Cytokine+ of TNF-α+ CD154+

90 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
**

*
*

50 Peripheral blood
80
15
CD4+ cells (%)

70 40
60
50 30 10 ns ns ns ns ns ns
*
*

40
30 20
5
20 10
10
0 0 0
lb sis
lb sis
L. an um

C culo s
lb sis

F. op s

. t a ile
m
C culo s
F. dop lis

i i
. t a ile
i i

au us
C culo s
F. dop lis

. t a ile
m

s
au us

i i
s

au us

B
s
im oli

ac im li
B

. d zi
. d zi

B
L. an oli

. d zi

er eu
id ali
er eu

er eu

an
an

o
an

iu

SE
SE

iu
a

C nit
SE

M S. ffic
C nit
M S. ffic

C nit
M S. ffic

pr hil
pr hil
ph . c

c
pr hil
c
i
ac im
B. ur

ub ur
ac im

ur
ub ur

ub ur
ur

ic
ic

ic

B. .

s
s

B. E.

s
E

E
im

n
m

.a
.a

.a
i

ph
ph
i

i
ty

ty
ty

L.
S.

S.
S.

Supplementary Figure S3
 ACCEPTED MANUSCRIPT
A B E. coli
** ns * ** ** ** ** 10
5
17.7 1.1 10 5 30.2 7.7 10
5
5.86 0.9 10
5
0.28 0.4
50 10 4 10
4
10 4 10 4

10 3 10 3

** *** ns ns ns ns ns 10 3

40
3
10

IL-17A
0 10 2 10
2

IFN-γ

IL-22
0

E. coli Nissle

IL-4
0 0

24 57.2 11.5 50.6 35.9 57.4 41.4 57.9

30 0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5

CFSE
20 B. animalis
CFSElow of CD4+ cells (%)

10 5 3.78 2.4 10 5 4.16 14.5 10 5 1.23 2.9 10 5 0.9 0.8

10 10
4
10
4
10
4
10
4

3
3 10 3 10 10 3
10

IL-17A
0 2 2 2

IFN-γ
0 10 10 10

IL-22
0 0

IL-4
0

8.2 85.7 6.3 75 10.2 85.7 11.1 87.2

PT
80 ** ** ** ** * ** * 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5

CFSE
ns ns ns ** ns ns ns F. prausnitzii
60 10
5
1.6 2.5 10
5
3.6 40.1 10
5
0.5 2.4 10
5
0.4 1.4
B. animalis 4 4 4 4

RI
10 10 10 10

3 10
3 10 3 10
3
10

40

IL-17A

IL-4
2
10

IFN-γ
2 2

IL-22
0 10 10
0 0
0

4.2 91.7 2.2 54.1 5.3 91.7 2.2 96

20
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10

CFSE

SC
0
D no microbe S. aureus Flu FMO control
be

in le

au s

ile
is

. d ii
ph co e

itz
u
ty E. siv

al
nt iss

iu
im li

ic
l
ro

0.07 2.95 48.2 0.14 0.25 0.6 0 0.02

hi

10 5 10 5 10 5 10 5
sn
im
ur

iff
va
re N
ic

op
an
m

FMO PE
RORγt
i

4 4 4 4
10 10 10 10
id
he ol

C
pr
no

U
ad E. c

ac
B.

10 3 10 3 10 3 10 3
F.
L.
S.

2 2 2 2
10 10 10 10

0 0 0 0

0.09 96.9 40.2 11.4 28.1 71.1 86.7 13.3

AN
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10

IL-17A IFN-γ
10 5 0.07 1.32 10 5 32.5 0.19 10 5 18.3 2 10 5 0.02 0

C
4 4 4 4
10 10 10

FMO PB
10
T-bet

ns ns ns ns 10 3 10 3 10 3 10 3
***
****

****
****
****

**

100 100
10 2 10 2 10 2 10 2
M

0 0 0 0

90 90 0.08
0 10
2
10
3
10
4
98.5
10
5
55.9
0 10
2
10
3
10
4
11.4
10
5
9.91
0 10
2
10
3
10
4
69.7
10
5
86.5
0 10
2
10
3
10
4
13.5
10
5

80 80
0 0.3 0.1 0.03 0 0.4 0 0
70 70
10 5 10 5 10 5 10 5
GATA-3

FMO APC
4 4 4
10 10 10 10 4

60 60 10 3 10 3 10 3
D

10 3

50 50 0 0 0 0

40 40
Cytokine+ of CFSElow CD4+ cells (%)

0.2 99.4 88.4 11.5 28.6 71 86.9 13.1

30 30
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5
TE

CFSE
20 20
10
0
10
0
E S. typhimurium L. acidophilus F. prausnitzii C. difficile

IL-22 IL-4
EP

ns ns ns ns ns ns ns
****
****

50 25
Flu S. aureus
40 20
C

RORγt + + + + - - - -
30 15 T-bet + + - - + + - -
GATA-3 + - + - + - + -
AC

20 10
10
***
ns

ns
ns

**
**

10 5 10 5 30.1 7.34
8
F
4
10

0 0 F. prausnitzii
10
3
IL-10+
IL-10+ (%)

5
10

6
ta ac igh

. t a id

C cul s
lb is
s

ta ac igh

. t a id

C cul s
lb is
s
s ine

s ine
er eu

an

er eu

an
. a os

. a os
Te a v SE h

M S. oxo

Te a v SE h

M S. oxo
nu c

nu c

4 0
ub ur

ic

ub ur

ic

10
IL-10

56.7 5.87
t

t
z F

z F
en C

en C

10 3
21.7 6.99
10 5
4
10 2
flu

flu

10 4
In

In

0 10
2
10
3
10
4
10
5 10 3 IL-10- 2
CD3
IFN-γ

65.1 6.2
0 10
3
10
4
10
5
0
IL-17A
ac an ium
pr ph lis
C usn us

ile
iff zii
ph E. B
L. B. ur oli
SE

F. ido ma

ic
a il
. d it
im c

i
ty
S.

Supplementary Figure S5
 ACCEPTED MANUSCRIPT
A B E S. typhimurium
S. typhimurium C. difficile S. typhimurium C. difficile
CD154+ of CD4+ cells (%)

0.08 0.03 0.6 0.08 70 IL-17A 100 IFN-γ 40 IL-22 100 IL-2
*

CD154+ TNF-α+ of
**** ns ns **** 90 *** ns 90
****

CD4+ cells (%)

60
0.06 0.06 80 80
50 30
0.02 0.4 70 70

40 60 60
0.04 0.04
50 20 50
0.01 0.2 30 40 40
0.02 0.02 30 30
20
10
20 20
0.0 0.00 10
0.00 0.00 10 10
Control (n=30) Control mucosa (n=17-20) 0 0 0 0

IBD (n=38) Inflamed mucosa (n=10-15) B. animalis

IL-17A IFN-γ IL-22 IL-2

PT
70 100 40 100
90 ns 90
60 *** ** ***
C 15
80 80
30
50 70 70

*** D 40 60 60
Memory CD4 T cell abundance

14 E. coli SEB 50 20 50

RI
13 100 100 30 40 40
IL-2+ of TNF-α+ CD154+

12 90 *** 90 * 30 30
20
(norm. to controls)

10
11 80 80
CD4+ cells (%)

20 20
10 10

Cytokine+ of TNF-α+ CD154+ CD4+ cells (%)

70 70 10 10
9 60 60 0 0 0 0

SC
8 50 50
7 L. acidophilus
40 40
6 30 30
70 IL-17A 70 IFN-γ 40 IL-22 100 IL-2
5 20 20 **** 60
ns ns 90
****
60
4 10 10 30
80
3 50 50 70
0 0
2

U
60
0 Control (n=23) 40 40
20 50
IBD (n=33) 30 30 40
Control Inflamed 20 20 30
AN
mucosa mucosa 10
20
10 10
10
0 0 0 0

F 100 IL-17A 100 IFN-γ

S. aureus
70 IL-22 100 IL-2 70 IL-17A 100 IFN-γ
F. prausnitzii
40 IL-22 100 IL-2
ns ** *** *
M

90 90 90 90 ns ns 90
60 60 * **
80 80 80 80 80
Cytokine+ of TNF-α+ CD154+ CD4+ cells (%)

50 30
70 70 70 50 70 70
60 60 40 60 60 60
40
50 50 50 50 20 50
30 30
D

40 40 40 40 40
30 30 20 30 20 30 30
10
20 20 20 20 20
10 10
10 10 10 10 10
TE

0 0 0 0 0 0 0 0
M. tuberculosis C. difficile
IL-17A IFN-γ IL-22 IL-2
70 ns 100 ns 40 ns 100 70 IL-17A 100 IFN-γ 40 IL-22 100 IL-2
***
90 90 90 ns ns 90
60 60 ** ****
80 80 80 80
30 30
EP

50 70 70 50 70 70
40 60 60 60 60
40
50 20 50 50 20 50
30 40 40 30 40 40
20 30 30 20 30 30
10 10
20 20 20 20
C

10 10
10 10 10 10
0 0 0 0 0 0 0 0
Control (n=23) Control (n=9-17)
AC

IBD (n=33)
IBD (n=15-22)

G
H I
+

50 50 50
IL-17A of TNF-α CD154 CD4

*** **** ** E. coli B. animalis

ns ns *** 4 Control (n=4)
*** ns S. typhimurium L. acidophilus
**** *** ns IBD (n=6)
+

40 40 40
2.5 2.5
Cytokine (% of control)

3 IL-17A ns IFN-γ
* *
cells (%)

30 30 30 ns 2.0 2.0
IL-10 (%)
+

2 *
1.5 1.5
20 20 20

1.0 1.0
10 10 10 1
+

0.5 0.5

0 0 0 0 0.0 0.0
Control Control Control
Ulcerative colitis Active IBD Control (n=9) IL-1β − + + − + +
Aminosalicylates
Crohn’s disease IBD remission Purine synthesis IBD (n=11) IL-6 − + + − + +
inhibitors
IL-23 − + + − + +
Biologics

Supplementary Figure S6
 Steady state Inflammation

Inner mucus Outer mucus Inner mucus 5

layer layer layer Recruitment of myeloid cells
CXCL2/5/8/9/10/11
AC
Tc Dc CCL2/ CCL7

Dc

Tc
C IL-17A
IFN-γ
TNF-α
mLNs Tc
Tc Dc 4
Tc Tc Tc
Activation of
Tc Tc tissue-resident cells
EP
Tc
Tc Dc
Tc Symbiosis Dysbiosis
Tc
Bc
Bc
Tc Bc
Tc Tc
TE
3
Tc Accumulation of
Tc Tc Tc T cells in tissue
Tc
D Epithelial Tc
Tc
Tc leakage Tc
IL-17A Tc
IL-10
Tc
Tc
IFN-γ
TNF-α
M Tc
Tc Tc 2
CCR6 Tc Tc Change in T cell
CCR4 phenotype
CCR7 Tc
Integrin α4β7
AN
Dc IL-1β Tc Tc
CCR9 Tc Tc
IL-6
ACCEPTED MANUSCRIPT

Dc
Tc IL-23 1
Circulation
U Tc
Recruitment of
Circulation
microbiota-specific
T cells
SC
Tc Naive
Dc Dendritic cells
CD4+ T cells

Tc
Activated
Epithelial cells
CD4+ T cells
Activated
RI
Tc CD4+ T cells Stromal cells
(in inflammation)

Neutrophils Microbiota
PT
Macrophages Bc B cells

Supplementary Figure S7