Arthritis & Rheumatology

Vol. 72, No. 1, January 2020, pp 179–191

DOI 10.1002/art.41075
© 2019, American College of Rheumatology

Proinflammatory Histidyl–Transfer RNA

Synthetase–Specific CD4+ T Cells in the Blood and Lungs of
Patients With Idiopathic Inflammatory Myopathies
Angeles S. Galindo-Feria,1 Inka Albrecht,1 Cátia Fernandes-Cerqueira,1 Antonella Notarnicola,1 Eddie A. James,2
Jessica Herrath, Maryam Dastmalchi,1 Tatyana Sandalova,3 Lars Rönnblom,4 Per-Johan Jakobsson,1
1

Maryam Fathi,5 Adnane Achour,3 Johan Grunewald,1 Vivianne Malmström,1 and Ingrid E. Lundberg1

Objective. Autoantibodies targeting histidyl–transfer RNA synthetase (HisRS; anti–Jo-­1) are common in the
i­diopathic inflammatory myopathies (IIMs) and antisynthetase syndrome. This study was undertaken to investigate
immunity against HisRS in the blood and lungs of patients with IIM/antisynthetase syndrome.
Methods. Bronchoalveolar lavage (BAL) fluid, BAL fluid cells, and peripheral blood mononuclear cells (PBMCs)
from patients with IIM/antisynthetase syndrome (n = 24) were stimulated with full-­length HisRS protein or a HisRS-­
derived peptide (HisRS11–23). BAL fluid and PBMCs from patients with sarcoidosis (n = 7) and healthy subjects (n = 12)
were included as controls. The CD4+ T cell response was determined according to levels of CD40L up-­regulation
and cytokine expression using flow cytometry. Anti–Jo-­1 autoantibody responses in the serum and BAL fluid were
assessed by enzyme-­linked immunosorbent assay. Lung biopsy samples from patients with IIM/antisynthetase syn-
drome (n = 14) were investigated by immunohistochemistry.
Results. In BAL fluid, CD4+ T cells from 3 of 4 patients with IIM/antisynthetase syndrome responded to stimula-
tion with HisRS protein, as measured by the median fold change in CD40L expresssion in stimulated cells compared
to unstimulated cells (median fold change 3.6, interquartile range [IQR] 2.7–14.7), and 2 of 3 patients with IIM/antisyn-
thetase syndrome had the highest responses to HisRS11–23 (median fold change 88, IQR 27–149). In PBMCs, CD4+ T
cells from 14 of 18 patients with IIM/antisynthetase syndrome responded to HisRS protein (median fold change 7.38,
IQR 2.69–31.86; P < 0.001), whereas a HisRS11–23 response was present in 11 of 14 patients with IIM/antisynthetase
syndrome (median fold change 3.4, IQR 1.87–10.9; P < 0.001). In the control group, there was a HisRS11–23 response in
3 of 7 patients with sarcoidosis (median fold change 2.09, IQR 1.45–3.29) and in 5 of 12 healthy controls (median fold
change 2, IQR 1.89–2.42). CD4+ T cells from patients with IIM/antisynthetase syndrome displayed a pronounced Th1
phenotype in the BAL fluid when compared to the PBMCs (P < 0.001), producing high amounts of interferon-­γ and
interleukin-­2 following stimulation. Anti–Jo-­1 autoantibodies were detected in BAL fluid and germinal center (GC)–like
structures were seen in the lung biopsy samples from patients with IIM/antisynthetase syndrome.
Conclusion. The results of this study demonstrate a pronounced presence of HisRS-­reactive CD4+ T cells in
PBMCs and BAL fluid cells from patients with IIM/antisynthetase syndrome as compared to patients with sarcoido-
sis and healthy controls. These findings, combined with the presence of anti–Jo-­1 autoantibodies in BAL fluid and
GC-­like structures in the lungs, suggest that immune activation against HisRS might take place within the lungs of
patients with IIM/antisynthetase syndrome.

Supported by L’Association française contre les myopathies (AFM-
Téléthon), Mexican National Council for Science and Technology (Conacyt),
Karolinska Institutet (KID funding), King Gustaf V 80 Year Foundation, Konung
Gustaf V:s och Drottning Victorias Frimurarestiftelse, Reumatikerförbundet,
Stockholm County Council (ALF project), Myositis Association, Swedish Heart-
Lung Foundation, Swedish Research Council (grant K2014-52X-14045-14-3),
and Swedish Rheumatism Association.
1
Angeles S. Galindo-Feria, MD, Inka Albrecht, PhD, Cátia Fernandes-
Cerqueira, PhD, Antonella Notarnicola, MD, Jessica Herrath, PhD, Maryam
Dastmalchi, MD, PhD, Per-Johan Jakobsson, MD, PhD, Johan Grunewald,
MD, PhD, Vivianne Malmström, PhD, Ingrid E. Lundberg, MD, PhD:
Karolinska Institutet, Karolinska University Hospital, and Center for
Molecular Medicine, Stockholm, Sweden; 2Eddie A. James, PhD: Benaroya
Research Institute at Virginia Mason Hospital, Seattle, Washington;
3
Tatyana Sandalova, PhD, Adnane Achour, PhD: Science for Life Laboratory,
Department of Medicine Solna, Karolinska Institutet, and Department
of Infectious Diseases, Karolinska University Hospital, Solna, Stockholm,
Sweden; 4Lars Rönnblom, MD, PhD: Science for Life Laboratory, Stockholm,
Sweden, and Uppsala University, Uppsala, Sweden; 5Maryam Fathi, MD,
PhD: Karolinska University Hospital, Stockholm, Sweden.
Drs. Galindo-Feria and Albrecht contributed equally to this work.
Dr. Albrecht owns stock in Sanofi Genzyme. Drs. Albrecht and Lundberg
have filed a patent application on a diagnostic test to identify patients with
anti-FHL1 autoantibodies. Dr. Rönnblom has received research support from

179
180 | GALINDO-­FERIA ET AL
INTRODUCTION
Idiopathic inflammatory myopathies (IIMs) represent a group
of chronic autoimmune disorders that are characterized by mus-
cle weakness and systemic extramuscular manifestations in the
joints, skin, heart, and, particularly, the lungs (1). The identification
of autoantibodies unique to IIMs, also known as myositis-­specific
autoantibodies (MSA), has allowed recognition of correlations
with distinct clinical phenotypes (2,3). Among the MSA, anti–Jo-­1
autoantibodies targeting histidyl-­transfer RNA synthetase (HisRS)
are the most common (found in 25–35% of patients with IIM) (4–6)
and are associated with interstitial lung disease (ILD) and antisyn-
thetase syndrome (3,7).
Insights from studies of several autoimmune diseases have
placed the presence and presentation of the autoantigen in the lung,
where innate immunity and genetics play important roles in the ini-
tiation of the autoimmune response, as seen in rheumatoid arthritis
(RA) (8–10), sarcoidosis (11), and multiple sclerosis (MS) (12). Sev-
eral genetic and experimental findings support the role of adaptive
immunity in anti–Jo-­1 autoantibody formation. Thus, HLA–DRB1*03
has been found to be strongly associated with anti–Jo-­1 autoan-
tibodies (13–15). Furthermore, observations of gene–environment
interactions suggest an additional risk of anti–Jo-­1 autoantibodies
in HLA–DRB1*03–positive patients who are ever smokers (14), indi-
cating the importance of environmental exposures in the develop-
ment of anti–Jo-­1 autoantibodies. In addition, the presence of class
switching, epitope spreading, and affinity maturation of anti–Jo-­1
autoantibodies points toward a significant contribution of antigen-­
specific T cells in the development of these antibodies (16–19).
A question that has been difficult to elucidate is, how might
HisRS, a ubiquitous cytoplasmic protein, induce an autoimmune
reaction? The high expression of HisRS in specific microenviron-
ments, such as in normal lung tissue (20), combined with altered
expression of HisRS and/or an enriched conformation of highly sen-
sitive HisRS in response to granzyme B cleavage in the lung (21),
supports the role of the lung as a potential site where immune toler-
ance could be broken. A previous study demonstrated the presence
of T cells reactive to both intact HisRS protein and HisRS fragments,
most of which were ~150 amino acids in length, in peripheral blood
mononuclear cells (PBMCs) from patients with IIM as well as PBMCs
from healthy controls (22). Other observations revealed that both
intact and granzyme B–cleaved HisRS, corresponding to residues
1–48, can drive specific immune responses, including recruitment
of CD4+ and CD8+ T cells, as well as immature dendritic cells and
activated monocytes (23). This finding, together with identification of
a B cell epitope among the 60 amino acids of the N-­terminal region
AstraZeneca. Dr. Lundberg has received consulting fees, speaking fees, and/or
honoraria from Corbus (less than $10,000 each), consulting fees and honoraria
from Bristol-Myers Squibb (less than $10,000), consulting fees from aTyr (less
than $10.000), honoraria from MedImmune (less than $10,000), research
support from Bristol-Myers Squibb and AstraZeneca, and owns stock or stock
options in Roche. No other disclosures relevant to this article were reported.
of the HisRS protein, makes this region an attractive part of the
protein to search for T cell–reactive peptides.
An additional question is, in which body compartment might
HisRS-­reactive T cells be triggered? An interesting possibility is
that such immune activation may take place in the lungs, as indi-
cated by the detection of expanded T cell clones in bronchoal-
veolar lavage (BAL) fluid and peripheral blood from patients with
IIM-­
associated interstitial pneumonitis (24), and the finding of
expansions in BAL fluid CD4+ and CD8+ T cells and correspond-
ing biased T cell receptor V-­gene usage in the muscle of patients
with IIM (25). These observations suggest the involvement of a
shared antigen–driven immune response in the inflammatory pro-
cess between the lungs and muscles of these patients. However,
antigen-­specific T cell responses in cells derived from the lungs of
patients with IIM have hitherto not been demonstrated.
The present study aimed to address T cell specificity and trig-
gering in anti–Jo-­1+ patients with IIM/antisynthetase syndrome.
Specifically, we aimed to investigate 1) T cell reactivity and phe-
notype upon stimulation with full-­ length HisRS protein and an
HLA–DRB1*03 binding HisRS-­ derived peptide in PBMCs from
anti–Jo-­1+ patients with IIM/antisynthetase syndrome, 2) whether
such antigen-­ specific T cells are present in the lungs of anti–­
Jo-­1+ patients with IIM/antisynthetase syndrome, and 3) whether
immune activation and the presence of anti–Jo-­1 autoantibodies
can be assessed in the lungs of anti–Jo-­1+ patients with IIM/anti-
synthetase syndrome.
PATIENTS AND METHODS
Patients and selection criteria. Samples were collected
between November 2010 and June 2013 at the Rheumatology
Unit and Respiratory Medicine Unit of Karolinska University Hospital
(Stockholm, Sweden). Patients with IIM fulfilled the criteria for definite,
probable, or possible polymyositis or dermatomyositis according to
the Bohan and Peter criteria (26,27). The diagnosis of ILD, meeting
the American Thoracic society criteria (28), was based on findings
from chest radiography/high-­resolution computed tomography and/
or pulmonary function tests. The diagnosis of antisynthetase syn-
drome was based on the presence of anti–Jo-­ 1 autoantibodies
in addition to one of the following features: ILD, myositis, arthritis,
Raynaud’s phenomenon, fever, or mechanic’s hands (7).
Sample collection. PBMCs were obtained from anti–Jo-­1+
patients with IIM/antisynthetase syndrome (n = 15; 6 male and 9
female) and anti–Jo-­1− patients (n = 3; all female) (corresponding
to patients P1–P18 in Table 1).
Address correspondence to Angeles S. Galindo-Feria, MD, Karolinska
University Hospital, Karolinska Institutet, Department of Medicine, Division
of Rheumatology, Center for Molecular Medicine, L8:04 SE-171 76 Stockholm,
Sweden. E-mail: angeles.galindo.feria@ki.se.
Submitted for publication November 23, 2018; accepted in revised form
August 6, 2019.
Table 1. Clinical and laboratory characteristics of the 24 patients with idiopathic inflammatory myopathy/antisynthetase syndrome according to type of experiment*
T cell reactivity†
HLA–DRB1
PBMCs BAL fluid
Muscle Anti–Jo-­1 status genotype
Full-­length Full-­length inflammatory
Patient Diagnosis protein HisRS11–23 protein HisRS11–23 infiltrates Serum BAL fluid Allele 1 Allele 2
P1 PM/antisynthetase syndrome + Not tested Nonviable cells Nonviable cells Present Positive‡§¶ Negative‡ 04 15
P2 Antisynthetase syndrome/ILD ++ ++ ++ +++ Absent Positive‡ Positive‡ 01 13
P3 Antisynthetase syndrome/ILD ++ +++ + +++ Absent Positive‡§¶ Positive‡ NA NA
P4 DM ++ +++ −# +# Present Negative‡§ Negative‡ 09 13
P5 PM +++ Not tested +++ Not tested Present Negative‡ Negative‡ 03 10
ANTIGEN-­SPECIFIC T CELLS IN INFLAMMATORY MYOPATHIES

P6 DM/ILD − + Nonviable cells Nonviable cells Absent Negative‡ NA 03 03

P7 Antisynthetase syndrome/ILD +++ + NA NA Absent Positive‡ NA 14 15
P8 Antisynthetase syndrome/ILD ++ ++ NA NA Absent Positive‡§¶ Positive‡ 03 13
P9 Antisynthetase syndrome/ILD +++ + NA NA Absent Positive‡ Positive‡ 03 04
P10 Antisynthetase syndrome/ILD ++ ++ NA NA Present Positiveद NA 03 15
P11 Antisynthetase syndrome/ILD ++ − NA NA Present Positive‡§¶ NA 03 13
P12 Antisynthetase syndrome/ILD +++ ++ NA NA Present Positive‡ NA 03 04
P13 Antisynthetase syndrome/ILD +++ + NA NA Present Positiveद NA 03 04
P14 Antisynthetase syndrome/ILD +++ − NA NA Present Positive‡§¶ NA 03 04
P15 Antisynthetase syndrome + Not tested NA NA Absent PositiveঠNA 01 03
P16 Antisynthetase syndrome/ILD − Not tested NA NA Present Positive§ NA 03 15
P17 Antisynthetase syndrome/ILD − − NA NA Absent Positive‡¶ NA 03 15
P18 DM − − NA NA Present Positive‡ NA 03 13
P19 Antisynthetase syndrome/ILD NA NA NA NA Present Positive‡§¶ Positive‡ 01 11
P20 DM/ILD NA NA NA NA Present Negative‡§¶ Negative‡ 03 15
P21 DM NA NA NA NA Absent Negative‡§¶ Negative‡ 04 07
P22 PM NA NA NA NA Present Negative‡§¶ Negative‡ 01 16
P23 DM/ILD NA NA NA NA Absent Negative‡ Negative‡ 03 04
P24 PM/antisynthetase syndrome NA NA NA NA Present Positive‡¶ Positive‡ 03 03
* HisRS11–23 = histidyl–transferase RNA synthetase peptide 11–23; PM = polymyositis; ILD = interstitial lung disease; NA = not available; DM = dermatomyositis.
† In peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage (BAL) fluid cells, + = low reactivity, ++ = medium reactivity, +++ = high reactivity, and − = nonreactive.
‡ Detected by enzyme-­linked immunosorbent assay.
§ Detected by immunoprecipitation.
¶ Detected by line blotting.
# Analysis of BAL fluid cells with short-­term stimulation (1 day), and in low-­viability cells with long-­term stimulation (5 days).
|
181
182 | GALINDO-­FERIA ET AL
From 6 of these patients, paired BAL fluid and BAL fluid
cell pellets were obtained by bronchoscopy (from 3 anti–Jo-­1+
patients and 3 anti–Jo-­1− patients [patients P1–P6 in Table 1])
(29). Additional cell-­depleted BAL fluid samples were obtained
from 4 anti–Jo-­ 1+ patients with IIM/antisynthetase syndrome
(1 male and 3 female) and 4 anti–Jo-­1− patients (1 male and 3
female) (patients P8 and P9 and P19–P24 in Table 1). All samples
were stored at −80°C at the Human Laboratory Area of the Center
for Molecular Medicine (Solna, Stockholm, Sweden). In addition,
serum samples were obtained from anti–Jo-­1+ patients with IIM/
antisynthetase syndrome (n = 17; 6 male and 11 female) and anti–
Jo-­1− patients (n = 7; 1 male and 6 female) (patients P1–P24 in
Table 1).
Patients included in the experiments were clustered in groups
according to the availability of samples. Group 1 included patients
with available PBMCs, BAL fluid, BAL fluid cell pellets, and paired
serum samples (patients P1–P6). Group 2 included patients with
available PBMCs and paired serum samples (patients P7–P18).
Group 3 included patients with serum samples and cell-­depleted
BAL fluid samples (patients P8 and P9 and P19–P24). T cell
experiments included patients from group 1 and group 2. Anti–
Jo-­1 antibody experiments included patients from group 1 and
group 3. For technical reasons (low cell numbers, low cell viability
of BAL fluid cells, or low volume of BAL fluid), some samples were
excluded. Details regarding the distribution of patients accord-
ing to the type of analysis, degree of T cell reactivity, presence or
absence of muscle inflammatory infiltrates, antibody status, and
HLA genotyping are provided in Table 1.
Lung tissue specimens from patients with IIM/antisynthetase
syndrome (n = 14; 4 anti–Jo-­1+ and 10 anti–Jo-­1−) and from
comparator cases (patients with chronic obstructive lung disease
[COPD]; n = 10) were retrieved from regional biobanks.
Age, sex, smoking status, diagnosis, disease duration from
onset of symptoms, clinical manifestations, anti–Jo-­1 antibody
status, and treatment at the time of blood/BAL fluid sampling
are summarized in Table 1 and Supplementary Table 1 (available
on the Arthritis & Rheumatology web site at http://onlin​elibr​ary.
wiley.com/doi/10.1002/art.41075/​abstract). Features of the mus-
cle biopsy specimens obtained at the time of disease onset from
patients with IIM/antisynthetase syndrome, as detailed in pathol-
ogy reports, are summarized in Supplementary Table 2 (http://
onlin​elibr​ary.wiley.com/doi/10.1002/art.41075/​abstract).
Control groups. For the disease control T cell experi-
ments, paired PBMCs and BAL fluid cell pellets were retrieved
from patients with sarcoidosis (n = 7; 6 male and 1 female)
(patients Sarc1–Sarc7 in Supplementary Table 3, avail­able at
http://onlin​elibr​ary.wiley.com/doi/10.1002/art.41075/​abstract),
since this disease is characterized by the presence of auto-
immune features along with ILD, and is associated with HLA–
DRB1*03:01. For detection of anti–Jo-­ 1 autoantibodies,
additional control patients with sarcoidosis and with avail­
able paired serum samples and cell-­depleted BAL fluid were
collected (n = 9; 4 male and 5 female). All samples were pro-
vided by the Respiratory Medicine Unit, according to a published
protocol (29). Descriptions of the lung and lymph node biopsy
samples from patients with sarcoidosis, as detailed in pathology
reports, are presented in Supplementary Table 4 (available at
http://onlin​elibr​ary.wiley.com/doi/10.1002/art.41075/​abstract).
For the healthy control T cell experiments, fresh PBMCs were
isolated from buffy coats and were obtained from healthy donors
who expressed at least one copy of the HLA–DRB1*03 allele
(n = 12; 3 male and 9 female). Blood samples from healthy sub-
jects were provided by Uppsala Bioresource at Uppsala University
Hospital in Sweden. For the autoantibody analysis, additional paired
serum and cell-­depleted BAL fluid from healthy controls (n = 18; 8
male and 10 female) were provided by the Respiratory Medicine
Unit.
Ethics. The study was approved by the Regional Ethics
Committee of Stockholm at Karolinska Institutet and Uppsala
University, and performed in accordance with the Declara-
tion of Helsinki guidelines on studies with human subjects. All
­subjects provided written informed consent prior to any study
procedure.
Functional T cell assay. Fresh samples were used for all
analyses. PBMCs were obtained from heparinized blood and
were prepared by centrifugation over Ficoll-­Hypaque gradi-
ents. PBMCs were resuspended in 96-­well plates in RPMI 1640
medium supplemented with 10% heat-­inactivated fetal bovine
serum (1 × 106 cells/well) according to an adapted protocol
(30). Cells were incubated for 5 days with recombinant human
full-­length HisRS (20 μg/ml; Bio Supply), with the candidate
peptide HisRS11–23 (20 μg/ml, sequence VKLQGERVRGLKQ;
GenScript) or with RPMI medium as a negative control.
Cells were restimulated on day 5 for 8 hours, and brefeldin
A (10 μg/ml; Sigma-­Aldrich) was added for the last 6 hours.
Stimulation with staphylococcal enterotoxin B (10 μg/ml,
Sigma-­Aldrich) was used as a positive control.
Following stimulation, cells were stained with Live/Dead
Fixable Green Dead Cell Stain as well as with antibodies toward
CD14, CD3, CD4, CD40L, interferon-­γ (IFNγ), interleukin-­2 ­(IL-­2),
and IL-­17A. In some experiments, blocking antibodies for HLA–
DR or HLA–DQ or staining with anti-­ CXCR3, anti-­ CCR5, and
anti-­CCR6 antibodies were used. Details on the antibodies used
are specified in Supplementary Materials and Methods and Sup-
plementary Table 5 (available at http://onlin​ elibr​
ary.wiley.com/
doi/10.1002/art.41075/​abstract).
Flow cytometry. Cells were run on a Gallios Analyzer
(Beckman Coulter), and data were analyzed using FlowJo soft-
ware, version 7.5.1 (Tree Star). The gating strategy is specified
in Supplementary Materials and Methods and Supplementary
ANTIGEN-­SPECIFIC T CELLS IN INFLAMMATORY MYOPATHIES
Figure 1 (available at http://onlin​elibr​ary.wiley.com/doi/10.1002/
art.41075/​abstract).
Identification of the HisRS T cell epitope. The predic-
tion of a CD4+ T cell epitope was based on the crystal struc-
ture of the HLA–DRB1*03:01 molecule in complex with class
II–­associated invariant chain peptide, which reveals the binding
pockets of HLA and how the anchor residues are docking into
HLA (31). Prior motif studies established that HLA–DRB1*03:01
epitopes most commonly contain an aliphatic residue in position
1, an acidic or polar residue in position 4, and either a basic
residue at position 6 paired with a small residue at position 9
(submotif 1) or a nonbasic residue at position 6 paired with a
large residue at position 9 (submotif 2) (32). In this way, a theo-
retical prediction of CD4+ T cell epitopes can be made from any
antigen. In our case, the sequence of the first 60 amino acids of
HisRS (UniProtKB identifier P12081) was examined to predict
peptides that can bind DRB1*03:01; residues 11–23 (HisRS11–23)
contained 2 such motifs and therefore had a high probability of
containing a bona fide DRB1*03:01 epitope. Thus, HisRS11–23
peptide was selected for further functional assays (Figure 1A).
BAL fluid and serum samples for detection of anti–Jo-­1
autoantibodies. Anti–Jo-­1 IgG and total IgG were measured by
enzyme-­linked immunosorbent assay (ELISA) in BAL fluid and serum
from patients with IIM/antisynthetase syndrome, control patients
with sarcoidosis, and healthy controls. Details on the protocol
used are presented in Supplementary Materials and Methods
(http://onlin​e​libr​ary.wiley.com/doi/10.1002/art.41075/​abstract).
Immunohistochemistry and histopathology. Paraffin-­
embedded lung tissue from patients with IIM/antisynthetase
syndrome and patients with COPD were analyzed by immuno-
histochemistry according to a previously published protocol (33).
The primary antibodies used were anti-­ CD3, anti-­
CD138, anti-­
CXCR3, and anti-­CCR5 or the isotype controls IgG, IgG2b, and
IgG1. Details on the protocol and antibodies used for ELISAs and
immunohistochemistry are specified in Supplementary Materials
and Methods, Supplementary Table 6, and Supplementary Figure
2 (available at http://onlin​elibr​ary.wiley.com/doi/10.1002/art.41075/​
abstract). Whole sections were evaluated independently by 2 inves-
tigators (AGF and CFC) on coded slides by semiquantitative scoring
for the extent of immunohistochemical staining on a 4-­point scale,
where 0 = none, 1 = low, 2 = intermediate, and 3 = high amount
of staining. Results are expressed as the mean of 2 observations.
Statistical analysis. Continuous variables are expressed as
the median with interquartile range (IQR; 25th–75th percentiles) or
minimum and maximum values. Categorical variables are presented
as frequencies and percentages. Differences between groups were
analyzed using Mann-­Whitney U test. For comparison of the per-
centage of CD40L up-­regulation between different stimulation con-
| 183
ditions, Wilcoxon’s signed-­rank test was used. Two-­tailed P values
less than 0.05 were considered significant. Up-­regulated expression
of CD40L in T cells was normalized to the value in unstimulated cells,
and presented as the median fold change according to the following
categories: low (<3-­fold), intermediate (3–10-­fold), or high (>10-­fold).
The analyses were performed using Prism 5 software (Graph Pad)
and IBM SPSS Statistics, version 25.
RESULTS
Clinical characteristics of the patients and controls.
A total of 17 anti–Jo-­1+ and 7 anti–Jo-­1− patients with IIM/anti-
synthetase syndrome were included in our analyses (Table 1). The
anti–Jo-­1+ patients (median age 55 years [IQR 53–55 years]) had a
longer disease duration and higher frequency of ILD compared to
the anti–Jo-­1− patients (median age 57 years [IQR 54–57 years])
(P = 0.033 and P = 0.021, respectively). There was no difference
in sex, age, or smoking status between the 2 groups of patients
(P = 0.625, P = 0.425, and P = 0.724, respectively). A compari-
son of the characteristics of the 2 groups of patients is presented
in Supplementary Table 7 (available at http://onlin​elibr​ary.wiley.com/
doi/10.1002/art.41075/​abstract).
Group 1 (T cell group, patients P1–P6) had a median dis-
ease duration of 5 months (IQR 1–48 months). Five patients in
this group were untreated and 1 patient was receiving immu-
nosuppressive treatment. Group 2 (T cell group, patients P7–
P18) had a median disease duration of 64 months (IQR 1–193
months), and 9 of 12 patients were receiving immunosuppres-
sive treatment. Group 3 (antibody group, patients P19–P24) had
a median disease duration of 13 months (IQR 4–60 months),
and 4 of 6 patients were receiving immunosuppressive treat-
ment (see Supplementary Table 1 [http://onlin​elibr​ary.wiley.com/
doi/10.1002/art.41075/​abstract]).
The control patients with sarcoidosis and healthy controls
assessed in T cell and antibody analyses had a median age of
55 years (IQR 43–62 years) and 33 years (IQR 28–49 years),
respectively. Detailed clinical information on the patients with
sarcoidosis is presented in Supplementary Table 3 (http://onlin​e​
libr​ary.wiley.com/doi/10.1002/art.41075/​abstract).
Presence of HisRS-­T cell reactivity in the peripheral
blood of patients with IIM/antisynthetase syndrome. To
identify the potential presence of antigen-­specific T cells against
HisRS in the blood of patients with IIM/antisynthetase syndrome, we
assessed the up-­regulation of CD40L in CD4+ T cells following stim-
ulation with HisRS protein or HisRS11–23 in patients from group 1 and
group 2. After stimulation of PBMCs with HisRS protein, we iden-
tified up-­regulation of CD40L on CD4+ T cells in 14 of 18 patients
with IIM/antisynthetase syndrome, with a median fold change of
7.38 (IQR 2.69–31.86) relative to unstimulated cells (P = 0.0002).
High (>10-­fold) up-­regulation of CD40L in CD4+ T cells fol-
lowing stimulation with the HisRS protein was observed in 6 of 14
184 | GALINDO-­FERIA ET AL

Figure 1. Anti–histidyl–transfer RNA synthetase (HisRS)–specific T cells are enriched in the blood of patients with idiopathic inflammatory
myopathy (IIM)/antisynthetase syndrome. A, A 13-­mer peptide within the first 60 amino acids of HisRS was predicted to represent a potential
T cell epitope, HisRS11–23. B, Representative plots from fluorescence-­activated cell sorter analysis (gated on lymphocytes and CD4+CD3+ T
cells, with exclusion of dead cells and monocytes) show up-­regulation of CD40L in CD4+ T cells as well as production of interferon-­γ (IFNγ),
interleukin-­2 (IL-­2), and IL-­17 on peripheral blood mononuclear cells (PBMCs) from an anti–Jo-­1+ patient with IIM/antisynthetase syndrome,
in unstimulated (un-­stim) conditions or after stimulation with full-­length HisRS protein or HisRS11–23. C–E, Up-­regulation of CD40L expression
was examined on CD4+ T cells in PBMCs from anti–Jo-­1+ patients with IIM/antisynthetase syndrome (n = 18) (patients P1–P18 in Table 1)
(C), patients with sarcoidosis (n = 6) (D), and HLA–DRB1*03–positive healthy controls (HCs) (n = 12) (E). Solid symbols in C–E represent the
median fold change in PBMCs from these groups, while gray-­shaded symbols represent the median fold change in PBMCs from anti–Jo-­
1− patients (C), patients with sardoidosis who showed reactivity toward HisRS protein and HisRS11–23 (D), and a healthy control subject who
showed reactivity to HisRS protein (E). Categories of fold change were low (<3-­fold), intermediate (3–10-­fold), or high (>10-­fold). Cells were
left unstimulated or stimulated with full-­length HisRS protein, HisRS11–23, or staphylococcal enterotoxin B (SEB) as a positive control. F, IFNγ
production was assessed on CD40L+CD4+ T cells in PBMCs from patients with IIM/antisynthetase syndrome. Solid symbols represent anti–
Jo-­1+ patients and gray-­shaded symbols represent anti–Jo-­1− patients; horizontal lines show the median. Cells were left unstimulated or
stimulated with full-­length HisRS recombinant protein, HisRS11–23, or SEB. * = P < 0.05 by 2-­tailed Wilcoxon’s matched-­pairs signed rank test.

patients with IIM/antisynthetase syndrome (5 anti–­Jo-­1+ and 1
anti–Jo-­1−), with a median fold change of 35 (IQR 22.76–48.53)
relative to unstimulated cells (Figures 1B and C). The highest
responses were present in the PBMCs from patients with IIM/
antisynthetase syndrome, specifically those who expressed at
least one copy of the HLA–DRB1*03 allele and were anti–Jo-­1+
as compared to anti–Jo-­1+ patients without the HLA–DRB1*03
allele (mean fold change in CD40L expression relative to unstim-
ulated cells 16.05 versus 7.57; P not significant) (see Supple-
mentary Figures 3A and C, available at http://onlin​elibr​ary.wiley.
com/doi/10.1002/art.41075/​abstract).
After stimulation of PBMCs with HisRS11–23, we detected
­up-­regulation of CD40L on CD4+ T cells in 11 of 14 patients with
IIM/antisynthetase syndrome, with a median fold change of 3.4
(IQR 1.87–10.9) relative to unstimulated cells (P = 0.0012). The
highest response toward HisRS11–23 (>10-­ fold change versus
unstimulated cells) was observed in 3 of 11 patients with IIM/
antisynthetase syndrome (2 anti–Jo-­1+ and 1 anti–Jo-­1−), with
a median fold change of 12.75 (IQR 10.9–37) (Figures 1B and C).
In the sarcoidosis group, up-­ regulation of CD40L was
detected in PBMCs from 2 of 7 patients after stimulation
with HisRS protein, with a median fold change of 5.08 (IQR
ANTIGEN-­SPECIFIC T CELLS IN INFLAMMATORY MYOPATHIES | 185

A B Anti DR/DQ block

100

% CD40L upregulation
50

0
HisRS anti DQ anti DR
[11-23]

Figure 2. HisRS reactivity is restricted to HLA–DR. A, Representative plots from fluorescence-­activated cell sorter analysis show up-­regulation
of CD40L on CD4+ T cells in PBMCs from anti–Jo-­1+ patients with IIM/antisynthetase syndrome after stimulation with HisRS11–23 compared
to unstimulated cells. This up-­regulation was abolished in the presence of anti–HLA–DR but not in the presence of anti–HLA–DQ blocking
antibodies. B, Effects of anti–HLA–DQ and anti–HLA–DR blocking antibodies on CD40L up-­regulation by HisRS11–23 were quantified in PBMCs
from anti–Jo-­1+ patients with IIM/antisynthetase syndrome (n = 2; patients P2 and P12 in Table 1). Bars show the median percentage change
(with upper limit of maximum range) relative to HisRS11–23 (set at 100%). See Figure 1 for definitions.

3.7–6.47) relative to unstimulated cells, and detected in 3 of 7
patients following stimulation with HisRS11–23, with a median fold
change of 2.09 (IQR 1.45–3.29) relative to unstimulated cells
(Figure 1D). The HisRS-­reactive patients with sarcoidosis were
positive for either HLA–DRB1*04/*15 or HLA–DRB1*01/*03 and
were former smokers (see Supplementary Table 3 [http://onlin​e​
libr​ary.wiley.com/doi/10.1002/art.41075/​abstract]).
In the healthy control group who were positive for HLA–
DRB1*03, up-­regulation of CD40L was observed in PBMCs stim-
ulated with HisRS protein in 1 of 12 subjects (5-­fold change relative
to unstimulated cells). After stimulation of PBMCs with HisRS11–23,
up-­regulation of CD40L was observed in 5 of 12 healthy controls,
with a median fold change of 2 (IQR 1.89–2.42) relative to unstim-
ulated cells (P = 0.06) (Figure 1E).
We next evaluated the proinflammatory effector functions of
HisRS-­specific T cells by examining cytokine production within the
CD4+CD40L+ T cell population. The HisRS-­specific CD4+CD40L+
T cells in peripheral blood mainly produced IL-­2 and IFNγ. In the
patients with IIM/antisynthetase syndrome, a median of 12% (IQR
5.39–25.6%) of CD4+CD40L+ T cells produced IFNγ after antigenic
stimulation with HisRS protein, and a median of 4% (IQR 2–18%)
produced IFNγ after stimulation with HisRS11–23 (P = 0.0084 and
P = 0.02 respectively, compared to unstimulated cells) (Figure 1F).
To confirm an HLA–DR dependency of the observed T cell
responses, PBMCs from 2 anti–Jo-­1+ patients were stimulated
with HisRS11–23 in the presence of anti–HLA–DR or anti–HLA–DQ
blocking antibodies. Anti–HLA–DR blocking efficiently abrogated
the up-­regulation of CD40L upon stimulation with HisRS11–23, while
anti–HLA–DQ blocking did not (Figures 2A and B), thereby indicat-
ing that the HisRS11–23 epitope is presented by HLA–DR.
Presence of HisRS-­specific T cells in the lungs of
patients with IIM/antisynthetase syndrome display a
­pronounced Th1 phenotype as compared to PBMCs. To
investigate whether the presence and functionality of antigen-­
specific CD4+ T cells may vary in different compartments, we
selected BAL fluid cells and corresponding PBMCs from anti–­
Jo-­ 1+ patients with IIM/antisynthetase syndrome (n = 2) and
anti–Jo-­1− patients (n = 2) (group 1, patients P2–P5 [Table 1])
and patients with sarcoidosis (n = 7) (see Supplementary Table
3 [http://onlin​elibr​ary.wiley.com/doi/10.1002/art.41075/​abstract]),
and stimulated the cells with either HisRS protein or HisRS11–23.
A CD40L response of BAL fluid cells stimulated with HisRS full-­
length protein was present in 3 of 4 patients with IIM/antisynthetase
syndrome (patients P2, P3, and P5), with a median fold change in
CD40L expression of 3.6 (IQR 2.68–14.7) relative to unstimulated
cells. The corresponding CD4+ T cells from PBMCs had a similar
up-­regulation of CD40L in response to the HisRS protein in all 4
patients tested (patients P2–P5), with a median fold change of 4.58
(IQR 3.25–25.36) relative to unstimulated cells (Figures 3A and B).
When the response to HisRS11–23 stimulation was analyzed
in the lung compartment of patients with IIM/antisynthetase syn-
drome, we identified a high CD40L up-­regulation in BAL fluid
cells from 2 of 3 patients (patients P2 and P3) when compared
to unstimulated conditions, with a median fold change of 88 (IQR
27–149). Furthermore, the CD40L responses in PBMCs were also
high in all 3 patients evaluated (patients P2–P4), with a median
fold change of 12.7 (IQR 4.18–37) relative to unstimuated cells.
Moreover, the highest frequencies of CD4+CD40L+ T cells
were found in BAL fluid after stimulation with HisRS11–23, with a
median fold change of 7.83% (IQR 1.37–29.9%), whereas in
PBMCs, the median fold change was 0.38% (IQR 0.02–5.89%),
suggesting an enrichment of antigen-­specific T cells in the lung
compartment (see Supplementary Table 8, available at http://onlin​e​
libr​ary.wiley.com/doi/10.1002/art.41075/​abstract). In addition, the
highest reactivity to HisRS11–23 was seen in the 2 anti–Jo-­ 1+
patients with IIM/antisynthetase syndrome, whereas BAL fluid
cells from 1 anti–Jo-­1− patient (patient P4) showed no response
upon HisRS11–23 stimulation.
In order to characterize the phenotype of CD4+ T cells in
BAL fluid and PBMCs, we selected unstimulated, paired CD4+
T cells from both compartments, obtained from 3 anti–Jo-­ 1+
186 | GALINDO-­FERIA ET AL

Figure 3. Anti–HisRS–specific T cells are detected in the lungs of patients with IIM/antisynthetase syndrome. A, Representative plots from
fluorescence-­activated cell sorter analysis (gated on lymphocytes and CD4+CD3+ T cells, with exclusion of dead cells and monocytes) show
up-­regulation of CD40L as well as production of IFNγ, IL-­2, and IL-­17 on CD4+ T cells obtained from bronchoalveolar lavage (BAL) fluid
(BALF) from an anti–Jo-­1+ patient with IIM/antisynthetase syndrome. BAL fluid T cells were left unstimulated or stimulated with full-­length
HisRS recombinant protein or HisRS11–23. B and C, CD40L up-­regulation on CD4+ T cells, relative to unstimulated cells, was compared
between BAL fluid cells and corresponding PBMCs from patients with IIM/antisynthetase syndrome (n = 4; patients P2–P5 in Table 1) (B) and
in BAL fluid CD4+ T cells between patients with IIM/antisynthetase syndrome (n = 4) and patients with sarcoidosis (n = 7) (C). Cells were left
unstimulated or stimulated with full-­length HisRS protein, HisRS11–23 peptide, or SEB for 5 days. Solid symbols represent the median fold change
in individual anti–Jo-­1+ patients with IIM/antisynthetase syndrome or patients with sarcoidosis; gray-­shaded symbols represent the median fold
change in anti–Jo-­1− patients. Categories of fold change were low (<3-­fold), intermediate (3–10-­fold), or high (>10-­fold). D, IFNγ production
on CD40L+CD4+ T cells was compared between BAL fluid cells and corresponding PBMCs from anti–Jo-­1+ patients with IIM/antisynthetase
syndrome after stimulation with full-­length HisRS recombinant protein or HisRS11–23. Solid symbols represent anti–Jo-­1+ patients and gray-­
shaded symbols represent anti–Jo-­1− patients; horizontal lines show the median. * = P < 0.05; *** = P < 0.001, by 2-­tailed Mann-Whitney
U test. See Figure 1 for definitions.

patients with IIM/antisynthetase syndrome (patients P1–P3) and 2
anti–Jo-­1− patients (patients P4 and P5). Almost all of the CD4+ T
cells in BAL fluid were positive for the Th1-­associated chemokine
receptors CXCR3 (97%) (Figure 4A) and CCR5 (93%) (Figure 4B),
compared to 46% and 50% of PBMCs, respectively (P < 0.01 and
P < 0.05, respectively). Moreover, ~60% of the BAL fluid T cells
expressed CCR6, compared to 20% of PBMCs (P < 0.01) (Fig-
ure 4C). In BAL fluid, >90% of CCR6+CD4+ T cells were positive
for CXCR3, indicating that CD4+ T cells in BAL fluid had a Th1/
Th17 phenotype in anti–Jo-­1+ and anti–Jo-­1− patients.
Furthermore, HisRS-­specific BAL fluid CD4+CD40L+ T cells
presented a pronounced proinflammatory Th1 phenotype that
was characterized by an increased production of IFNγ, when com-
pared to the corresponding CD4+CD40L+ T cells from PBMCs, in
both anti–Jo-­1+ and anti–Jo-­1− patients. On average, 60% of the
CD4+CD40L+ T cells from BAL fluid produced IFNγ in response to
antigenic stimulation with either HisRS full-­length protein or HisRS11–23,
compared to 10% of the CD4+CD40L+ T cells in the corresponding
blood samples (P < 0.001 and P < 0.05, respectively) (Figure 3D).
In the disease control group of patients with sarcoidosis, BAL
fluid cells from 2 of 7 patients with sarcoidosis (patients Sarc1 and
Sarc4) displayed a high (>10-­fold) CD40L response to the HisRS
protein, with a median fold change of 20 (IQR 15.75–25.5) relative
to unstimulated cells. Two other control patients with sarcoidosis (of
the 7 tested) presented high CD40L up-­regulation in response to
HisRS11–23 (patients Sarc3 and Sarc7), with a median fold change
of 59 (IQR 22.7–95) relative to unstimulated conditions (Figure 3C).
CD40L up-­regulation in BAL fluid cells from the group of patients with
sarcoidosis who responded to HisRS full-­length protein was 4 times
higher compared to that in the corresponding PBMCs (Figure 1D
ANTIGEN-­SPECIFIC T CELLS IN INFLAMMATORY MYOPATHIES | 187

Figure 4. Th1 cells and anti–Jo-­1 antibodies are present in the lungs of patients with IIM/antisynthetase syndrome. A–C, Representative plots
from fluorescence-­activated cell sorter analysis (bottom) and quantification of the results (top) show the percentage of CD4+CD3+CXCR3+ T cells
(A), CD4+CD3+CCR5+ T cells (B), and CD4+CD3+CXCR3+CCR6+ T cells (C) in PBMCs and bronchoalveolar lavage (BAL) fluid (BALF) cells from
patients with IIM/antisynthetase syndrome (n = 5). D and E, Presence of anti–Jo-­1 autoantibodies was analyzed by enzyme-­linked immunosorbent
assay (ELISA) in the BAL fluid (D) and corresponding serum (E) of patients with IIM/antisynthetase syndrome, patients with sarcoidosis, and healthy
controls. The broken horizontal line indicates the cutoff value for positivity, calculated using the values in healthy controls (mean absorbance in healthy
controls + 3SD). Results are expressed as the normalized (Norm.) values for the absorbance at 405 nm (optical density ratio of anti–Jo-­1 IgG to total
IgG in the BAL fluid and serum). Symbols in A–E show individual subjects; horizontal lines show the mean. F, Presence of anti–Jo-­1 IgG autoantibodies
was compared between the BAL fluid and serum of anti–Jo-­1+ (n = 6) and anti–Jo-­1− (n = 7) patients with IIM/antisynthetase syndrome, assessed by
ELISA with values normalized to the total IgG levels in corresponding compartments. Patients were classified as anti–Jo-­1+ if they were seropositive
by at least one method (ELISA, immunoprecipitation, or line blotting, as described in Table 1). The broken horizontal line indicates the cutoff value for
seropositivity. * = P < 0.05; ** = P < 0.01, by 2-­tailed Mann-Whitney U test. See Figure 1 for definitions.

and Figure 3C). The frequencies of CD4+CD40L+ T cells in patients
with IIM/antisynthetase syndrome, control patients with sarcoidosis,
and healthy controls are presented in Supplementary Table 8 (http://
onlin​elibr​ary.wiley.com/doi/10.1002/art.41075/​abstract).
Anti–Jo-­1 autoantibodies in BAL fluid. To further dis-
sect the HisRS-­specific immune response in the lung compart-
ment, cell-­depleted BAL fluid samples from patients with IIM/
antisynthetase syndrome (n = 13), corresponding to group 1
and group 3 (patients P1–P5 in group 1, and patients P8 and
P9 and P19–P24 in group 3), patients with sarcoidosis (n = 9),
and healthy controls (n = 18) were analyzed for the presence of
anti–Jo-­1 autoantibodies. We detected anti–Jo-­1 IgG autoan-
tibodies in BAL fluid from 6 of 7 anti–Jo-­1+ patients and in
2 of 9 patients with sarcoidosis, but not in healthy controls or in
anti–Jo-­1− individuals (Figure 4D). After normalizing the amount
of anti–Jo-­1 autoantibodies to the values for total IgG, all of the
anti–Jo-­1+ patients who showed reactivity in the BAL fluid were
also reactive in the serum (Figures 4E and F).
Infiltration of lung tissue by T cells and plasma cells
in patients with IIM/antisynthetase syndrome. Germinal
center (GC)–like structures characterized by T cells and sur-
rounding plasma cells were observed in the lung tissue from 2
of 4 anti–Jo-­1+ patients with IIM/antisynthetase s­ yndrome but
in none of the lung tissue samples from anti–Jo-­1− patients
188 | GALINDO-­FERIA ET AL

Figure 5. Histologic examination of the lungs of a representative anti–Jo-­1+ patient with idiopathic inflammatory myopathy/antisynthetase
syndrome and interstitial lung disease. A, Immunohistochemical analysis of the lung tissue sections reveals T cell infiltrates (CD3 staining) (upper
panels) and infiltration of plasmablasts (CD138 staining) (lower panels). Boxed areas in the left panels are shown at higher magnification on
the right. Original magnification × 10 on left; × 25 on right. B, Immunohistochemical staining of the lung tissue shows several germinal center–
like formations on CD3 staining (left and upper right panels) and CD138 staining (lower right panel). Boxed area in the left panel is shown at
higher magnification on the upper right. Original magnification × 5 on left; × 10 on right. Isotype controls for CD3, CCR5, and CXCR3 with
semiquantitative analysis are presented in Supplementary Figure 2 (http://onlinelibrary.wiley.com/doi/10.1002/art.41075/abstract).

or patients with COPD (Figures 5A and B). In addition,
we observed infiltrating CD3+ T cells in all patient groups
(patients with IIM/antisynthetase syndrome and patients with
COPD), with a higher number of infiltrating CD3+ T cells in
patients with COPD than in patients with IIM/antisynthetase syn-
drome (P = 0.009). No differences between groups were seen
for CD138+ plasma cell expression (P = 0.37) (see Supplemen-
tary Figures 2A and C, available at http://onlinelibrary.wiley.com/
doi/10.1002/art.41075/abstract). There was no difference in the
expression of CXCR3 and CCR5 between anti–Jo-­1+ and anti–
Jo-­1− patients (staining not performed in lung biopsy samples
from patients with COPD). Transbronchial lung biopsy samples
from patients with sarcoidosis did not show the presence of GC
formation (see Supplementary Table 4).
ANTIGEN-­SPECIFIC T CELLS IN INFLAMMATORY MYOPATHIES
DISCUSSION
In this study, we describe activation of antigen-­specific CD4+
T cells in peripheral blood and BAL fluid cells from patients with
IIM/antisynthetase syndrome by an HLA–DRB1*03-­associated
T cell epitope corresponding to the stretch of residues 11–23
within the HisRS protein. The stimulation of CD4+ T cells with the
full-­length HisRS protein and the HisRS11–23 peptide induced up-­
regulation of CD40L in CD4+ T cells in both the lung and blood
compartments, indicating the presence of HisRS-­specific T cells
in patients with IIM/antisynthetase syndrome.
To our knowledge, the phenotype of T cells involved in
the immune reactivity toward a specific peptide in IIM/antisyn-
thetase syndrome has not been previously addressed. The
present description of T cell reactivity is directed against a
candidate HisRS-­derived peptide (HisRS11–23) that we selected
based on binding predictions to HLA–DRB1*03:01. The high
reactivity observed in patients with IIM/antisynthetase syn-
drome compared to patients with sarcoidosis and healthy
controls indicates that the HisRS11–23 epitope is an important
target for autoreactive T cells in IIM/antisynthetase syndrome.
Furthermore, our results demonstrate that the activation of
HisRS-­specific T cells was abrogated by blocking HLA–DR
but not by blocking HLA–DQ. We thus demonstrate a T cell
activation bias in HLA–DRB1*03:01–positive patients toward
HisRS11–23. These findings confirm previous results demon-
strating T cell proliferation following stimulation with substan-
tially larger fragments of HisRS in T cell cultures (22).
Nevertheless, the observed T cell activation was not entirely
limited to the anti–Jo-­ 1+ subset of patients with IIM/antisyn-
thetase syndrome, but occurred in some anti–Jo-­1− patients,
although to a substantially lower degree. While the genetic asso-
ciation between IIM with anti–Jo-­1 antibody positivity and HLA–
DRB1*0301 is well established, we find it interesting that patients
with other class II major histocompatibility complex alleles could
also be implicated in the presentation of HisRS-­derived peptides.
Our findings of an increased reactivity against the HisRS
protein, and in particular the HisRS-­derived peptide HisRS11–23 in
lung-­derived T cells compared to blood-­derived cells, are, in our
opinion, striking, and despite the limited number of cases with BAL
fluid–derived T cells, this observation may point to the lung as a
potential site of primary activation of T cells against HisRS, similar
to what has been proposed in other autoimmune diseases such
as RA (8,9,34) and MS (12). We also identified the presence of
anti–Jo-­1 autoantibodies in BAL fluid as well as GC-­like structures
in lung tissue from anti–Jo-­1+ patients, supporting the hypothesis
of the lungs as a potential site for immune activation and anti–Jo-­1
autoantibody production. As some patients displayed reactivity to
the HisRS full-­length protein but not to the HisRS11–23 epitope,
other HisRS-­derived peptides are likely to be implicated.
Interestingly, 2 patients with sarcoidosis with the highest
HisRS T cell reactivity were former smokers, indicating a possible
| 189
gene–environment interaction. In this context, the previously
described higher expression of HisRS in lung tissue compared
to other types of tissue is interesting (20). Also, a granzyme
B–sensitive form of HisRS has been identified exclusively in the
lung (21). Therefore, increased amounts of granzyme B triggered
by proinflammatory environmental stimuli, such as infections or
smoke exposure (35), may contribute to the release of N-­terminal
HisRS fragments and the formation and enhanced processing of
neoepitopes that can be recognized by HisRS-reactive T cells in
patients with IIM/antisynthetase syndrome (21,36).
The N-­ terminal HisRS fragments have chemotactic
properties that can induce migration of CCR5-­ expressing
lymphocytes, activated monocytes, and immature dendritic
cells (23). The aforementioned mechanism suggests that
the HisRS protein or fragments released from damaged
lungs may be involved in the recruitment of activated T cells,
including HisRS-­activated cells to inflammatory sites in the
lungs, thereby providing costimulatory signals to B cells and
antigen-­presenting cells. In this hypothesis, the muscle might
become targeted in later stages of the disease (20).
Consistent with previous reports (37–40), the present study
demonstrated high expression of the chemokine receptors CCR5
and CXCR3 (markers of Th1 cells) and CCR6 (Th17 marker) in
nearly 100% of CD4+ T cells isolated from the BAL fluid of both
anti–Jo-­1+ and anti–Jo-­ 1− patients. The enriched expression
of these chemokine receptors in BAL fluid T cells compared to
peripheral blood T cells was not unexpected, as several studies
have shown that these receptors are known to regulate antigen-­
induced T cell homing in lung diseases, such as asthma (41,42),
COPD (43), and sarcoidosis (44), and can even be found in
healthy smokers (45). Moreover, the identification of a Th1/Th17
phenotype (Th17.1 cells) in BAL fluid from patients with IIM/anti-
synthetase syndrome, characterized by an excessive secretion of
IFNγ, has also been reported in patients with chronic sarcoidosis
(38,46,47) and in other autoimmune diseases such as Crohn’s
disease (48) and arthritis (49).
The design of the present study has a number of limitations.
One limitation was the low availability of paired samples from
BAL fluid and PBMCs from untreated patients with inflammatory
active IIM/antisynthetase syndrome. Acquiring BAL fluid sam-
ples requires an invasive procedure, and it may be unethical to
delay the start of treatment until the bronchoscopy can be per-
formed. Nevertheless, the results obtained from this small num-
ber of patients were consistent, contributing to the significance
of our observations.
In conclusion, the results of the present study indicate that
specific HisRS-­derived peptides in HLA–DRB1*03–positive indi-
viduals, including the HisRS11–23 peptide investigated herein, may
be presented to locally available T cells, which become activated
and subsequently adopt a proinflammatory phenotype. These
T cells may promote B cell maturation and activation, GC-­like
structure formation, and subsequent production of anti–Jo-­
­ 1
190 | GALINDO-­FERIA ET AL
autoantibodies. One possible site for the initial activation of the
immune system could be in the lungs, where environmental
factors such as smoking or infections may cause an unspecific
inflammation in the lung tissue, leading to granzyme B release
and HisRS cleavage that may then lead to the recruitment of T
cells. Further studies on other relevant peptides with a broader
coverage of the antigenic sites of HisRS, as well as studies on
the phenotypes of locally infiltrating T cells, in patients with
IIM/antisynthetase syndrome remain a priority.
ACKNOWLEDGMENTS
We thank Eva Lindroos and Yvonne Sundström for their
excellent technical assistance, as well as nurse Christina
Ottosson for collecting blood samples, Julia Norkko and Gloria
Rostvall for handling serum samples, and Benita Dahlberg for
handling BAL fluid samples. We also thank Barbro Larsson and
Leonid Padyukov for the genotyping of the samples, as well
as Karine Chemin for assistance with the T cell assays, and
Karolina Tandre for providing blood samples from healthy
controls. Finally, we thank Associate Professor Inger Nennesmo
for providing the muscle pathology reports, and Karine
Chemin, Valérie Leclair, and Lars Klareskog for constructive
criticism of the manuscript.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it criti-
cally for important intellectual content, and all authors approved the final
­version to be published. Dr. Galindo-­Feria had full access to all of the
data in the study and takes responsibility for the integrity of the data and
the accuracy of the data analysis.
Study conception and design. Galindo-­ Feria, Albrecht, Fernandes-­
Cerqueira, Notarnicola, James, Dastmalchi, Rönnblom, Jakobsson, Fathi,
Grunewald, Malmström, Lundberg.
Acquisition of data. Galindo-­ Feria, Albrecht, Fernandes-­ Cerqueira,
Notarnicola, Herrath, Dastmalchi, Fathi, Lundberg.
Analysis and interpretation of data. Galindo-­Feria, Albrecht, Fernandes-­
Cerqueira, Notarnicola, James, Dastmalchi, Sandalova, Fathi, Achour,
Grunewald, Malmström, Lundberg.
ADDITIONAL DISCLOSURES
Author Albrecht is currently an employee of Sanofi Genzyme. Author
Herrath is currently an employee of Pfizer.
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