Human MAIT Cells Are Devoid of Alloreactive Potential: Prompting Their Use As Universal Cells For Adoptive Immune Therapy

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1 Human MAIT cells are devoid of alloreactive potential:
2 prompting their use as universal cells for adoptive immune therapy.
3
4
5 Marie Tourret1*, Nana Talvard-Balland1*, Marion Lambert1*, Ghada Ben Youssef1, Mathieu
6 F. Chevalier1, Armelle Bohineust1, Thomas Yvorra2, Florence Morin3, Saba Azarnoush4,
7 Olivier Lantz5,6,7, Jean-Hugues Dalle1,4 and Sophie Caillat-Zucman1,3.
8
9 Original article
10
11 1- Institut national de la santé et de la recherche médicale (INSERM) UMR976, Human
12 Immunology, Pathophysiology and Immunotherapy, Université de Paris, Paris, France
13 2- INSERM UMR3666/U1143, Université PSL, Institut Curie, Paris, France
14 3- Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris (AP-HP), Université de Paris,
15 Laboratoire d’Immunologie, Paris, France
16 4- Hôpital Robert Debré, AP-HP, Université de Paris, Département d’Immuno-Hématologie,
17 Paris, France
18 5- INSERM U932, Université PSL, Institut Curie, Paris, France.
19 6- Institut Curie, Laboratoire d'immunologie clinique, Paris, France.
20 7- Institut Curie, Centre d'investigation Clinique en Biothérapie (CIC-BT1428), Paris, France
21
22 * M.T, N.T-B and M.L contributed equally to this work.
23
24 Correspondence to :
25 Sophie Caillat-Zucman, Laboratoire d’Immunologie, Hôpital Saint-Louis,
26 1 Avenue Claude Vellefaux, 75010, Paris, France
27 Phone: 33-1-42 49 90 81
28 Fax: 33-1-42 38 52 47
29 E-mail: sophie.caillat-zucman@aphp.fr
30
31 Running title: MAIT cells and allogeneic response
32
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
1
medRxiv preprint doi: https://doi.org/10.1101/2021.04.29.21256184; this version posted April 30, 2021. The copyright holder for this preprint
1 Keywords
2 Mucosal associated invariant T (MAIT) cells; allogeneic response; adoptive immune therapy;
3 graft-versus-host disease.
5 Declarations
7 Ethics approval and consent to participate
8 The study was carried out with the approval of the Robert Debré Hospital Ethics Committee
9 (HREC 2013/49) and the CPP Ile de France IV (2015/03NICB), in agreement with the
10 principles of the Declaration of Helsinki and French legislation. All subjects (or their parents
11 for the children cohort) provided written informed consent. The study was registered in a public
12 trial registry: ClinicalTrials.gov number NCT02403089.
13 Mice were used after approval of all procedures by the Institutional animal Care and Use Ethics
14 Committee (CE121#16624).
15
16 Availability of data:
17 Data are available on reasonable request to sophie.caillat-zucman@aphp.fr
18
19 Competing interests:
20 none declared
21
22 Funding
23 This work was supported by grants from Agence Nationale de la Recherche (ANR) and
24 Direction Générale de l’Offre de Soins (DGOS) (PRTS 14-CE15-0005, acronym NEOMAIT),
25 Agence de la Biomédecine, and Cent pour Sang la vie. N.T-B was supported by Ligue contre
2
1 le Cancer and Fondation pour la Recherche Médicale (FRM). Access to samples from the
2 CRYOSTEM collection was made possible thanks to the financial support of Association
3 Laurette Fugain.
5 Author contributions and consent for publication
6 M. Tourret, N Talvard-Balland, M. Lambert, G. Ben Youssef, M. Chevalier, A. Bohineust, F
7 Morin and S. Azarnoush designed research studies, conducted experiments, analyzed data,
8 and/or provided patient samples. T. Yvorra produced synthetic 5-OP-RU. O Lantz and J-H.
9 Dalle analyzed data and wrote the manuscript. S. Caillat-Zucman designed research studies,
10 supervised the study, analyzed data, and wrote the manuscript with the help of other coauthors.
11 All authors provided input, edited and approved the final version of the manuscript.
12
13
14
15
16 Abbreviations
17 CFSE: Carboxyfluorescein succinimidyl ester; GVHD: graft-versus-host disease; GVL:
18 graft-versus-leukemia; HSCT: hematopoietic stem cell transplantation; MAIT: Mucosal
19 associated invariant T; MA: myeloablative; NMA: non myeloablative; MSD: matched sibling
20 donor; MUD: matched unrelated donor; TCR : T cell receptor; Tconv : conventional T cells;
21 5-OP-RU: 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil.
22
23
24
25
3
1 ABSTRACT
2 Background: Mucosal associated invariant T (MAIT) cells are semi-invariant T cells that
3 recognize microbial antigens presented by the highly conserved MR1 molecule. MAIT cells
4 are predominantly localized in the liver and barrier tissues and are potent effectors of anti-
5 microbial defense. MAIT cells are very few at birth and accumulate gradually over a period of
6 about 6 years during infancy. The cytotoxic potential of MAIT cells, as well as their newly
7 described regulatory and tissue repair functions, open the possibility of exploiting their
8 properties in adoptive therapy. A prerequisite for their use as “universal” cells would be a lack
9 of alloreactive potential, which remains to be demonstrated.
10 Methods: We used ex vivo, in vitro and in vivo models to determine if human MAIT cells
11 contribute to allogeneic responses.
12 Results: We show that recovery of MAIT cells after allogeneic hematopoietic stem cell
13 transplantation recapitulates their slow physiological expansion in early childhood, independent
14 of recovery of conventional T cells. In vitro, signals provided by allogeneic cells and cytokines
15 do not induce sustained MAIT cell proliferation. In vivo, human MAIT cells do not expand nor
16 accumulate in tissues in a model of T-cell mediated xenogeneic graft-versus-host disease
17 (GVHD) in immunodeficient mice.
18 Conclusions: Altogether, these results provide evidence that MAIT cells are devoid of
19 alloreactive potential and pave the way for harnessing their translational potential in universal
20 adoptive therapy overcoming barriers of HLA disparity.
21
22
23
24
25
4
1 Introduction
3 MAIT cells are innate-like T cells expressing a semi-invariant TCR which recognizes
4 microbial-derived riboflavin (vitamin B2) precursors such as 5-OP-RU presented by MR1, a
5 monomorphic non-classical MHC class-1 like molecule (1-3). Upon TCR engagement, MAIT
6 cells proliferate, release proinflammatory cytokines and kill target cells, supporting their role
7 in antimicrobial defense (4-6). Most bacteria and yeasts, but not mammal cells, are able to
8 synthesize riboflavin and hence provide MR1 ligands (7). This TCR-MR1 recognition pathway
9 therefore represents a sophisticated discriminatory mechanism to target microbial antigens
10 while sparing the host. MAIT cells can also be activated in a TCR-independent way in response
11 to inflammatory cytokines such as IL-12 and IL-18 (8-10). MAIT cells are predominantly
12 localized in the liver and barrier tissues, in agreement with their expression of several
13 chemokine receptors (4, 11). They are also abundant in the adult blood, but very few in cord
14 blood. We previously showed that the postnatal expansion of MAIT cells was a very slow
15 process requiring at least 5-6 years to reach adult levels, and likely resulted from repeated
16 encounters of a few MAIT cell clones with MR1-restricted microbial antigens (12).
17 The curative effect of allogeneic hematopoietic stem cell transplantation (HSCT) in
18 hematological malignancies is based on the capacity of donor T cells to eliminate residual tumor
19 cells (graft-versus-leukemia (GVL) effect). A serious drawback is that donor T cells may also
20 recognize normal nonhematopoietic cells, leading to graft-versus-host disease (GVHD), which
21 is characterized by activation, expansion and migration to target tissues of donor alloreactive T
22 cells (13-17). In the first weeks after HSCT, the T cell compartment recovers through peripheral
23 expansion of graft-derived T cells in response to increased levels of homeostatic cytokines and
24 to host’s allogeneic antigens. Reconstitution of a fully diversified T-cell repertoire occurs only
25 later by resumed thymic output of newly-generated naïve T cells (18). Even under favorable
5
1 conditions, it takes at least 2 months to produce naive T cells, and a plateau of thymic output is
2 reached only after 1- 2 years.
3 We observed that MAIT cell recovery was delayed up to 6 years after allogeneic cord
4 blood transplantation, mimicking the long postnatal expansion period (12). Other studies
5 confirmed that MAIT cell numbers failed to normalize for at least a year after HSCT (19-21)
6 and suggested that a low MAIT cell number was associated with an increased risk of GVHD,
7 although the mechanisms remain unclear (19, 21, 22). In mouse HSCT models, recipient’s
8 residual MAIT cells protected mice from acute intestinal GVHD through microbial-induced
9 secretion of IL-17 promoting gastrointestinal tract integrity and reducing alloantigen
10 presentation, ultimately limiting the proliferation of donor-derived alloreactive T cells (23).
11 Recently, transcriptomic and functional studies have revealed new tissue repair and regulatory
12 functions of MAIT cells (24-28). Altogether, these results open the possibility of exploiting the
13 effector or tissue repair/regulatory properties of MAIT cells in adoptive immunotherapy,
14 provided that they do not cause alloreactivity. Here, we used in vitro and in vivo models to
15 provide evidence that human MAIT cells are devoid of alloreactive potential. Our results pave
16 the way for harnessing the translational potential of MAIT cells in universal adoptive therapy
17 overcoming barriers of HLA disparity.
18
19
20 Patients and Methods
21
22 Patients
23 Three independent cohorts of patients undergoing allogeneic HSCT for hematological
24 malignancy were studied. Patient and graft characteristics are described in Table 1.
6
1 Cohort 1 included 40 children recipients, for whom peripheral blood mononuclear cells
2 (PBMCs) samples were prospectively collected at Robert Debré Hospital between January
3 2013 and December 2015. Blood samples were collected prior to conditioning (± day -15 before
4 HSCT) and at 1, 3, 6, 12 and 24 months after HSCT as the standard of care for assessment of
5 immunologic recovery. Patients who died before day 180 were not included in the analysis.
6 Cohort 2 included 64 additional HSCT children in stable remission for whom PBMCs samples
7 were collected at time of a routine visit at Robert Debré Hospital 2 to 16 years after HSCT.
8 All children from cohorts 1 and 2 received unmanipulated bone marrow transplant from HLA-
9 matched sibling donor (MSD) or unrelated donor (MUD). Myeloablative conditioning was
10 provided with VP16 and total body irradiation (TBI), or with cyclophosphamide and busulfan.
11 In vivo T-cell depletion by ATG was given in the majority of MUD recipients. Primary
12 prophylaxis of GVHD consisted of a calcineurin inhibitor alone (MSD recipients) or with
13 methotrexate (MUD recipients).
14 Cohort 3 included 49 adult donor/recipient pairs for whom frozen annotated PBMCs were
15 provided by the CRYOSTEM consortium (https://doi.org/10.25718/cryostem-collection/2018)
16 and SFGM-TC (Société Francophone de Greffe de Moelle-Thérapie Cellulaire). Patients
17 received bone marrow or peripheral blood stem cells from a matched sibling donor and were
18 given myeloablative or non-myeloablative conditioning. Blood samples were collected before
19 conditioning, and at 3 and 12 months post-HSCT in the absence of aGVHD. In case of aGVHD,
20 samples were collected at the time of diagnosis before any treatment, one month later (i.e
21 towards 3 months post-HSCT) and at 12 months.
22
23 Cells and reagents
24 PBMCs were isolated and used immediately or frozen. 5-OP-RU was synthesized as described
25 in (29-31). Human MAIT cells were expanded for 6 days in human T-cell culture medium
7
1 (RPMI-1640, Invitrogen, Life Technologies) containing 10% human AB serum (EuroBio), IL-
2 2 (100 U/mL, Miltenyi) and 300 nM 5-OP-RU.
4 Flow cytometry
5 MAIT cells were analyzed in fresh whole blood, or in isolated PBMCs where indicated.
6 Multiparametric 14-color flow cytometry analyzes were performed as described in
7 Supplementary data. MAIT cells were defined as CD3+CD4-CD161highV7.2+ cells in the first
8 part of the study (HSCT patients). This population fully overlapped with the population labeled
9 by MR1:5-OP-RU tetramers (31). Thereafter, we used the specific MR1:5-OP-RU tetramer
10 when it became available (NIH tetramer core facility).
11
12 In vitro stimulations
13 Human carboxyfluorescein succinimidyl ester (CFSE)-labeled (5M) PBMCs were cultured in
14 RPMI-1640 supplemented with IL-2 (100 U/mL), IL-15 (50 ng/mL), IL-7 (50 ng/mL, all from
15 Miltenyi Biotec), or IL-12/IL-18 (50 ng/mL each, R&D Systems) and/or 300 nM 5-OP-RU.
16 For mixed lymphocyte reactions, CFSE-labeled PBMCs used as responders (1x106/ml) were
17 incubated with -irradiated allogeneic stimulator PBMCs (1:1 ratio) in 96-well round-bottom
18 plates. Cells were harvested at day 6 and stained before flow cytometry analysis.
19
20 Adoptive transfer of xenogeneic cells
21 NOD-Scid-IL-2Rnull (NSG) mice (Jackson laboratory, Bar Harbor, MI) were bred in the animal
22 facility of St-Louis Research Institute and housed under specific pathogen-free conditions.
23 Eight- to 10-week-old female mice were used after approval of all procedures by the
24 Institutional animal Care and Use Ethics Committee (CE121#16624). Mice were irradiated (1.3
25 Gy) 24 hours prior to injection of 5x106 human PBMCs in the caudal vein. Development of
8
1 GVHD was monitored 3 times per week based on weight loss, hunching posture, reduced
2 mobility and hair loss. Human chimerism in peripheral blood (percent of human CD45+ cells)
3 was assessed weekly. Where indicated, mice received 5-OP-RU (1 nmol i.p. every 3 days from
4 the day of PBMCs infusion) and/or human IL-15 (100 ng i.p. every 3 days). Mice were
5 sacrificed at the indicated time, or when weight loss was >15%. Peripheral blood, spleen, liver,
6 lungs and intestine were harvested, and cells were isolated as described in Supplementary data.
8 Statistics
9 Differences between groups were analyzed using non-parametric tests for paired (Wilcoxon)
10 and unpaired (Mann-Whitney or Kruskall-Wallis) groups, or two-way ANOVA. Correlations
11 were assessed using the Spearman’s rank correlation. Two-sided p values < 0.05 were
12 considered significant. Analyzes were performed using Prism software v.9 (GraphPad).
13 All data including outliers were included with one pre-determined exception: flow cytometry
14 cell-subset percentages were considered non-evaluable if the parent subset contained <100
15 events.
16
17
18 Results
19
20 1/ MAIT cell reconstitution is delayed for several years after HSCT
21 Our previous findings have shown that it takes up to 6 years to recover normal MAIT cell values
22 after cord blood transplantation, suggesting that MAIT cells do not proliferate in response to
23 allogeneic stimulation (12). However, MAIT cells in cord blood are naïve, and their number is
24 1- 2 logs lower than in adult blood, which could contribute to this slow recovery. We therefore
25 analyzed the kinetics of MAIT cell reconstitution in other HSCT settings.
9
1 Forty children who received an unmanipulated bone marrow transplant after myeloablative
2 conditioning were studied longitudinally up to 24 months after HSCT. While the number of
3 conventional T cells (Tconv) gradually increased from 1 month after transplantation and
4 returned almost to normal after one year, there was virtually no increase in the number of MAIT
5 cells during the study period (Figure 1A). Two years after HSCT, MAIT cell values remained
6 5 times lower than in age-matched donors (Figure 1A). The number of Tconv and MAIT cells
7 before and up to 3 months after HSCT was lower in matched unrelated donor (MUD) than in
8 matched sibling donor (MSD) recipients, likely due to a longer time to transplant and more
9 frequent use of in vivo T-cell depletion in MUD recipients. However, while Tconv reached
10 comparable values after 3 months regardless of the donor type, MAIT cell numbers remained
11 around 2 times lower in MUD recipients than in MSD recipients during the 24-months follow-
12 up (Figure 1B).
13 To extend our findings to a longer follow-up period, we analyzed 64 additional children at time
14 of a routine visit 2 to 16 years after transplantation. As observed after cord blood transplantation
15 (12), the number of MAIT cells increased very slowly (even more slowly in MUD than MSD
16 recipients), to reach a plateau approximately 6 years after HSCT (Figure 1C). This slow
17 recovery was not related to the underlying malignancy, gender or age of the recipient, pre-
18 HSCT conditioning (with or without TBI), severe microbial infection during the first months
19 after HSCT, or duration of immunosuppressive treatment (data not shown).
20 T-cell reconstitution is impaired in patients with aGVHD, at least in part because of defective
21 thymic production of HSC-derived T cells (18). We previously observed that thymus-derived
22 naïve MAIT cells appeared in cord blood recipients 6 months after transplantation (12). Here
23 we found that the number of MAIT cells after 6 months was lower in patients with severe (grade
24 3-4) aGVHD compared to those without or mild (grade 0-2) aGVHD, as also observed for
10
1 Tconv cells (Figure 1D). These data suggest that the alteration of thymic function associated
2 with aGVHD participates in the defective recovery of MAIT cells after HSCT.
4 2/ The reconstitution of MAIT cells after HSCT is affected by their number in the donor and
5 by the existence of aGVHD
6 To further explore the potential link between MAIT cell numbers aGVHD and MAIT cell
7 numbers, we retrospectively analyzed 49 adult HSCT donor/recipient pairs from the national
8 Cryostem consortium, including 25 patients with aGVHD. As observed in children, MAIT cells
9 did not significantly expand during the one-year study period, while the number of Tconv
10 increased sharply. Moreover, absolute numbers of MAIT and Tconv cells at 3 months were
11 lower in patients with severe aGVHD than in those without or mild aGVHD, whereas
12 subsequent reconstitution was not affected (Figure 2A). The proportion of Ki67+ cells at the
13 time of aGVHD, which could represent alloreactive proliferating cells, was higher in Tconv
14 cells than in MAIT cells. One year after HSCT, Ki67 expression was hardly detectable in both
15 cell types (Figure 2B).
16 MAIT cell recovery was not influenced by the conditioning regimen (myeloablative or non-
17 myeloablative) or by the source of HSC (peripheral blood or bone marrow) (data not shown).
18 Notably, the number of MAIT cells in the donor was significantly correlated to their number in
19 the recipient after HSCT, whereas no correlation was observed for Tconv (Figure 2C).
20 However, there was no association between the number of MAIT cells in the donor and the
21 presence of severe aGVHD (Figure 2D).
22 Altogether, these data indicate that the number of MAIT cells in the donor influences the early
23 reconstitution of MAIT cells in the recipient, but do not provide evidence for a role of MAIT
24 cells in aGVHD.
25
11
1 3/ MAIT cells do not proliferate in response to alloantigen stimulation, except in the presence
2 of MR1 ligand.
3 Graft-derived conventional T cells expand through lymphopenia-induced homeostatic
4 proliferation and response to host’s allogeneic antigens. However, not all T cells respond with
5 the same efficiency to these proliferative cues (32).
6 We first analyzed the in vitro responsiveness of MAIT cells to homeostatic cytokines. CFSE-
7 labeled PBMCs were treated with IL-7 or IL-15, or IL-2 as control, alone or in combination
8 with the microbial-derived MR1 ligand, 5-OP-RU, and the proliferation of MR1-tetramer+
9 MAIT cells was monitored according to CFSE dilution (Figure 3A, B). In the absence of
10 cytokine, MAIT cells did not respond to 5-OP-RU alone. Most MAIT cells proliferated in
11 response to IL-15 or IL-7 (but not to IL-2) alone, but the number of divisions was significantly
12 higher when 5-OP-RU was added to IL-15 or IL-2.
13 Chemotherapy and radiation induce the secretion of IL-12 and IL-18, which may trigger early
14 TCR-independent activation of MAIT cells transferred with the graft. We observed a low
15 proliferative response of MAIT cells to IL-12/IL-18 combination, which increased in the
16 presence of 5-OP-RU but remained lower than proliferation induced by IL-15 (Figure 3A, B).
17 We next explored the capacity of MAIT cells to respond to allogeneic cells in a mixed
18 lymphocyte reaction (MLR). Unlike Tconv, MAIT cells barely proliferated in response to
19 allogeneic stimulation (Figure 3C). However, the addition of 5-OP-RU to the MLR induced a
20 strong proliferation of MAIT cells (Figure 3D), suggesting that cytokines (IL-2 or other)
21 produced by neighboring alloreactive T cells during the culture period allowed MAIT cells to
22 proliferate only in response to the MR1 ligand.
23 Overall, these results suggest that signals provided by allogeneic cells and cytokines present in
24 the post-HSCT period are not sufficient to induce sustained expansion of MAIT cells.
25
12
1 4/ Human MAIT cells do not expand in immunodeficient mice and do not cause xenogeneic
2 GVHD (xeno-GVHD)
3 To further explore the potential of human MAIT cells to expand in vivo and participate to
4 GVHD tissue lesions, we used a model of xenogeneic GVHD (xeno-GVHD) in which low
5 doses of human PBMCs (huPBMCs) are injected into irradiated immunodeficient NSG mice.
6 In this model, human T cells consistently expand in the mouse and mediate an acute GVHD-
7 like syndrome with extensive T-cell tissue infiltration and damage of mouse skin, liver, intestine
8 and lungs, resulting in death by 30-50 days (33, 34).
9 Mice were injected with 5x106 huPBMCs, among which MAIT cells represented around 3% of
10 T cells. The presence of CD45+ huPBMCs was determined at different times in tissues of
11 recipient mice, including those where MAIT cells are known to preferentially reside. Four
12 weeks after transfer, variable proportions of CD45+ cells were found in peripheral blood and
13 tissues, almost all of which were Tconv and less than 0.05% were MAIT cells (Figure 4A).
14 Next, mice injected with huPBMCs were monitored to evaluate aGVHD progression and
15 euthanized when weight loss was >15% (±45 days after transfer). A massive accumulation of
16 Tconv was observed in tissues, in particular in the spleen, lungs and liver. However, MAIT
17 cells were very few in all compartments (Figure 4B).
18 Human MAIT cells efficiently recognize the murine MR1 molecule (35), ruling out a defective
19 presentation of MR1 ligands to human MAIT cells in NSG mice. However, since MAIT cells
20 do not proliferate significantly in vitro in the absence of 5-OP-RU, the availability of MR1
21 ligand could be a factor limiting their expansion or survival in mice housed under specific-
22 pathogen-free conditions. Mice were thus injected with 1 nmol 5-OP-RU intraperitoneally
23 every 3 days from the day of huPBMC injection, a dose previously shown to activate
24 endogenous MAIT cells (36). This did not result in any increase in MAIT cells in peripheral
25 blood or tissues (Figure 4C).
13
1 It is not clear whether mouse cytokines can sustain homeostatic proliferation and survival of
2 human MAIT cells in NSG mice due to a species barrier between human lymphoid cells and
3 the murine recipient microenvironment (37-39). This is a key question for IL-15, as it is mostly
4 mouse-derived in the xeno-GVHD model given the low human myeloid chimerism. Indeed,
5 we found that human MAIT cells cultured with mouse IL-15 did not proliferate at all in vitro
6 (Figure 5A). Proliferation was increased when 5-OP-RU was added to mouse IL-15, although
7 it remained lower than with human IL-15. Murine and human IL-7 had similar effects on MAIT
8 cell proliferation, regardless of the presence of 5-OP-RU (Figure 5A). Thus, IL-15 availability
9 may be a factor limiting the proliferation of MAIT cells in NSG mice.
10 We therefore sought to determine the division rate of MAIT cells after transfer of CFSE-labeled
11 huPBMCs into NSG mice. One week after transfer, large proportions of both Tconv and MAIT
12 cells from the liver and spleen displayed low CFSE fluorescence, indicating that they were able
13 to divide (Figure 5B). These proportions increased when mice were treated with human IL-15
14 from the day of transfer, leading to a significant accumulation of Tconv and MAIT cells in the
15 liver and spleen (figure 5B).
16 We, therefore, investigated whether human IL-15 would enhance MAIT cell accumulation in
17 target tissues during xeno-GVHD. NSG mice were treated with human IL-15 three times per
18 week from the day of huPBMC transfer and euthanized when they developed severe signs of
19 aGVHD (25 days after transfer, i.e. earlier than control mice). A massive infiltration of T cells
20 was observed in all compartments, but MAIT cells still remained barely detectable (Figure 5C).
21 Altogether, these results indicate that human MAIT cells are able to divide and survive in
22 immunodeficient mice, but do not participate in T cell-mediated tissue lesions during xeno-
23 GVHD even in the presence of human IL-15.
24
25
14
1 Discussion
3 The newly described tissue repair and immunoregulatory functions of MAIT cells open
4 fascinating perspectives for their use in adoptive therapy to control immune-mediated damage
5 in tissues where these cells are known to accumulate. However, such strategy can be considered
6 in unrelated recipients only if MAIT cells are devoid of GVH potential. As anticipated by the
7 very limited diversity of their TCR that recognizes microbial antigens presented by the highly
8 conserved MR1 molecule, our results demonstrate that MAIT cells do not proliferate in
9 response to allogeneic signals and do not participate to aGVHD lesions, in line with our
10 previous observation that MAIT cells were undetectable in target tissues at time of aGVHD
11 (12).
12 Human MAIT cells are very few at birth and accumulate gradually during infancy, with
13 about a 30-fold expansion to reach a plateau around 6 years of age (12). Several pieces of
14 evidence suggest that the drivers of this peripheral expansion are related to successive
15 encounters with microbes leading to an accumulation of MAIT cell clonotypes that will
16 constitute the future MAIT cell pool (40). Indeed, MAIT cells are absent in germ-free mice and
17 are very few in laboratory mice, but dramatically expand following challenge with riboflavin-
18 producing microbes (35, 36, 41). Moreover, their development in mice depends on early-life
19 exposure to defined microbes that synthesize riboflavin-derived antigens (27). In human
20 subjects with controlled infection, MAIT cells show evidence of expansion of select MAIT cell
21 clonotypes (42), but longitudinal analyzes of MAIT cell numbers in relation to the composition
22 of the microbial environment are still lacking.
23 Here, we show that the reconstitution of MAIT cells after HSCT recapitulates their
24 physiological expansion in the infancy and takes place over a period of at least 6 years,
25 regardless of recipient- or donor-related factors such as age, underlying disease, donor type,
15
1 stem cell source or conditioning regimen. The number of MAIT cells in the early post-transplant
2 period is at least in part dependent on the number of MAIT cells transferred with the graft.
3 Subsequent MAIT cell recovery relies upon thymus-derived naïve MAIT cells. The low
4 absolute MAIT cell counts in patients with grade 3-4 aGVHD, which associate with low
5 absolute counts of conventional T cells, may be a consequence of a defective thymic function.
6 Indeed, aGVHD-associated thymic damage results in the loss of the large population of double-
7 positive (DP) thymocytes (43). This may decrease the thymic output of naïve MAIT cells,
8 which undergo positive selection by recognizing MR1 at the surface of these DP thymocytes
9 (44). In addition, loss of diversity and increased bacterial domination early after HSCT, in
10 particular by Enterococcus, has been associated with increased risk of aGVHD (45-47). One
11 might speculate that blooming of Enterococcus (a strain unable to synthesize riboflavin) could
12 prevent from early expansion of donor-derived MAIT cells due to lack of MR1-ligands. So far,
13 the relationship between abundance and diversity of gut microbiota and post-transplant MAIT
14 cell reconstitution remains controversial (19, 22). Investigating the gut metagenome or
15 metatranscriptome of HSCT recipients for the presence of riboflavin biosynthesis genes should
16 answer this question.
17 In mouse HSCT models, conditioning-resistant host residual MAIT cells promote
18 gastrointestinal tract integrity and limit the proliferation of donor-derived alloreactive T-cells,
19 thus protecting from aGVHD (23). Although these findings are not directly translatable to
20 human aGVHD due to specificities of HSCT models in mice and differences between mouse
21 and human MAIT cells (23, 48, 49), they support our observations that human MAIT cells do
22 not contribute to aGVHD. Using a classical in vitro model of alloreactivity, we show that
23 MAIT cells do not proliferate in response to allogeneic cells, unless combined MR1 ligand and
24 cytokine signals are present. Moreover, MAIT cells do not participate in the development of
25 xeno-GVHD in NSG mice infused with huPBMCs. It is likely that the xeno-GVHD is caused
16
1 by a fraction of T cells having a low frequency in donor PBMCs, which subsequently expand
2 in mouse organs upon recognition of murine MHC (50). One cannot exclude that the NSG host
3 with an ablated immune compartment may be less likely to provide conditions for presentation
4 of MR1-ligands to MAIT cells or the delivery of costimulatory signals. However, MR1 is
5 highly conserved across various species, with 90% of sequence similarity between mice and
6 humans, so that murine MR1 can present 5-OP-RU to human MAIT cells as efficiently as
7 human MR1 (35). That donor T cells may outcompete MAIT cells by limiting the availability
8 of IL-15 seems unlikely, as demonstrated by the failure of exogenous human IL-15 to allow
9 MAIT cell accumulation in tissues at time of xeno-GVHD. Whether MAIT cells fail to traffic
10 or find their niche in the host due to species barriers between chemokine receptors and their
11 ligands is also unlikely since MAIT cells were found in the spleen and liver one week after
12 transfer. Rather, it appears that MAIT cells do not expand upon recognition of murine MHC
13 and consequently do not participate to GVHD tissue lesions.
14 Due to ubiquitous expression of MR1 and abundance of human MAIT cells in tissues,
15 MAIT cell functions need to be tightly regulated to control the balance between healthy and
16 pathological processes. Following HSCT, homeostatic cytokines may provide early but limited
17 proliferation signals to graft-derived MAIT cells, at least ensuring their survival in the recipient
18 in the early post-transplant period. However, sustained expansion of mature and/or thymus-
19 derived naïve MAIT cells will only occur if MR1 ligands are present together with
20 inflammatory signals. Our data showing the lack of alloreactive potential of MAIT cells pave
21 the way for safely harnessing their functions in adoptive immune therapy. MAIT cells are
22 excellent candidates for engineering universal chimeric antigen receptor cells (CAR-MAIT), as
23 they are easy to expand in vitro, can traffic to tissues, have potent effector functions and are
24 unlikely to cause GVHD. In addition, the novel tissue repair and regulatory functions of MAIT
25 cells open up new avenues for exploiting these cells for clinical benefit.
17
1 Acknowledgments
2 The authors thank all the patients and their physicians and the nurse and technician staff from
3 Hôpital Robert Debré who helped with this study, as well as all members of the CRYOSTEM
4 Consortium for providing patients samples used in this study : University Hospital of Angers,
5 University Hospital of Dijon Bourgogne, University Hospital of Besançon, University Hospital
6 of Grenoble, University Hospital of Lille, University Hospital of Lyon, University Hospital of
7 Bordeaux, Paoli-Calmettes Institute, AP-HM, University Hospital of Nantes, AP-HP (Saint
8 Louis Hospital, La Pitié-Salpêtrière Hospital, Saint-Antoine Hospital, Robert Debré Hospital,
9 Necker Hospital, Groupe Hospitalier Henri Mondor), INSERM, University Hospital of
10 Toulouse, University Hospital of Tours, University Hospital of Rennes, University Hospital of
11 Clermont-Ferrand, University Hospital of Saint-Etienne, Lucien Neuwirth Cancer Institute,
12 University Hospital of Poitiers, University Hospital of Nice, University Hospital of Brest,
13 Military Hospital of Percy, University Hospital of Montpellier, Gustave Roussy Institute,
14 University Hospital of Limoges, University Hospital of Caen, Etablissement Français du
15 Sang. CRYOSTEM project has been funded by ANR, the Institut National du Cancer (INCa)
16 and some patient associations.
17 The authors are grateful to Véronique Parietti (Plateforme d'expérimentation animale, IRSL,
18 Hôpital Saint-Louis) for her help.
19
20
21
22
23
24
25
18
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23
24
25
24
1 Table 1. Characteristics of HSCT recipients

2
Children Children Adults
Longitudinal Cross-sectional Retrospective
cohort cohort cohort
Patients (n) 40 64 49
Sex, n (%)
- Female 17 (42.5) 23 (36) 18 (36.7)
- Male 20 (57.5) 41 (64) 31 (63.3)
Age, mean years (range) 9.4 (0.75-16) 7 (0.9-17.8) 44.4 (17-65.8)
Primary disease, n (%)
- ALL 23 (57.5) 25 (39.1) 6 (12.2)
- AML 11 (27.5) 11 (17.2) 19 (38.8)
- JMML 3 (7.5) 2 (3.1) -
- NHL 1 (2.5) 1 (1.6) 13 (26.5)
- MDS 1 (2.5) 2 (3.1) 5 (10.2)
- CML 1 (2.5) 3 (4.7) 2 (4.1)
- Others - 20 (31.2) 4 (8.2)
Donor, n (%)
- MSD 19 (47.5) 32 (50) 49 (100%)
- MUD 21 (52.5) 32 (50) -
Stem cell sources, n (%)
- Peripheral blood - - 30 (61.2)
- Bone marrow 40 (100) 64 (100) 19 (38.8)
Conditioning, n (%)
- MA 40 (100) 64 (100) 24 (49)
- NMA - - 25 (51)
In vivo T-cell depletion, n 12 (30) 18 (28) 20 (40.8)
(%)
Acute GVHD
- Grade 1-2 24 (60) NA 21 (42.8)
- Grade 3-4 9 (22.5) NA 4 (8.2)
3
4 ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; CLL: chronic
5 lymphoblastic leukemia; CML: chronic myeloid leukemia; JMML: juvenile myelomonocytic
6 leukemia ; MDS : myelodysplasic syndrome ; NHL : non-Hodgkin lymphoma. MA :
7 myeloablative ; NMA : non-myeloablative.
8
25
1 Figure Legends
3 Figure 1: MAIT cell recovery takes several years after HSCT in children
4 (A) Absolute counts (left panel) and percentages (right panel) of CD3 T cells (Tconv) and
5 MAIT cells detected in the peripheral blood of 40 children before conditioning (pre) and from
6 month 1 to month 24 after HSCT. Data are shown as mean ± SEM.
7 (B) Recovery dynamics of MAIT cells (left panel) and CD3 T cells (right panel) in transplant
8 recipients of matched sibling donors (MSD, red line) and unrelated donors (MUD, black line).
9 Results show the mean absolute numbers ± SEM during the first 24 months after HSCT. 2-way
10 ANOVA, *** P = 0.0005; ns: not significant.
11 (C) MAIT cell recovery takes at least 6 years to reach normal values. Left panel: Relationship
12 between log10-transformed MAIT cell absolute numbers and time from transplantation up to
13 16 years after HSCT in MSD (red dots) and MUD (black dots) recipients. Right panel:
14 Recovery is slower in MUD than MSD recipients for at least to 2 years after HSCT. Mann-
15 Whitney ** P ≤ 0.002; ns: not significant
16 (D) Comparison of MAIT (left panel) and Tconv (right panel) cell recovery in patients without
17 or with mild (grade 0-2) or severe (grade 3-4) aGVHD. Results show the mean absolute
18 numbers ± SEM. 2-way ANOVA, * P= 0.04, ***P = 0.0006.
19 Light gray and red areas show the reference physiological T cell and MAIT cell count interval,
20 respectively, obtained from 36 age-matched healthy donors.
21
22 Figure 2: MAIT cell recovery in adult HSCT recipients
23 (A) Kinetics of MAIT (left panel) and Tconv (right panel) cell recovery in patients from the
24 CRYOSTEM cohort (n= 49) according to the absence or the presence of mild (grade 1-2) or
25 severe (grade 3-4) aGVHD. Patients with aGVHD were sampled at the time of disease diagnosis
26
1 and 1 month later, i.e. towards M3. Results show the mean absolute numbers ± SEM. Shaded
2 areas show MAIT and T cell count intervals in the donors. 2-way ANOVA, not significant.
3 (B) Correlation between the number of MAIT (left) or Tconv (right) cells in the donor and that
4 in the recipient 3 months (top panels) and 12 months (bottom panels) after HSCT. Spearman
5 correlation r and P values are indicated.
6 (C) Quantification of Ki67+ proliferating cells among MAIT, CD4 and CD8 T cells at time of
7 aGVHD (left panel) and 12 months after HSCT (right panel). Scatter plots with bar showing
8 the mean percentage of Ki67+ cells. Wilcoxon non parametric paired test, ** P= 0.004; *** P=
9 0.0008; ns: not significant.
10 (D) Comparison of the number of MAIT cells in the donor according to the absence or presence
11 of mild (grade 1-2) or severe (grade 3-4) aGVHD. Kruskall-Wallis non-parametric paired test,
12 ns: not significant
13
14 Figure 3: MAIT cell proliferative response to cytokines and allogeneic cells in vitro
15 Carboxyfluorescein succinimidyl ester (CFSE)-labeled human PBMCs (106/mL) were cultured
16 for 6 days in the indicated conditions. Proliferation of MR1 tetramer+ MAIT (red lines) and
17 conventional T (Tconv, black lines) cells was quantified by CFSE dilution at the end of the
18 culture.
19 (A, B) Proliferative response to stimulation by IL-7 (50ng/mL), IL-15 (50 ng/mL), IL-2 (100
20 U/mL) and IL-12/18 (50 ng/mL each) in the presence or absence of the synthetic MR1 ligand,
21 5-OP-RU (300 nM). (A) Representative CFSE staining gated on MAIT and Tconv cells after a
22 6-day culture. (B) Division index (mean number of divisions in the total population) of MAIT
23 and Tconv cells. Mean ± SEM of 3 independent experiments. Paired t test, * P= 0.045; ** P=
24 0.004.
27
1 (C) Mixed lymphocyte reaction: CFSE-labeled responder PBMCs were cultured with irradiated
2 allogeneic PBMCs at 1:1 ratio. Responder T cells were identified at the end of the 6-day culture
3 by gating on CD3+ T cells following exclusion of dead cells.
4 Left panel: Representative CFSE staining gated on MAIT (red) and Tconv (black) cells after 6-
5 day culture in the presence of allogeneic (upper panel) or autologous (lower panel) PBMCs.
6 Numbers in the quadrants indicate the percentage of proliferating (CFSElow) cells among each
7 population. Right panel: Individual values and means ± SEM of proliferating Tconv and MAIT
8 cells (n= 11 experiments using different recipient/donor pairs). Paired t test, **** P<0.0001
9 D) Mixed lymphocyte reaction was performed in the absence (upper panel) or presence (lower
10 panel) of 300 nM 5-OP-RU. Representative CFSE staining gated on MAIT (red) and Tconv
11 (black) cells after a 6-day culture.
12
13 Figure 4: Absence of MAIT cell expansion in a xenogeneic GVHD model
14 Irradiated (1.3 Gy) NSG mice injected with 5.106 huPBMCs were monitored to evaluate
15 aGVHD progression and euthanized at the indicated time. Peripheral blood, spleen, liver, lungs
16 and colon were harvested, and cells were isolated.
17 (A-C) The proportion of human CD45+ leukocytes among viable cells (i.e. human chimerism,
18 upper panels), and the proportion of Tconv (black dots) and MAIT (red dots) cells among
19 human CD45+ cells (lower panels) were determined by flow cytometry. Results show
20 individual values and median (horizontal bar). (A) Mice were sacrificed at day 28 after injection
21 (n= 5 mice). (B) Mice were euthanized when they exhibited signs of aGVHD (± day 45, n= 11
22 diseased mice). (C) Mice received 5-OP-RU every 3 days (1 nmol i.p.) from the day of
23 huPBMC injection and were euthanized at time of aGVHD (n=7 diseased mice).
24
28
1 Figure 5: Human IL-15 does not increase MAIT cell accumulation in the target tissues in
2 the xeno-GVHD model
3 (A) CFSE-labeled human PBMCs were cultured for 6 days with mouse (open black histograms)
4 or human (shaded grey histograms) IL-15 (50 ng/mL) or IL-7 (10 ng/mL) in the absence or
5 presence of 5-OP-RU. Representative experiment showing CFSElow proliferating MAIT cells
6 (gated on CD3 T cells).
7 (B) NSG mice were injected with 20x106 CFSE-labeled human PBMCs without or with human
8 IL-15 (100 ng i.p. every other day from the day of transfer). The spleen and liver were harvested
9 7 days later, and cells were analyzed by flow cytometry for CFSE expression (left panel) and
10 numbers of MAIT and Tconv cells (right panel).
11 (C, D) Mice were treated with huIL-15 from the day of transfer of 5.106 huPBMCs, and
12 euthanized at the time of aGVHD (day 25, n= 3 diseased mice). Results show the proportion of
13 human CD45+ leukocytes among viable cells (C), and absolute counts of Tconv and MAIT
14 cells (D) in the indicated compartments. Individual values and median (horizontal bar) are
15 shown.
16
17
18
19
20
29
Figure 1
A 10 100
100
CD3+ T cells /µL
% CD3+ T cells
80 2000 8 80
MAIT cells /µL
% MAIT cells
60 6 60
Tconv
Tconv 40
40 1000 4
20 MAIT 2 20
MAIT
0 0 0 0
pre 1 3 6 9 12 18 24 pre 1 3 6 9 12 18 24
time from HSCT (months) time from HSCT (months)
B 50 3000
CD3+ T cells /µL

40
MAIT cells /µL
2000
30 MUD ns
MSD
20
1000
10 MUD
*** MSD
0 0
pre 1 3 6 9 12 18 24 pre 1 3 6 9 12 18 24
C 1000 ns ns
1000 ** **
100
MAIT cells /µL
MAIT cells /µL
100
10
10
1
1 MUD 0.1 MUD
MSD MSD
0.1 0.01
0 50 100 150 200 <1 1-2 2-6 >6
time from HSCT (months) time from HSCT (years)
D 50 3000 grade 0-2

grade 3-4
CD3+T cells /µL
40
MAIT cells /µL
2000
30 grade 0-2
20 grade 3-4 ***
1000
10
*
0 0
pre 1 3 6 9 12 18 pre 1 3 6 9 12 18
Figure 2
CD3+ T cells /µL

MAIT cells /µL
B
25 ns
20
ns ns
% Ki67+ cells
% Ki67+ cells
15
10
0
MAIT CD4+ CD8+
C
CD3+ T cells /µL at M3
r=0,53 r=-0,01
MAITcells /µL at M3
p=0,0008 ns
CD3+ T cells /µL at M12
r=-0,15
MAITcells /µL at M12
r=0,3505
p=0,0361 ns
D
ns
MAIT cells /µL in the donor
125 ns ns
100
75
50
25
0
0 1-2 3-4
acute GVHD (stage)
Figure 3
A MAIT B MAIT
Tconv Tconv
4
*
none IL-7 IL-15 IL-2 IL-12/18 **
3
Division index
without
2
5-OP-RU
1
with
0
IL-12/
IL-7 IL-15 IL-2 IL-18
cytokine - + + + + + + + +
CFSE
5-OP-RU + - + - + - + - +
C D
Tconv MAIT Tconv MAIT
****
80
allogeneic
without
35.7%
26%
60
% of CFSElow
1.84% 1.45%
5-OP-RU
CFSE 40 CFSE
94.6%
autologous
20
with
23.3%
3.65% 1.15% 0
Tconv MAIT
CFSE CFSE
Figure 4
A B C
Day 28 ± Day 45 (time of aGvHD) ± Day 45 (time of aGvHD)
+5-OP-RU
100 100 100

100
among viable cells
among viable cells
among viable cells

80 80 80
80
% of huCD45+
% of huCD45+
% of huCD45+
60 60 60
60
40 40 40
40
20 20 20
20
0 0 00
er
PB
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gs
r
r
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o
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ool
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le
ol
103
Li
Li
Lu
Lu
3
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103Tconv
SSp
C
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C
C
10
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Tconv Tconv

102 Tconv Tconv
102MAIT
MAIT 102MAIT MAIT
MAIT MAIT
101 101 101
100 100 100

huCD45+ cells
% Bamong huCD45+ cells

100 90 10 0
90 100 90
10-1 80 10-1 80 10-1 80
10-2 70 -2 70 70
10 10-2
10-3 0.10 -3 0.10 0.10
10 10-3
% among
10-4 0.05 10 -4 0.05 10 -4 0.05

en
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s
C
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s ive
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hu
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PB
hu
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xeno-GvHD mice
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Figure 5
A IL-7 + IL-15 +
IL-7 IL-15
5OP-RU 5-OP-RU
human
mouse
CFSE
B (D7)
ctrl + huIL-15
MAIT 95.1% MAIT
46.8% 3000 * 60000
of MAIT cells (cells/organ)

*
of Tconv (cells/organ)
absolute number
absolute number
2000 40000
liver
Tconv Tconv 1000

62.7% 93.5% 20000
0 0
ctrl IL-15 ctrl IL-15
CFSE
MAIT 81.1% MAIT 6000 300000 *

of MAIT cells (cells/organ)
24.3%
of Tconv (cells/organ)
absolute number
absolute number
4000 200000
spleen
Tconv Tconv 2000 100000

91.1%
48.1%
0 0
ctrl IL-15 ctrl IL-15
CFSE
C (D25)
absolute number (cells/organ) absolute number (cells/organ)
absolute number (cells/organ) absolute number (cells/organ)
100 800000 100 800000

80 80
600000 600000
60 60
lungs
400000 400000
liver
40 40
200000 20 200000
20
0 0 0 0
MAIT Tconv MAIT Tconv MAIT Tconv MAIT Tconv
100 400000 100 300000

80 80
300000
200000
60 60
spleen
colon
200000
40 40
100000
20 100000 20
0 0 0 0
MAIT Tconv MAIT Tconv MAIT Tconv MAIT Tconv

Human MAIT Cells Are Devoid of Alloreactive Potential: Prompting Their Use As Universal Cells For Adoptive Immune Therapy

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Human MAIT Cells Are Devoid of Alloreactive Potential: Prompting Their Use As Universal Cells For Adoptive Immune Therapy

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medRxiv preprint doi: https://doi.org/10.1101/2021.04.29.21256184; this version posted April 30, 2021.

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1 Human MAIT cells are devoid of alloreactive potential:

2 prompting their use as universal cells for adoptive immune therapy.

7 Ethics approval and consent to participate

12 trial registry: ClinicalTrials.gov number NCT02403089.

17 Data are available on reasonable request to sophie.caillat-zucman@aphp.fr

24 Direction Générale de l’Offre de Soins (DGOS) (PRTS 14-CE15-0005, acronym NEOMAIT),

5 Author contributions and consent for publication

6 M. Tourret, N Talvard-Balland, M. Lambert, G. Ben Youssef, M. Chevalier, A. Bohineust, F

17 CFSE: Carboxyfluorescein succinimidyl ester; GVHD: graft-versus-host disease; GVL:

18 graft-versus-leukemia; HSCT: hematopoietic stem cell transplantation; MAIT: Mucosal

9 of alloreactive potential, which remains to be demonstrated.

11 contribute to allogeneic responses.

13 transplantation recapitulates their slow physiological expansion in early childhood, independent

16 accumulate in tissues in a model of T-cell mediated xenogeneic graft-versus-host disease

17 (GVHD) in immunodeficient mice.

20 adoptive therapy overcoming barriers of HLA disparity.

4 microbial-derived riboflavin (vitamin B2) precursors such as 5-OP-RU presented by MR1, a

9 therefore represents a sophisticated discriminatory mechanism to target microbial antigens

17 The curative effect of allogeneic hematopoietic stem cell transplantation (HSCT) in

20 recognize normal nonhematopoietic cells, leading to graft-versus-host disease (GVHD), which

21 is characterized by activation, expansion and migration to target tissues of donor alloreactive T

23 expansion of graft-derived T cells in response to increased levels of homeostatic cytokines and

2 reached only after 1- 2 years.

9 secretion of IL-17 promoting gastrointestinal tract integrity and reducing alloantigen

10 presentation, ultimately limiting the proliferation of donor-derived alloreactive T cells (23).

13 effector or tissue repair/regulatory properties of MAIT cells in adoptive immunotherapy,

17 overcoming barriers of HLA disparity.

20 Patients and Methods

23 Three independent cohorts of patients undergoing allogeneic HSCT for hematological

12 prophylaxis of GVHD consisted of a calcineurin inhibitor alone (MSD recipients) or with

13 methotrexate (MUD recipients).

15 provided by the CRYOSTEM consortium (https://doi.org/10.25718/cryostem-collection/2018)

16 and SFGM-TC (Société Francophone de Greffe de Moelle-Thérapie Cellulaire). Patients

18 given myeloablative or non-myeloablative conditioning. Blood samples were collected before

21 towards 3 months post-HSCT) and at 12 months.

23 Cells and reagents

2 2 (100 U/mL, Miltenyi) and 300 nM 5-OP-RU.

6 Multiparametric 14-color flow cytometry analyzes were performed as described in

9 by MR1:5-OP-RU tetramers (31). Thereafter, we used the specific MR1:5-OP-RU tetramer

10 when it became available (NIH tetramer core facility).

13 Human carboxyfluorescein succinimidyl ester (CFSE)-labeled (5M) PBMCs were cultured in

20 Adoptive transfer of xenogeneic cells

10 and unpaired (Mann-Whitney or Kruskall-Wallis) groups, or two-way ANOVA. Correlations

20 1/ MAIT cell reconstitution is delayed for several years after HSCT

25 analyzed the kinetics of MAIT cell reconstitution in other HSCT settings.

19 after HSCT, or duration of immunosuppressive treatment (data not shown).

21 thymic production of HSC-derived T cells (18). We previously observed that thymus-derived

5 by the existence of aGVHD

15 cell types (Figure 2B).

21 presence of severe aGVHD (Figure 2D).

3 Graft-derived conventional T cells expand through lymphopenia-induced homeostatic

5 the same efficiency to these proliferative cues (32).

12 higher when 5-OP-RU was added to IL-15 or IL-2.

15 proliferative response of MAIT cells to IL-12/IL-18 combination, which increased in the

22 proliferate only in response to the MR1 ligand.

8 and lungs, resulting in death by 30-50 days (33, 34).

17 cells were very few in all compartments (Figure 4B).

25 blood or tissues (Figure 4C).

9 may be a factor limiting the proliferation of MAIT cells in NSG mice.

15 liver and spleen (figure 5B).

23 GVHD even in the presence of human IL-15.