Abstract
Invariant natural killer T (iNKT) cells play a role in preventing graft-versus-host disease (GVHD) in bone marrow transplantation (BMT), but it is not known whether and how the NKT10 and NKT17 subsets prevent GVHD. Here, we investigated the anti-GVH effects of iNKT cell subsets in a major-MHC mismatched murine BMT in which BALB/c recipients (H-2d) received grafts from C57BL/6 (H-2b) donors. The graft consisted of bone marrow and T cells from Traj18KO (iNKT cell deficient) mice supplemented with iNKT cells purified from donors with various genetic alterations of iNKT subsets. First, NKT17-enriched CD4- iNKT cells showed anti-GVH effects similar to those of NKT2-enriched CD4+ iNKT cells, while NKT2/17 deficient iNKT cells failed to prevent GVHD, suggesting that either or both of these subsets have anti-GVH function. Furthermore, IL10KO iNKT cells completely lost their protective effects, whereas IL17AFDKO iNKT cells demonstrated partially abrogated anti-GVH effects, supporting the indispensable role of NKT10 in preventing GVHD after BMT. Using IL-17 fate-mapping mice, we demonstrated that NKT17 can trans-differentiate into NKT10 in vitro after antigenic stimulation under T regulatory type 1 (Tr1)-promoting conditions. In conclusion, NKT10 plays a role in preventing GVHD in BMT and NKT17 may contribute to the anti-GVH effects via trans-differentiation into NKT10.
Introduction
Allogeneic stem cell transplantation (ASCT) is a curative immunotherapy for hematologic malignancies that achieves clinical efficacy through the graft versus leukemia (GVL) effect mediated by reconstituted donor T cells (). While insufficient GVL effects lead to leukemia relapse, dysregulated donor T cells recognizing alloantigen can cause graft-versus-host disease (GVHD), the major complication after ASCT (). Thus, optimal regulation of donor immunity is key to enhancing the GVL effects of donor T cells and preventing GVHD in ASCT.
CD1d-restricted invariant Natural Killer (iNKT) cells are rare but powerful regulatory cells known to exert a spectrum of functions ranging from immune-regulation to direct anti-tumor activity (). Unlike classical regulatory T cells (Treg), iNKT cells are functionally (NKT1, NKT2, NKT9, NKT10, NKT17) and phenotypically (CD4+ vs CD4-) diverse, and this functional heterogeneity may contribute to their unique ability to control various forms of immune-dysregulation (–). Thus, iNKT cells have potential to play a master-regulator role to maintain a fine balance between the anti-GVH and GVL effects of donor T cells in ASCT.
iNKT cell functional subtypes are differentially distributed among CD4+ and CD4- iNKT cells, and thus the CD4+ and CD4- populations are associated with different functions (–). Human CD4+ iNKT cells express more FoxP3 and Th2 cytokines than CD4- iNKT cells and may exert superior immune-regulation. On the other hand, CD4- iNKT cells have higher expression of cytolytic granules and NK receptors and demonstrate increased cytolytic activity. Thus, they may have better antitumor effector function (–). Of note, in clinical studies of matched donor ASCT, a higher graft dose of total iNKT or NKT1/NKT17-enriched CD4- iNKT cells was associated with better GVHD outcome (, ). This is an unexpected observation given the bona fide pro-inflammatory properties of CD4- iNKT cells. In murine Bone Marrow Transplantation (BMT) model, the addition of FACS-isolated CD4+ iNK T cells to the donor graft suppressed murine GVHD via the expansion of Tregs (–). However, it is not known how effective their anti-GVH effects are compared with CD4- iNK T cells. Lastly, an evaluation of the anti-GVH and anti-tumor effects of NKT1, NKT2, and NKT17 subsets in murine BMT reported that NKT2 and NKT17 displayed better immunosuppressive function, while NKT1 displayed superior anti-tumor effects without significant anti-GVH effects ().
Recently, a murine IL-10-producing iNKT subset, NKT10, has been described in mice pretreated with the agonist glycolipid α−galactosyl ceramide (αGalCer) (). This NKT10 subset is reported to play a regulatory role in the pathogenesis of obesity (–), diabetes (, ), experimental autoimmune encephalomyelitis (), tumor progression (), and colitis (). However, it is not known whether and how NKT10 contributes to the iNKT-mediated anti-GVH effects after ASCT. Here, we investigated the potential role of NKT10 cells in preventing GVHD using a major MHC-mismatched murine BMT model.
Materials and methods
Animals
Animal experiments were conducted in compliance with the protocols approved by the MD Anderson Institutional Animal Care and Use Committee. Six to eight-week-old female BALB/c, C57BL/6, B6(Cg)-Traj18tm1.1Kro/J (Traj18KO), and B6.129P2-Il10tm1Cgn/J (IL-10KO) mice were purchased from Jackson Laboratories (Bar Harbor, ME), acclimatized for three weeks prior to the experimental procedures and maintained under specific pathogen-free conditions. IL-17RBKO/B6 mice were provided by Dr. Masaro Taniguchi and IL-17AFDKO/B6 mice were generated by crossing IL-17AFKO mice () and IL17dtm1Lex from Genetech-Lexicon Pharmaceuticals (). IL-17Fate+ mice were generated by breeding Foxp3RFP IL-10eGFP IL-17AKata triple reporter mice with IL-17A Fate reporter mice (IL-17ACRE Rosa26 STOPfl/fl (R26YFP)), both of which were provided by Drs. Richard Flavell and Nicola Gagliani ().
Materials
T cell media (TCM) was made of Roswell Park Memorial Institute Medium (RPMI)-1640 supplemented with L-glutamine (Gibco, #11875-093), 10% heat-inactivated fetal calf serum (FCS) (Hyclone, #A-1115-L), 0.001 mg/ml gentamicin (Gibco, #15710-015), 0.1 mM nonessential amino acids (Gibco, #11140-050) and essential amino acids (Gibco, #11130-051), 10 mM HEPES buffer solution (Gibco, #15630-080), and 5.5 μM 2-mercaptoethanol (2-ME) (Gibco, #21985-023). The freezing media consisted of 45% TCM, 45% FCS, and 10% Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, #67-68-5). Phosphate-Buffered Saline (PBS, #10010-023) was purchased from Gibco (Grand Island, New York). Recombinant murine IL-2 (#575408), human IL-6 (#570806), and IL-23 (#574102) were purchased from Biolegend (San Diego, California). Recombinant TGFβ1 (7754-BH-100) was purchased from R&D (Minneapolis, Minnesota). Alpha-GalactosylCeramide (αGalCer, #867000) was obtained from Avanti Polar Lipids (Alabaster, Alabama) and solubilized in dimethyl sulfoxide (DMSO) at concentrations ranging from 100 to 500 μM. The following antibodies against specific targets were purchased from BioLegend (San Diego, California), BD Bioscience (San Jose, California), or R&D systems (Minneapolis, Minnesota): CD16/32 (2.4G.2), CD3e (145-2C11), CD4 (RMA-4), CD122 (TM-B1), RoRγt (G31-378), PLZF (R17-809), IL-10 (JESS-16E3), IL-17A (TC11-18H10), IL-4 (11B11), and IFNγ (XMG1.2). APC and PE-conjugated αGalcer/mCD1d tetramers were provided by the National Institutes of Health (NIH) Tetramer facilities. Anti-mouse CD4-microbeads (#130-104-451), and CD8α-microbeads (#130-104-076) were purchased from Miltenyi Biotech (San Jose, California). GolgiStop protein transport inhibitor (#554724), GolgiPlug protein transport inhibitor (#555029), Fixable Viability Stain 620 (#564996), and BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (#554714) were purchased from BD Bioscience. The eBioscience FoxP3/Transcription Factor Staining buffer set (00-5523-00) was purchased from Invitrogen. The Ghost dye UV 450 (13-0868) was purchased from Tonbo Bioscience (San Diego, CA). Paraformaldehyde (PFA) 16% w/v aq solution (#043368) and LIVE/DEAD Fixable Red (L34971) were purchased from ThermoFisher (Waltham, Massachusetts).
Cell preparation for donor grafts
First, bone marrow (BM) cells were prepared by flushing the murine tibiae and femurs from the donor wild type (WT) or Traj18KO C57BL/6 (H-2b) with PBS. Conventional T cells (Tcon) were isolated from donor splenocytes using Magnetic Activated Cell Sorting (MACS) with anti-CD4 and CD8 microbeads according to the manufacture’s instruction. For the isolation of iNKT cells, splenocytes from eight to twelve-week-old donor C57BL/6 mice with various genetic backgrounds (WT, IL-17RBKO, IL-10KO, IL-17AFDKO, αGalCer pre-treated mice) were stained for iNKT cells with αGalcer/CD1d-PE tetramer, followed by MACS with anti-PE microbeads. Subsequently, enriched iNKT cells were stained for CD3 and/or CD4 and further purified on FACS Aria II cell sorter (BD Bioscience). To obtain iNKT cells from αGalCer-pre-treated mice, C57BL/6 mice were intraperitoneally injected once with 4μg αGalCer in DMSO/PBS or vehicle, and iNKT cells were purified 1 month after the αGalCer treatment as described above.
Murine bone marrow transplantation
Animal experiments were conducted in compliance with the approved protocols of the MD Anderson Institutional Animal Care and Use Committee (IACUC). Briefly, recipient BALB/c mice (H-2d) between age of 8–12 weeks were irradiated with 800 cGy using the Cesium-137 irradiator on day -1, received donor grafts consisting of bone marrow (BM, 5x106) or BM + conventional T cells (Tcon, 1x106) from either WT or Traj18KO C57BL/6 (B6, H-2b) ± iNKT cells (5x104 or 1x105) purified from WT, IL-17RBKO, IL-10KO, IL-17AFDKO, or αGalCer pretreated WT B6 mice on day 0. All recipient mice were monitored daily for survival and 2–3 times weekly for clinical signs of GVHD (skin (0:normal, 1: presence of scaling paws or tails, 2: presence of the denuded lesions), posture (0:normal, 1: kyphosis at rest, 2:kyphosis impairing movement), weight (0: weight loss <10%, 1: weight loss <25%, 2: weight loss ≥25%), activity (0:normal, 1: stationary more than 50% of the time, 2: stationary unless stimulated), fur (0:normal, 1: mild to moderate ruffling, 2: severe ruffling) as previously reported (, ).
In vitro stimulation of iNKT cells
A single-cell suspension of splenocytes was prepared from IL-10eGFP or IL-17Fate+ mice, and 2x106 splenocytes were stimulated with 100 nM αGalCer in 200 μl TCM in one well of a 96-well plate containing murine IL-2 (mIL-2, 20 ng/ml) and additional Treg-promoting TGFβ1 (5 ng/ml) or Type 1 regulatory T cell (Tr1)-promoting cytokines IL-6 (25 ng/ml), IL-23 (25 ng/ml), and TGFβ (5 ng/ml). After the indicated time for culture, iNKT cells were stained for Ghost dye V450, CD3, CD4, and iNKTCR with αGalCer/CD1d tetramer in the presence of FcR blocker (αCD16/32), fixed with 2% PFA, and acquired using LSRFortessa X-20 (BD Bioscience, Franklin Lakes, NJ) or Cytek Aurora (Cytek Biosciences). The expression of IL-10eGFP, IL-17AKatuschka, and IL-17A FateYFP of iNKT cells was assessed using FlowJo v10 (FlowJo, LLC). The experiments were performed in quadruplicate.
In vivo stimulation of iNKT cells
C57BL/6 mice were injected peritoneally with 4 μg of αGalCer in 200 μl PBS or PBS only. A month later, iNKT cells in the mice were restimulated in vivo by intraperitoneal injection of 1 μg of αGalCer in 200 ul PBS. After 90 min of in vivo restimulation, isolated splenocytes were further incubated in TCM containing GolgiStop (1:1500) and GolgiPlug (1:1000). Subsequently, the cells were stained for following surface markers: TCRβ CD4, CD8α, the iNKTCR with αGalCer/CD1d tetramer in the presence of the LIVE/DEAD Fixable Red dye and FcR blocker (αCD16/32) for 45 min at 4 °C, followed by washing. The cells were fixed with BD Fixation/permeabilization buffer for 20 min, followed by washing. The cells were stained for 45 min with BD Perm/Wash buffer containing various anti-cytokine antibodies. After washing, the cells were resuspended in PBS and subjected to acquisition using Cytek Aurora (Cytek Biosciences). The cytokine expression of iNKT cells was assessed using FlowJo v10 (FlowJo, LLC). A total of 4 mice were used per treatment group.
Analysis of iNKT cell subsets
BALB/c thymocytes were stained for TCRβ, iNKTCR, and CD4 in the presence of the LIVE/DEAD Fixable Red dye and FcR blocker (αCD16/32) for 45 min at 4 °C, washed, and fixed/permeabilized with FoxP3/Transcription factor staining kit. The cells were then stained for RORγt and PLZF in the presence of FcR blocker (αCD16/32) with perm/wash buffer for an additional 45 min, followed by acquisition using BD LSRFortessa X-20. The expression of transcription factors was assessed using FlowJo v10 (Flowjo, LLC).
Statistical analysis
The log-rank test was used to compare the differences in survival between the control and treatment groups, and Analysis of Variance (ANOVA) as well as Tukey’s Honestly Significant Difference (HSD) test was used to assess differences in variables between control and treatment groups. All statistical analyses were performed using GraphPad Prism 10 software. Statistically significant differences were deemed for any P-value less than 0.05.
Results
In ASCT, both CD4+ and CD4- iNK T cells have been shown to exert differing mechanisms of immune-regulation governed by the presence of distinct iNKT cell functional subsets (–, –). For example, CD4+ iNKT cells are enriched for NKT2 while CD4- iNKT cells are enriched for NKT17 (Figure 1A). However, their relative contribution in preventing GVHD has not been investigated in murine BMT. Therefore, we first validated the anti-GVH effects of CD4+ and CD4- iNKT cells in a major MHC-mismatched murine bone marrow transplantation (BMT) model (Figures 1B, C). Here, lethally-irradiated BALB/c recipients received donor grafts from B6 donors consisting of BM plus Tcon ± highly purified CD4+ or CD4- iNKT cells and were monitored for clinical GVHD and survival (Figure 1B). Both CD4+ and CD4- iNKT cells were similarly effective in ameliorating murine GVHD when supplemented to the donor graft, mirroring the anti-GVH effects of human CD4- iNK T cells (Figure 1C) (, ).
Figure 1
Recently, Maas-Bauer et al. reported that highly purified NKT2 and NKT17 cells controlled murine GVHD, while NKT1 showed better anti-tumor effects without significant anti-GVH effects (). Specifically, NKT2 improved clinical GVHD and GVHD-related mortality more robustly than NKT17 (). In our study, the absolute amount of NKT2 from NKT2-enriched CD4+ iNKT cells was lower than that of NKT17 in NKT17-enriched CD4- iNKT cells as indicated in examples in Figure 1A. However, NKT2-enriched CD4+ iNKT cells (lower NKT2 number) showed similar anti-GVH effects compared with NK17-enriched CD4- iNKT cells (higher NKT17 number). Thus, the results may confirm superior anti-GVH effects of NKT2 to NKT17 subset in line with the previous report ().
Independently, we confirmed the anti-GVH effects of NKT2 and NKT17 in murine BMT. Here we used donor grafts derived from Traj18(iNKT)KO C57BL/6 donors, supplemented with iNKT cells isolated from wild-type or IL-17RBKO C57BL/B6 mice (Figure 2A) (). IL-17RB+CD4+ iNKT cells produce Th-2 cytokines (IL-4, IL-9, IL-10, IL-13), whereas IL-17RB+CD4- iNKT cells produce IL-17A in an e4BP4-dependent fashion (). Thus, IL-17RBKO iNKT cells are deficient in the production of both Th2 and Th17-type cytokines and showed a complete loss of anti-GVH effects of iNKT cells when supplemented to donor grafts. This finding indicates that IL-17RB+CD4+ NKT2 and IL17RB+CD4- NKT17 subsets are responsible for preventing GVHD after BMT (Figure 2B). In other words, the remaining NKT1 in IL-17RBKO iNKT cells did not have significant anti-GVH effects, in line with the previous study ().
Figure 2
e4BP4 can also regulate the production of IL-10 in CD4+ T cells and IL17RB+ iNKT cells (, ). Thus, we investigated the relative role of NKT10 to NKT17 in preventing murine GVHD. Here, we purified iNKT cells from IL-10KO or IL17AFDKO donor C57BL/6 mice and supplemented them with Trja18KO/B6 donor grafts (BM+T) for BALB/c recipients in murine BMT (Figure 2C). The deletion of IL-10 in donor iNK T cells obliterated their anti-GVH effects, while the absence of IL17AFD in donor iNKT cells only partially compromised the anti-GVH effects.
To further confirm the anti-GVH effects of NKT10, we first enriched NKT10 in donor C57BL/6 mice with a single injection of αGalCer as previously reported (), and then purified NKT10-enriched iNKT cells and supplemented them to Traj18KO/B6 donor grafts (Figure 3A). A single injection of αGalCer has been shown to increase murine IL-10+ iNKT cells in the spleen to an average of 5% (). We also demonstrated that a single injection of αGalCer led to a significant expansion of IL-10+ iNKT cells and concurrent alteration of other NKT subsets such as a trending increase in NKT17 (p = 0.1349) and, reciprocally, a trending decrease in NKT1 and NKT2 (p = 0.2364, 0.1330, respectively) (Figure 3B). Thus, these αGalCer-pretreated iNKT cells consisted of altered iNKT subsets favoring immunosuppression and showed significantly improved clinical GVHD scores and a trend of improved survival (p = 0.1018) of the recipient mice (Figure 3C). Although the enriched NKT10 in αGalCer-pretreated iNKT cells may have significantly contributed to the improved anti-GVH effects, it may be difficult to accurately assess how altered NKT1, NKT2, and NKT17 subsets synergistically attributed to their anti-GVH effects of polyclonal iNKT cells.
Figure 3
NKT17-enriched CD4- iNKT cells exhibited similar anti-GVH effects to NKT2-enriched CD4+ iNKT cells (Figure 1C), and the loss of donor NKT10 was detrimental to the murine BMT (Figure 2C). Here, we investigated whether CD4- iNKT cells regulate the anti-GVH effects via IL-10 production. To test this hypothesis, we first assessed whether CD4+ or CD4- iNKT cells from IL-10eGFP+ splenocytes could produce IL-10 after antigenic stimulation in vitro and observed that CD4- iNK T cells produced IL-10 at a level similar to that of CD4+ iNKT cells through day 3 from the stimulation but in a lesser degree than CD4+ iNKT cells on day 5 (Figures 4A, B). Thus, NKT17 subset in CD4- iNKT cells may acquire the ability to produce IL-10 during GVHD. Similarly, this phenomenon has been described in conventional Th17 cells, which can transdifferentiate into Type 1 regulatory T cells (Tr1) after chronic inflammation ().
Figure 4
Various markers have been used to identify iNKT subsets such as transcription factors (), a combination of CD4 and CD122 (), CD43 and ICOS (), CD138 for NKT17 (), or NRP1 for NKT10 (). However, these markers may not be the best when studying the differentiation of iNKT subsets during activation as the expression may be altered upon activation. To investigate the potential transition of NKT17 to NKT10 upon activation, we utilized IL-17A Fate-mapping mice (IL-17ACre.Rosa26STOPf/fYFP) in which Cre-recombinases are transiently expressed under the IL-17A promoter and delete stop-cassettes to allow the permanent expression of YFP. In these mice, YPF marks T cells with prior and current production of IL-17 (). We generated IL17A Fate+ mice (L-17ACre.Rosa26STOPf/fYFPxIL-17AKatushkaIL-10eGFPFoxp3RFP) by crossing IL-17A Fate-mapping mice with IL-17AKatushkaIL-10eGFPFoxp3RFP triple reporter mice (). This IL17A Fate+ mice allow us to characterize IL-17AKatushka+ iNKT cells as NKT17 that currently express IL-17, IL-17A-FateYFP+ iNKT cells with a prior history of IL-17 production (exNKT17), and IL-10GFP+ iNKT cells as NKT10.
Splenocytes from IL17A Fate+ mice were stimulated with αGalCer and murine IL-2 under Treg-promoting conditions (TGFβ1), or Tr1-promoting conditions (IL-6, IL-23, and TGFβ) for 4 days. The presence of IL-17AKatushka(NKT17), IL-10eGFP(NKT10), IL-17-FateYFP(exNKT17) in iNKT cells was assessed (Figures 4C, D). Both Treg and Tr1-promoting conditions significantly expanded NKT10 after antigenic stimulation, while only Tr1-promoting conditions increased NKT17. Interestingly, the fractions of the NKT10/NKT17 hybrid subset (IL-10eGFP+ IL-17AKatushka+) and NKT10/exNKT17 (IL-10eGFP+ IL-17AKatushka- FateYFP+) were significantly increased under the Tr1-promoting condition. Our results suggest that NKT17s can transdifferentiate into NKT10s in vitro after antigenic stimulation under Tr1-promoting conditions mimicking chronic inflammation, suggesting a novel mechanism of immune-regulation by NKT17.
Discussion
iNKT cells are thought to play a role in preventing GVHD in ASCT through IL-4 production (, ), elimination of alloantigen-presenting dendritic cells (), and expansion of Tregs (). Here, we present evidence that IL-10 production by iNKT cells may contribute to their anti-GVH effects. IL-10 is a potent anti-inflammatory cytokine that controls proinflammatory gene expression on various immune cells via IL-10/IL-10R/STAT3 pathways, modulating their immune-regulating function (, ). More specifically, IL-10 plays a critical role in the immunosuppressive function of classical Tregs (). In addition, IL-10 produced by Tregs can enhance their expansion and suppressive function in an autocrine fashion (, ). Similarly, IL-10 produced by iNKT cells may help differentiate Tregs and reinforce their suppressive function to prevent GVHD. Previously, iNKT cells have been shown to facilitate donor Treg expansion in an IL-4-dependent manner in murine BMT (), suggesting that iNKT cells mediate Treg-dependent anti-GVH effects through redundant pathways. However, the relative contributions of IL-4 by NKT2 and IL-10 by NKT10 to the anti-GVH effects of iNKT cells remain unanswered and require further investigation.
ASCT is a therapy in which recipients receive donor hematopoietic stem cells from either the bone marrow or peripheral blood that contain varying fractions of mature donor immune cells (, ). Thus, the early peripheral tolerance induction of mature T cells is likely a key to reducing GVHD risk in ASCT. Graft engineering with a precise control of donor T cells and Tregs is one promising approach to achieve early peripheral tolerance (). However, iNKT cells will likely have advantages over Tregs as they can mediate additional GVL effects through innate natural killer-like properties, direct cytolysis of CD1d+ tumors, or promoting GVL effects of donor T cells (, –). Although we and others have shown that human iNKT cells in donor grafts mitigated GVHD in preclinical studies (, ), there is no clinical trial evaluating iNKT cell-based therapy to improve transplant outcome. This gap in clinical translation is due in large part to the paucity and functional heterogeneity of human iNKT cells. Here, we demonstrated that NKT10-enriched murine iNKT cells can be generated via antigenic stimulation under Treg and Tr1-promoting conditions in vitro (Figure 4). Likewise, one can potentially obtain NKT10-enriched human iNKT cells from ex vivo expansion via antigenic stimulation under similar conditions (). Thus, it may be feasible to modify donor grafts with additional NKT10-enriched human iNKT cells at a minimum dose of 0.5-1x105/kg, a number proven to be effective in improving ASCT outcomes (, ).
In summary, we provided novel insights into the regulatory role of NKT10 in BMT and the potential mechanisms through which NKT17-enriched CD4- iNKT cells prevent GVHD. Further, we demonstrated that the NKT10, NKT10/NKT17, NKT10/exNKT17 subsets could be generated by antigenic stimulation under Tr1-promoting conditions in vitro. With further investigation, this could inform a clinical strategy of modifying ASCT donor grafts with NKT10/NKT17-like subsets generated by ex vivo expansion of human iNKT cells under Tr1-promoting conditions (, ). Thus, our findings can facilitate the clinical translation of iNKT-based cellular therapy for transplantation and other autoimmune diseases.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by the MD Anderson Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
AT-O: Validation, Conceptualization, Writing – review & editing, Formal analysis, Investigation, Methodology. DB: Investigation, Writing – review & editing, Formal analysis, Data curation, Conceptualization. MG: Investigation, Writing – review & editing. JC: Writing – review & editing, Investigation. HH: Writing – review & editing, Investigation. LY: Investigation, Writing – review & editing. DL: Investigation, Writing – review & editing. QM: Conceptualization, Writing – review & editing. SC: Writing – review & editing, Conceptualization. JI: Data curation, Validation, Conceptualization, Project administration, Supervision, Funding acquisition, Writing – original draft, Methodology, Formal analysis, Investigation, Writing – review & editing, Visualization.
Funding
The author(s) declared that financial support was received for this work and/or its publication. JI was supported by New Investigator Award from American Society of Blood and Marrow Transplantation, Amy Strelzer Manasevit Scholar award from BeTheMatch, Cancer Prevention and Research Institute of Texas (RP200023), MD Anderson Cancer Start-up Fund, Careers in Immunology Fellowship from American Association of Immunology. AT-O was supported by New Investigator Award from American Society of Transplantation and Cellular Therapy, and Careers in Immunology Fellowship from American Association of Immunology. Flow Cytometry and Cellular Imaging Core Facility was supported in part by The University of Texas MD Anderson Cancer Center and P30CA016672.
Acknowledgments
We thank Drs Richard Flavell and Nicola Gagliano for providing Foxp3RFP IL-10eGFP IL-17AKata triple reporter mouse and IL-17A Fate reporter mouse (L-17ACRE Rosa26 STOPfl/fl (R26YFP), and Dr Masaru Taniguchi for providing IL17RBKO mice.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author JI declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Summary
Keywords
bone marrow transplantation, GvHD, invariant natural killer T cells, NKT10, NKT17
Citation
Trujillo-Ocampo A, Boagni D, Grefe M, Clinton J, He H, Yu L, Li D, Ma Q, Chang SH and Im JS (2026) The IL-10-producing NKT10 subset plays a critical role in preventing graft-versus-host-disease. Front. Immunol. 17:1817755. doi: 10.3389/fimmu.2026.1817755
Received
25 February 2026
Revised
07 June 2026
Accepted
18 June 2026
Published
01 July 2026
Volume
17 - 2026
Edited by
Abdel Rahim A. Hamad, Johns Hopkins University, United States
Reviewed by
Carolina P. Pacini, Universidade de Lisboa, Portugal
Ahmed Fouda, PhD, McGill University, Canada
Updates
Copyright
© 2026 Trujillo-Ocampo, Boagni, Grefe, Clinton, He, Yu, Li, Ma, Chang and Im.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Jin S. Im, jim@mdanderson.org
Disclaimer
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