Key Points
Loss of thymic ectopic self-antigen expression during murine acute GVHD is responsible for the de novo generation of autoreactive T cells.
Functional impairment of the thymus medulla mechanistically links acute GVHD to posttransplantation autoimmunity.
Abstract
During acute graft-versus-host disease (aGVHD) in mice, autoreactive T cells can be generated de novo in the host thymus implying an impairment in self-tolerance induction. As a possible mechanism, we have previously reported that mature medullary thymic epithelial cells (mTEChigh) expressing the autoimmune regulator are targets of donor T-cell alloimmunity during aGVHD. A decline in mTEChigh cell pool size, which purges individual tissue-restricted peripheral self-antigens (TRA) from the total thymic ectopic TRA repertoire, weakens the platform for central tolerance induction. Here we provide evidence in a transgenic mouse system using ovalbumin (OVA) as a model surrogate TRA that the de novo production of OVA-specific CD4+ T cells during acute GVHD is a direct consequence of impaired thymic ectopic OVA expression in mTEChigh cells. Our data, therefore, indicate that a functional compromise of the medullary mTEChigh compartment may link alloimmunity to the development of autoimmunity during chronic GVHD.
Introduction
Acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD) remain primary complications of allogeneic hematopoietic stem cell transplantation (alloHSCT).1,2 Acute graft-versus-host disease is initiated by alloreactive donor T cells, which target a restricted set of tissues including the thymus.3,4 Human aGVHD predisposes to cGVHD with autoimmune manifestations that are integral components of the disease.5,6 It remains uncertain how autoimmunity is mechanistically linked to alloimmunity, but the thymus may play a role in this process.1,4,7,8
In the thymus, self-tolerance of the nascent T-cell receptor repertoire is attained through negative selection.9 Essential for clonal deletion is the exposure of developing T cells to self-antigens, including those with highly restricted tissue expression. Thymic ectopic expression of tissue-restricted peripheral self-antigens (TRA) is a distinct property of mature medullary thymic epithelial cells (mTEChigh) that express the transcription factor autoimmune regulator (Aire).10 Importantly, intimate associations exist between perturbations in TRA expression (independent of cause), and the susceptibility to autoimmunity in both animals and humans.10-12
We and others have demonstrated that mTEChigh are targets of donor T-cell alloimmunity during aGVHD,3,7,13 and that thymic aGVHD interferes with the capacity of Aire+mTEChigh to sustain TRA diversity.14 Mechanistic links between altered thymic TRA expression and hence deviations in the TRA repertoire, the thymic production of autoreactive T-cells, and ultimately their peripheral appearance during aGVHD have not yet been established. Here we provide direct evidence in transgenic mice that de novo production of TRA-specific T-cells during aGVHD is a consequence of impaired ectopic TRA expression that results from a diminished mTEChigh cell pool.
Study design
Female C57BL/6 (H-2b), Balb/c (H-2d), CBy.PL(B6)-Thy1a/ScrJ (Balb/c-Thy1.1;H-2d), B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II;H-2b), and C57BL/6-Tg(Ins2-TFRC/OVA)296Wehi/WehiJ (rat insulin promoter [RIP]-membrane-bound form of ovalbumin [mOVA];H-2b) were purchased from the Jackson Laboratory and were kept in accordance with institutional regulations. RIP-mOVA mice express a membrane-bound form of OVA (mOVA; residues139-385) under control of the RIP.15 These mice express mOVA in the pancreas, but also in the thymus specifically in mTEC.16 We bred Rag2-deficient OT-II mice, producing transgenic Vα2Vβ5 T-cell receptor (TCR) specific for OVA323-339, with B6.SJL-PtprcaPep3b/BoyJ (B6.CD45.1;H-2b) on a CD45.1+ congenic background at the Benaroya Research Institute (Seattle, WA). Thymic aGVHD (H-2d→H-2b) was induced by transplantation of Balb/c T-cells into total body irradiated and fully major histocompatibility complex (MHC)-mismatched RIP-mOVA recipients (d→RIP-mOVAb; Figure 1A; see the supplemental Methods on the Blood Web site). The thymic epithelial cell compartment was analyzed at 2 and 4 weeks after alloHSCT by flow cytometry (FACSAria; Becton Dickinson, Mountain View, CA). The mTECs were identified as cells with a CD45−EpCam+Ly51−UEA1+MHCIIlow (mTEClow) or MHCIIhigh (mTEChigh) phenotype, respectively, as described.14 To study negative thymic selection, the d→RIP-mOVAb recipients were reirradiated 4 weeks after the first alloHSCT and infused with syngeneic, rigorously (>2 log) T cell depleted OT-II bone marrow cells (TCDBM) mixed with C57BL/6 wild-type TCDBM (designated as OT-IIb→[d→RIP-mOVAb]; Figure 1A). Emergence and function of OVA-specific CD4+ T cells (CD45.1+) was tested after the second syngeneic HSCT by flow cytometry (supplemental Methods). Immunohistochemistry, polymerase chain reaction, T-cell function, and statistical analyses were performed as described before14 and in the supplement Data.
Acute GVHD reduces thymic ectopic expression of the surrogate self-antigen OVA in RIP-mOVA recipients. The mTEC compartment was analyzed in a transgenic murine model of H-2d→H-2b allo-HSCT. (A). Acute GVHD was induced in 8-week-old, lethally irradiated RIP-mOVA recipients by transfer of TCDBM mixed with Thy1.2+ splenic T-cells from Balb/c donors (TCDBM + T group). This alloHSCT setting was designated as [d→RIP-mOVAb]. As controls without aGVHD, mice received Balb/c-Thy1.1+ TCDBM only (TCDBM group). Four weeks after the first alloHSCT, [d→RIP-mOVAb] mice were lethally reirradiated and retransplanted in a second syngeneic HSCT with H-2b TCDBM from CD45.1+ OT-II mice mixed at a 1:4 ratio with cells from wild-type CD45.2+ C57BL/6 mice (H-2b). This approach generated OT-IIb→[d→RIP-mOVAb] chimeric mice. (B) Flow cytometry analysis for identification of Epcam+Ly51− mTEClow and mTEChigh cells in [d→RIP-mOVAb] mice in the absence (TCDBM group; ○) and presence (TCDBM + T group; ●) of aGVHD at 2 and 4 weeks after the first alloHSCT. The numbers shown in each flow cytometry dot plot represent frequencies (%, mean ± standard deviation [SD]) of the respective population among total mTEC. Line graphs depict absolute cell numbers of mTEClow and mTEChigh. The figure represents data from 3 independent experiments with ≥3 mice per group analyzed. *P < .05, Mann-Whitney U test. (C) Expression of mOVA mRNA was determined by quantitative polymerase chain reaction in mTEChigh, which was purified from the total residual TEC pools isolated from mice with (●) or without (○) aGVHD at 2 and 4 weeks after the first alloHSCT. Expression is shown as relative expression normalized to GAPDH. Dashed lines indicate normal mOVA mRNA expression in naïve untransplanted RIP-mOVA mice. *P < .05, Mann-Whitney U test. (D) Expression of Aire mRNA was analyzed in purified mTEChigh cells in the alloHSCT groups above. Aire expression is shown as relative expression normalized to GAPDH. *P < .05, Mann-Whitney U test. To detect Aire protein, immunohistochemistry and confocal microscope analysis was performed on thymic frozen sections taken from [d→RIP-mOVAb] mice with or without aGVHD (2 weeks). Cytokeratin-18 (CK18, blue) and CD14-positive cells (red) define cortical thymic epithelial cells (cTEC) and mTEC, respectively. Aire+ cells are shown in yellow and localize to the thymus medulla. Thymic architecture and Aire are lost during aGVHD (lower right panel).
Acute GVHD reduces thymic ectopic expression of the surrogate self-antigen OVA in RIP-mOVA recipients. The mTEC compartment was analyzed in a transgenic murine model of H-2d→H-2b allo-HSCT. (A). Acute GVHD was induced in 8-week-old, lethally irradiated RIP-mOVA recipients by transfer of TCDBM mixed with Thy1.2+ splenic T-cells from Balb/c donors (TCDBM + T group). This alloHSCT setting was designated as [d→RIP-mOVAb]. As controls without aGVHD, mice received Balb/c-Thy1.1+ TCDBM only (TCDBM group). Four weeks after the first alloHSCT, [d→RIP-mOVAb] mice were lethally reirradiated and retransplanted in a second syngeneic HSCT with H-2b TCDBM from CD45.1+ OT-II mice mixed at a 1:4 ratio with cells from wild-type CD45.2+ C57BL/6 mice (H-2b). This approach generated OT-IIb→[d→RIP-mOVAb] chimeric mice. (B) Flow cytometry analysis for identification of Epcam+Ly51− mTEClow and mTEChigh cells in [d→RIP-mOVAb] mice in the absence (TCDBM group; ○) and presence (TCDBM + T group; ●) of aGVHD at 2 and 4 weeks after the first alloHSCT. The numbers shown in each flow cytometry dot plot represent frequencies (%, mean ± standard deviation [SD]) of the respective population among total mTEC. Line graphs depict absolute cell numbers of mTEClow and mTEChigh. The figure represents data from 3 independent experiments with ≥3 mice per group analyzed. *P < .05, Mann-Whitney U test. (C) Expression of mOVA mRNA was determined by quantitative polymerase chain reaction in mTEChigh, which was purified from the total residual TEC pools isolated from mice with (●) or without (○) aGVHD at 2 and 4 weeks after the first alloHSCT. Expression is shown as relative expression normalized to GAPDH. Dashed lines indicate normal mOVA mRNA expression in naïve untransplanted RIP-mOVA mice. *P < .05, Mann-Whitney U test. (D) Expression of Aire mRNA was analyzed in purified mTEChigh cells in the alloHSCT groups above. Aire expression is shown as relative expression normalized to GAPDH. *P < .05, Mann-Whitney U test. To detect Aire protein, immunohistochemistry and confocal microscope analysis was performed on thymic frozen sections taken from [d→RIP-mOVAb] mice with or without aGVHD (2 weeks). Cytokeratin-18 (CK18, blue) and CD14-positive cells (red) define cortical thymic epithelial cells (cTEC) and mTEC, respectively. Aire+ cells are shown in yellow and localize to the thymus medulla. Thymic architecture and Aire are lost during aGVHD (lower right panel).
Results and discussion
We reported before that aGVHD causes a quantitative decline in the Aire+mTEChigh pool and consequently a less diverse TRA repertoire, thus impairing the molecular platform for central tolerance induction.14 It remained uncertain, however, whether such mechanism sufficed for the escape of TRA-specific TCR from thymic deletion. Because the precise antigen specificities of autoreactive effector T cells in cGVHD remain unidentified,17 we used mOVA as a surrogate self-antigen and tested whether loss of mOVA expression affected central deletion of OVA-specific T cells during aGVHD. We chose the OT-II→RIP-mOVA system because (1) thymic mOVA expression is restricted to mTEC16 ; (2) TCR selection against mOVA recapitulates physiological tolerance induction to TRA in the thymus medulla16,18-21 ; and (3) a reduction of mOVA mRNA in mTEC by <30% suffices for RIP-mOVA thymi to fail to delete OT-II cells.22
We studied aGVHD in lethally irradiated RIP-mOVA recipients of fully MHC-mismatched Balb/c donors (designated [d→RIP-mOVAb]; Figures 1A and supplemental Figure 1). Consistent with previous data that reduction in mTEC compartment size is a universal manifestation of thymic aGVHD,14 total mTEClow, and mTEChigh, cells were diminished in numbers to ≤103 cells/mouse at 4 weeks after alloHSCT (Figure 1B). In addition, the presence of thymic aGVHD in [d→RIP-mOVAb] mice (supplemental Figure 1) reduced global OVA mRNA levels in total residual mTEChigh cell pools isolated after transplantation (Figure 1C). Our data also consistently demonstrated a reduction in the expression of both Aire mRNA and protein as a consequence of aGVHD-mediated TEC injury (Figure 1D). Because Aire regulates OVA expression19 and because the Aire+mTEChigh subset is reduced in numbers during aGVHD,14 our data argues that loss of Aire+mTEChigh was responsible for the deficiency in thymic OVA during aGVHD.
We postulated that aGVHD interfered with negative selection of the OVA TCR because (1) Aire−/−RIP-mOVA mice cannot efficiently delete OT-II T-cells19 and (2) total thymic mOVA expression levels correlate with deletion efficacy of OVA-reactive TCR.16,18,19,21,22 To test our hypothesis, transgenic recipients with or without aGVHD were reirradiated and transplanted with syngeneic OT-II TCDBM (designated as OT-IIb→[d→RIP-mOVAb]; Figure 1A). Thymic OT-II CD4+ T-cell development was monitored by assessment of CD45.1+ cells. An adequate ratio (7:1)16,21 between CD45.1+ immature CD4+8+ (DP) and mature CD4+CD8− thymocytes (CD4SP) indicated regular deletion of OVA-specific TCR in OT-IIb→[d→RIP-mOVA] mice without disease, as expected (Figure 2A, top left). Much lower DP/CD4SP ratios were observed in transgenic recipients with aGVHD (low thymic mOVA), indicating inefficient deletion of OT-II cells. DP/CD4SP ratios were in the majority of these mice not distinguishable from ratios in OT-IIb→[d→C57BL/6] nondeleting controls (no thymic mOVA). Deficient elimination of OT-II cells in transgenic mice with aGVHD was substantiated by twofold to threefold higher frequencies of CD45.1+CD4SP among total thymic CD4SP cells when compared with mice without aGVHD (Figure 2A, top right; supplemental Figure 2). Thus, an aGVHD-mediated loss of OVA expression in mTEChigh resulted in an unopposed escape of “forbidden” OVA-specific Vα2+Vβ5+CD4+ T-cell clones (Barnden et al.23 ; supplemental Figure 2) within the host thymus. OT-II cells were also present in the lymph nodes and spleens of transgenic mice with aGVHD (Figure 2A, bottom). Because mature OT-II T-cells were not passively transferred from donor grafts (supplemental Figure 2), formation of the peripheral OT-II pool was thymus-dependent.
OVA-specific T-cell clones escape negative selection during aGVHD. Four weeks after their first alloHSCT, the [d→RIP-mOVAb] mice with (●) or without (○) aGVHD received TCDBM (H-2b) from CD45.1+ OT-II and CD45.2+ C57BL/6 mice in a second syngeneic HSCT as described in Figure 1A. A third group included a second syngeneic HSCT into nontransgenic GVHD- recipients of a first alloHSCT (⩾ TCDBM OT-IIb→[d→C57BL/6b]). OT-II CD4+ T-cells were analyzed in primary and secondary lymphoid organs 4 weeks later in all 3 groups. (A) Upper panels: Thymic OT-II CD4+ T-cell development. Top left: the DP/CD4SP ratios between immature and mature thymocytes derived from CD45.1+ OT-II bone marrow-derived cells were calculated and are shown as mean ± SD. The figure represents data from 3 independent experiments. *P < .05, Kruskall-Wallis test with Dunn’s multiple comparison test. Top right: Flow cytometric analysis of CD4SP thymocytes (live gate defined by 4,6 diamidino-2-phenylindole− cells). The frequencies of CD45.1+ OT-II cells among total thymic CD4SP cells are shown as mean ± SD. Lower panels: Emergence of OT-II cells in the periphery. The frequencies of OT-II cells (CD45.1+CD4+) among total CD4+ T cells in the spleens and lymph nodes are shown as mean ± SD. The figure represents combined data from 3 independent experiments with ≥6 mice analyzed per group. *P < .05, Kruskall-Wallis test with Dunn’s multiple comparison test. (B) Intracellular Foxp3 expression was analyzed in splenic CD4+ T cells isolated from OT-IIb→[d→RIP-mOVAb] mice with or without aGVHD at 4 weeks after the second syngeneic HSCT. Flow cytometry plots depict surface CD45.1 and intracellular Foxp3 expression. (C) Quadrants [a], [b], [c], and [d] were further analyzed for surface expression of folate receptor 4 (FR4) and CD73. Data are representative of at least 2 independent experiments with ≥6 mice analyzed per group. (D) Cultures of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+ T-cells isolated from spleens and lymph nodes of transplanted mice were used to detect ex vivo the proliferative response to OVA323-339 peptide presented by syngeneic APC (see supplemental Methods). Histograms of CFSE fluorescence in CD4+ responder cells are shown (log fluorescence intensity and cell numbers). Data are representative for ≥6 mice analyzed per group. The data substantiate that peripheral OT-II cells are responsive to their cognate antigen and therefore do not enter into an anergic state.
OVA-specific T-cell clones escape negative selection during aGVHD. Four weeks after their first alloHSCT, the [d→RIP-mOVAb] mice with (●) or without (○) aGVHD received TCDBM (H-2b) from CD45.1+ OT-II and CD45.2+ C57BL/6 mice in a second syngeneic HSCT as described in Figure 1A. A third group included a second syngeneic HSCT into nontransgenic GVHD- recipients of a first alloHSCT (⩾ TCDBM OT-IIb→[d→C57BL/6b]). OT-II CD4+ T-cells were analyzed in primary and secondary lymphoid organs 4 weeks later in all 3 groups. (A) Upper panels: Thymic OT-II CD4+ T-cell development. Top left: the DP/CD4SP ratios between immature and mature thymocytes derived from CD45.1+ OT-II bone marrow-derived cells were calculated and are shown as mean ± SD. The figure represents data from 3 independent experiments. *P < .05, Kruskall-Wallis test with Dunn’s multiple comparison test. Top right: Flow cytometric analysis of CD4SP thymocytes (live gate defined by 4,6 diamidino-2-phenylindole− cells). The frequencies of CD45.1+ OT-II cells among total thymic CD4SP cells are shown as mean ± SD. Lower panels: Emergence of OT-II cells in the periphery. The frequencies of OT-II cells (CD45.1+CD4+) among total CD4+ T cells in the spleens and lymph nodes are shown as mean ± SD. The figure represents combined data from 3 independent experiments with ≥6 mice analyzed per group. *P < .05, Kruskall-Wallis test with Dunn’s multiple comparison test. (B) Intracellular Foxp3 expression was analyzed in splenic CD4+ T cells isolated from OT-IIb→[d→RIP-mOVAb] mice with or without aGVHD at 4 weeks after the second syngeneic HSCT. Flow cytometry plots depict surface CD45.1 and intracellular Foxp3 expression. (C) Quadrants [a], [b], [c], and [d] were further analyzed for surface expression of folate receptor 4 (FR4) and CD73. Data are representative of at least 2 independent experiments with ≥6 mice analyzed per group. (D) Cultures of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+ T-cells isolated from spleens and lymph nodes of transplanted mice were used to detect ex vivo the proliferative response to OVA323-339 peptide presented by syngeneic APC (see supplemental Methods). Histograms of CFSE fluorescence in CD4+ responder cells are shown (log fluorescence intensity and cell numbers). Data are representative for ≥6 mice analyzed per group. The data substantiate that peripheral OT-II cells are responsive to their cognate antigen and therefore do not enter into an anergic state.
In transgenic recipients with aGVHD, the fraction of C57BL/6 (CD45.1−) donor bone marrow–derived Foxp3+ regulatory T-cells (Treg) among total splenic CD4+ cells were reduced in frequency from a normal average of 10% to an average <1% (Figure 2B, upper left quadrants [a]). Among Foxp3+CD45.1− cells, some were FR4highCD73high, documenting their anergic phenotype24 (Figure 2C, far left panels [a]). In contrast, emerging OT-II (CD45.1+) cells were exclusively Foxp3− conventional T-cells whose FR4−CD73− phenotype suggested that they were nonanergic24 (Figure 2C, panels [c]). Indeed, CD45.1+CD4+ (OT-II) cells, but not CD45.1−CD4+ (non-OT-II) cells, isolated from aGVHD mice vigorously responded to OVA peptide in culture (Figure 2D).
Taken together, we provide direct evidence in transgenic mice using OVA as model TRA that intrathymic de novo production of TRA-specific CD4+ T-cells during aGVHD is triggered by impaired ectopic TRA expression. These OVA-reactive T cells are exported into a periphery that is characterized by Treg deficiency. We advocate that functional compromise of the mTEC compartment may provide a pathogenic link between alloimmunity and the development of autoimmunity.25 The identification of the specificities of autoreactive effector T cells in cGVHD will allow to test whether such a mechanism operates not only for a surrogate TRA, but is universal for thymic ectopic expression of those TRA that are present in tissues known to be targets of cGVHD.
The online version of this article contains a data supplement.
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Acknowledgments
The authors thank Dr Gabor Szinnai (University Children's Hospital, Basel) for his constructive review of our manuscript, Katrin Hafen (Basel) for expert technical help, and Nicole von Burg (Basel) for providing OVA peptide.
This work was supported by Swiss National Science Foundation (grants 310030-129838 [W.K.] and 310010-122558 [G.A.H.]), and by a grant from the Hematology Research Foundation, Basel, Switzerland (W.K.).
Authorship
Contribution: S.D. and M.H.H. designed and performed the study; M.V. performed the study; W.K. and G.A.H. shared senior authorship; W.K. and G.A.H. designed the work; and S.D. and W.K. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Simone Dertschnig, Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland; e-mail: simone.dertschnig@unibas.ch.