Impaired thymic expression of tissue-restricted antigens licenses the de novo generation of autoreactive CD4⁺ T cells in acute GVHD

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 (mTEC^high) 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 mTEC^high are targets of donor T-cell alloimmunity during aGVHD,^3,7,13  and that thymic aGVHD interferes with the capacity of Aire⁺mTEC^high 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 mTEC^high cell pool.

Female C57BL/6 (H-2^b), Balb/c (H-2^d), CBy.PL(B6)-Thy1^a/ScrJ (Balb/c-Thy1.1;H-2^d), B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II;H-2^b), and C57BL/6-Tg(Ins2-TFRC/OVA)296Wehi/WehiJ (rat insulin promoter [RIP]-membrane-bound form of ovalbumin [mOVA];H-2^b) 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; residues_139-385) 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 OVA_323-339, with B6.SJL-Ptprc^aPep3^b/BoyJ (B6.CD45.1;H-2^b) on a CD45.1⁺ congenic background at the Benaroya Research Institute (Seattle, WA). Thymic aGVHD (H-2^d→H-2^b) 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-mOVA^b; 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⁺MHCII^low (mTEC^low) or MHCII^high (mTEC^high) phenotype, respectively, as described.¹⁴  To study negative thymic selection, the d→RIP-mOVA^b 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-II^b→[d→RIP-mOVA^b]; 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.

We reported before that aGVHD causes a quantitative decline in the Aire⁺mTEC^high 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 medulla^16,18-21 ; 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-mOVA^b]; 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 mTEC^low, and mTEC^high, 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-mOVA^b] mice (supplemental Figure 1) reduced global OVA mRNA levels in total residual mTEC^high 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⁺mTEC^high subset is reduced in numbers during aGVHD,¹⁴  our data argues that loss of Aire⁺mTEC^high 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-II^b→[d→RIP-mOVA^b]; 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-II^b→[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-II^b→[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 mTEC^high 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.

In transgenic recipients with aGVHD, the fraction of C57BL/6 (CD45.1⁻) donor bone marrow–derived Foxp3⁺ regulatory T-cells (T_reg) 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 FR4^highCD73^high, 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 T_reg 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 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.).

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.