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-2^d→H-2^b 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-mOVA^b]. As controls without aGVHD, mice received Balb/c-Thy1.1⁺ TCDBM only (TCDBM group). Four weeks after the first alloHSCT, [d→RIP-mOVA^b] mice were lethally reirradiated and retransplanted in a second syngeneic HSCT with H-2^b TCDBM from CD45.1⁺ OT-II mice mixed at a 1:4 ratio with cells from wild-type CD45.2⁺ C57BL/6 mice (H-2^b). This approach generated OT-II^b→[d→RIP-mOVA^b] chimeric mice. (B) Flow cytometry analysis for identification of Epcam⁺Ly51⁻ mTEC^low and mTEC^high cells in [d→RIP-mOVA^b] 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 mTEC^low and mTEC^high. 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 mTEC^high, 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 mTEC^high 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-mOVA^b] 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).