OVA-specific T-cell clones escape negative selection during aGVHD. Four weeks after their first alloHSCT, the [d→RIP-mOVA^b] mice with (●) or without (○) aGVHD received TCDBM (H-2^b) 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-II^b→[d→C57BL/6^b]). 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-II^b→[d→RIP-mOVA^b] 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 OVA_323-339 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.