Protection from GvHD is encoded in NFATc1/ΔS⁺ T conv as well as in NFATc1/ΔS⁺ T regs. (A) Detection of CD25⁺ Foxp3⁺ cells as percentage of CD4⁺CD90.1⁺ in spleen and mesenteric LNs of aGvHD-induced mice on day 6. Transplanted allogenic luc⁺ T cells were derived from WT versus Nfatc1^deltaSUMO mice. Statistical significance was calculated using Student’s t test; *, P < 0.05; **, P < 0.01. (B) Evaluation of surface expression of TIGIT on CD4⁺CD90.1⁺ T conv (no TIGIT detectable) and CD4⁺CD90.1⁺ Foxp3⁺ T regs regained from spleen and mesenteric lymph nodes of aGvHD-induced mice on day 6 by flow cytometry. Statistical significance was calculated with Student’s t test; **, P < 0.01. (C–F) Allogenic pure CD90.1⁺ luc⁺ WT versus NFATc1/ΔS⁺ T conv from pretreated DEREG⁺ mice were transferred together with BM CD90.2⁺ luc⁻ and CD90.1⁺ luc⁻ WT versus NFATc1/ΔS⁺ T regs in all possible combinations. (C and D) Representative in vivo BLI (C) and ex vivo BLI data from internal organs (D). In vivo data were quantified on days 3 and 5 after aGvHD induction, ex vivo on day 6. Two-way ANOVA; *, P < 0.05; **, P < 0.005; ***, P < 0.001. (E) Estimation of CD25⁺ Foxp3⁺ (CD4 gate) T reg frequencies in the presence of WT versus NFATc1/ΔS⁺ T conv by flow cytometry 6 d after T conv transfer. (F) IL-2 expression per CD4⁺ T cell (mean fluorescent intensity [MFI]) 6 d after T conv transfer. (G) Frequencies of splenic IL-2⁺ CD4⁺ T cells 6 d after T conv transfer, analyzed by flow cytometry and quantified from n = 4; two-way ANOVA; *, P < 0.05.