Post–congenic BMT, donor Tregs show a naive, proliferative, and anti-inflammatory phenotype. Host (circles) and donor (squares) Tregs (TCRβ⁺CD4⁺CD25⁺Foxp3⁺) were isolated from spleen and LN 3 and 7 weeks post-BMT from BMT-treated mice with arthritis and PGIA controls (pooled data from 3 and 7 weeks). Each Treg set, circle, and square joined together by the line in-between derive from the same mouse. Results shown are from 2 combined experiments. (A) Percentages host vs donor naive Tregs (CD45RB^high, CD44^low). Spleen (left): PGIA control (N = 7) and BMT-treated mice (N = 4) at 3 weeks (N = 5) and 7 weeks post-BMT. LN (right): PGIA control (N = 8) and BMT-treated mice (N = 4) at 3 weeks (N = 3) and 7 weeks. (B) Percentages host vs donor proliferative Tregs (Ki-67⁺). Spleen (left): PGIA control (N = 3) and BMT-treated mice (N = 4) at 3 weeks (N = 5) and 7 weeks. LN (right): PGIA control (N = 3) and BMT-treated animals (N = 4) at 7 weeks. (C) Host vs donor-derived spleen Tregs from PGIA+BMT-treated animals. Seven weeks after BMT, spleen Tregs (TCRβ⁺CD4⁺CD25⁺) were sorted and relative messenger RNA expression of Helios (left; N = 7, 10 mice) and neuropillin-1 (right; N = 7, 10 mice) was measured by qPCR. (D) Relative expression of the anti-inflammatory cytokine marker IL-10 messenger RNA in spleen Tregs (N = 7, 10 mice). *P < .05 calculated using Wilcoxon rank test. N.D., not determined due to lack of cells.