Chronic GVHD is associated with reduced Treg cell numbers and immune suppression. (A) Lethally irradiated C57BL/6 mice were transplanted with 10 × 10⁶ TCD BM cells and no T cells or 2 × 10⁶ or 5 × 10⁶ CD3⁺ T cells from BALB/c donors (n = 4-12, 2 experiments). (B) Clinical scores of the recipients. (C) Frequency and number of splenic CD3⁺CD4⁺FoxP3⁺ were analyzed at day 28 (n = 1-6, 1 experiment). (D) Lethally irradiated B6D2F1 mice received BMT with 5 × 10⁶ TCD BM cells and no T cells or 0.1, 0.25, or 0.5 × 10⁶ CD3⁺ T cells from C57BL/6 donors (n = 8, 2 experiments). (E) Clinical scores of the recipients. (F) Frequency and number of splenic CD3⁺CD4⁺FoxP3⁺ were analyzed at day 28 (n = 3, 1 experiment). Representative images of H&E staining of skin taken at day 28 after BMT of the recipients from the BMT described in (G) A (T-cell dose: 3 × 10⁶) and (H) D (T-cell dose: 0.5 × 10⁶). (I) Lethally irradiated B6D2F1 recipients were transplanted with 5 × 10⁶ TCD BM cells from WT.B6 or B6.CD11c.OVA donor mice with or without 0.5 × 10⁶ CD3⁺ T cells from C57BL/6 donor mice. At day 28 after BMT, CFSE-labeled and -sorted OT-II Tg T cells (2 × 10⁶ cells) were injected in intravenous CFSE dilution and analyzed 3 days later in spleen. The index of proliferation is represented (n = 5-11) from 2 experiments. A 1-way analysis of variance was performed to compare mean proliferative indexes between groups. (J) Lethally irradiated B6D2F1 or FVB/N recipients were transplanted with 5 × 10⁶ TCD BM cells from B6 or B6D2F1, respectively, with or without supplementation with CD3⁺ T cells from the same donor. Splenic donor cDC reactivity with YAe antibody was assessed by flow cytometry on day 28 after BMT. t test with Welch’s correction was performed for each system. *P < .05; **P < .01; ***P < .001; ****P < .0001 (GraphPad Prism 6.01).