CD4⁺CD25⁺Treg numbers and function are similar in WT C57BL/6 and C57BL/6-Kit^W-sh/Wshmice and Tregs can suppress GVHD in the virtual absence of mast cells. (A) C57BL/6-Kit^W-sh/Wsh mice have similar numbers of Tregs compared with WT C57BL/6 mice as indicated by FACS analysis (n = 3). (B) In vitro proliferation of 2 × 10⁵ C57BL/6 Tcon monitored by ³H-thymidine incorporation indicates Tcon proliferation can be equally suppressed by Tregs isolated from C57BL/6-Kit^W-sh/Wsh or WT C57BL/6 mice. White bar, unstimulated responders (nonproliferation control of 2 × 10⁵ Tcon [CD4:CD8 = 2:1]); striped bar, Tcon responders with 4 × 10⁵ irradiated BALB/c splenocytes as stimulators (proliferation control); black/gray bars, Tcon + stimulators in the presence of C57BL/6 WT or C57BL/6-Kit^W-sh/Wsh Treg as indicated. Bars indicate mean ± standard error (n = 3). (C) Highly purified CD4⁺CD25⁺ Tregs (5 × 10⁵) from FVB/N donors (H-2^q) were adoptively transferred into lethally irradiated C57BL/6 mice (H-2^b) that had received TCD-BM and 1.0 × 10⁶ Tcon from FVB/N donors (to induce GVHD). WT Tregs are equally capable of suppressing GVHD (P < .05 for both groups receiving Treg vs groups receiving Tcon alone in both WT and C57BL/6-Kit^W-sh/Wsh recipients, indicating mast cells are not involved in Treg suppression of GVHD; survival data are combined from 3 independent experiments (n = 10 for Treg groups; n = 15 for all other groups).