SHIP-deficient CD25⁻ T cells exhibit multiple molecular features of an immunoregulatory cell and have profound immunosuppressive capacity. Single-cell suspensions were prepared from spleens and mesenteric LN of 6- to 9-week-old SHIP^−/−, SHIP^ΔIP/ΔIP, and their respective WT controls, and from polyI/C-treated MxCreSHIP^flox/flox and SHIP^flox/flox mice. (A) Representative histogram of FoxP3 expression levels in fixed CD25⁻ T cells from spleen and LN of the indicated genotype. (B) Western blot analysis of FoxP3 protein expression in FACS-purified CD25⁻ T cells from the indicated SHIP-deficient strain and WT counterpart. (C) Bar graph representing the percentage frequency of FoxP3⁺ T cells among CD25⁻ T cells and absolute numbers of CD25⁻FoxP3⁺ T cells in the spleen and LN of the indicated genotype. (D,E) FACS-purified C57BL/6J SHIP-deficient CD25⁻ T cells (labeled CD25⁻), SHIP-deficient or WT CD25⁺ T_regs (labeled CD25⁺) were mixed with FACS-purified C57BL/6J WT effector CD25⁻ T cells at the indicated ratios (suppressors/effectors) in a one-way MLR where irradiated BALB/C splenocytes were used as stimulators. This is representative of 3 independent MLR assays conducted at the indicated cell ratios. *P < .05. **P < .01. ***P < .001.