T cells are defective in the expression of PD-1, CTLA-4, and Foxp3 at the early stages of HP. (A) Freshly isolated spleen cells were transfused into irradiated mice. After 2 weeks, splenic CD4⁺CD25⁻ T cells were isolated and stimulated with anti-CD3/CD28 for 48 hours and analyzed for CD25 expression (IL-2α receptor). Almost all HP T cells were activated, as shown by up-regulation of CD25. Similar results were obtained with conventional (non-HP) T cells (data not shown). (B) Freshly isolated spleen cells were transfused into irradiated mice. After 2 weeks and 3 weeks, splenic CD4⁺CD25⁻ T cells were isolated and stimulated with anti-CD3/CD28 for 48 hours and analyzed for PD-1 expression. Few HP T cells up-regulated PD-1 expression at 2 weeks, but this was restored to normal levels at 3 weeks. (C) Same as panel B, but analyzed for cytoplasmic and membrane CTLA-4. In panels A-C, dotted line histogram indicates isotype control; solid line histogram, specific antibody. Low expression of CTLA-4 followed the same pattern as PD-1 expression. (D) Same as panel B, but stimulation was in the absence (dotted line histogram) or presence (solid line histogram) of 2 ng/mL TGF-β1, and analyzed for Foxp3 expression. At 2 weeks, Foxp3 was poorly induced in HP T cells compared with control T cells, but this was restored at 3 weeks. (E) Percentage of Foxp3⁺ cells induced by TGF-β as in panel D (mean ± SEM; n = 4), at 2 weeks (HP 2 wk) or 3 weeks (HP 3 wk) after cell transfer. The expression of PD-1, CTLA-4, and Foxp3 was severely deficient at 2 weeks (n = 4, P < .05), but completely restored at 3 weeks. In all cases, 4 mice per group were examined and a representative histogram is shown.