Impaired negative regulation of homeostatically proliferating T cells by Anna Shvets, Rabindranath Chakrabarti, Rosana Gonzalez-Quintial, Roberto Baccala, Argyrios N. Theofilopoulos, and Gérald J. Prud'homme Blood Volume 113(3):622-625 January 15, 2009 ©2009 by American Society of Hematology

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. 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. Anna Shvets et al. Blood 2009;113:622-625 ©2009 by American Society of Hematology

HP T cells are weakly suppressed by CTLA-4 and B7-H4. HP T cells are weakly suppressed by CTLA-4 and B7-H4. (A) Splenic CD4+CD25− T cells recovered 2 or 3 weeks after cell transfer and stimulated with plate-bound anti-CD3/CD28, to induce CTLA-4 expression, and restimulated with anti-CD3 in the presence of anti–CTLA-4 (or control IgG). Supernatants were assayed for IFN-γ and IL-2. Results are presented as percentage suppression = 100 × (cytokine release in the absence of anti–CTLA-4 − cytokine release in the presence of anti-CTLA-4)/cytokine release in the absence of anti–CTLA-4. indicates normal; □, HP 2 weeks; and ■, HP 3 weeks. Anti–CTLA-4 exerted significant inhibitory effect on normal and HP 3-week cells (P < .05). ** indicates IFNγ production was increased by 34% plus or minus 8%, rather than suppressed. (B-C) HP T cells (■) or control T cells (▲) were recovered 25 days following cell transfer and stimulated with anti-CD3/anti-CD28, in the presence or absence of B7-H4/Ig. Supernatants were assayed for IFNγ (B) and IL-2 (C). Similar results were obtained at 21 days after cell transfer (data not shown). * indicates HP T cells are significantly different from normal T cells (P < .05). (D) B7-H4/Ig costimulation enhances IL-10 production in normal (▲) but not HP T (■) cells (P < .05). The experimental protocol is same as panels B and C. The results are the mean plus or minus SEM of 3 mice. Anna Shvets et al. Blood 2009;113:622-625 ©2009 by American Society of Hematology