Impaired negative regulation of homeostatically proliferating T cells

Abstract

Acute lymphopenia-induced homeostatic proliferation (HP) of T cells promotes antitumor immunity, but the mechanism is unclear. We hypothesized that this is due to a lack of inhibitory signals that allows activation of T cells with low affinity for self-antigens. Tumors resist immunity in part by expressing inhibitory molecules such as PD-1 ligand 1 (PD-L1), B7-H4, and TGF-beta. In irradiated mice undergoing HP, we found that T cells displayed a severe deficit in the activation-induced expression of inhibitory molecules PD-1 and CTLA-4, and TGF-beta1-induced expression of Foxp3. HP T cells were also less suppressed by B7-H4/Ig and, unlike control T cells, failed to produce IL-10 in response to this molecule. This deficiency in regulation was reversed as normal T-cell numbers were restored. We conclude that T cells are weakly regulated by inhibitory molecules during the acute phase of HP, which could explain their increased effectiveness in cancer immunotherapy.

Figures

Figure 1

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.

Figure 2

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.