Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura

Abstract

Immune thrombocytopenic purpura (ITP) is characterized by the presence of antiplatelet autoantibodies as a result of loss of tolerance. CD4+CD25+ regulatory T cells (Tregs) are important for maintenance of peripheral tolerance. Decreased levels of peripheral Tregs in patients with ITP have been reported. To test whether inefficient production or reduced immunosuppressive activity of Tregs contributes to loss of tolerance in patients with chronic ITP, we investigated the frequency and function of their circulating CD4+CD25(hi) Tregs. We found a com-parable frequency of circulating CD4+CD25(hi)Foxp3+ Tregs in patients and controls (n = 16, P > .05). However, sorted CD4+CD25(hi) cells from patients with chronic ITP (n = 13) had a 2-fold reduction of in vitro immunosuppressive activity compared with controls (n = 10, P < .05). The impaired suppression was specific to Tregs as shown by cross-mixing experiments with T cells from controls. These data suggest that functional defects in Tregs contribute to breakdown of self-tolerance in patients with chronic ITP.

Figures

Figure 1

Treg frequency and function in patients with chronic ITP. (A) Representative dot plot of CD25 and Foxp3 reactivity of gated CD4+ T-cell population in PBMCs. Isotype control for anti-Foxp3 antibody was used to set the gates for calculating the percentage of the Foxp3+CD25hi cells within the CD4+ subset. (B) The percentages of Foxp3+CD25hi cells in controls and patients with chronic ITP as calculated by the gating strategy shown in panel A. (C) Representative dot plot of CD4 and CD25 in total PBMCs with the box showing the CD4+CD25hi population. Below the dot plot is the histogram showing the levels of Foxp3 reactivity in the CD4+CD25hi subset expressed as percentage. The gating was set using isotype control for anti-Foxp3 antibody. (D) The levels of Foxp3 reactivity in controls and patients shown as percentages based on gating strategy in panel C. Horizontal bars represent the medians in panels B and D. (E) The mean percentage inhibition of the proliferative response of stimulated CD4+CD25− cells by autologous CD4+CD25hi cells from 13 of the chronic ITP patients (■) and 10 healthy controls (□) was calculated (“Proliferation assay”). CD4+CD25hi and CD4+CD25− sorted cells were stimulated with plate-bound 0.1 μg/mL anti-CD3 antibodies and allogeneic accessory cells, alone or cocultured at various suppressor-responder ratios (1:1, 1:4, and 1:16). Addition of CD4+CD25hi cells at 1:1 ratios to CD4+CD25− cells inhibited proliferative responses and decreasing the number of CD4+CD25hi cells resulted in less inhibition of proliferation. For comparison, percentage inhibition of proliferation of CD4+CD25hi when stimulated alone (1:0) is also shown. Regulatory T cells from patients with chronic ITP showed significantly less suppressor activity. Error bars depict standard error of the mean. (F) CD4+CD25− responder T cells from healthy donors (n = 5) were cocultured with CD4+CD25hi cells from patients with chronic ITP (n = 5) using the same conditions as in panel E, and mean percentage inhibition was calculated for the various responder-suppressor ratios (△). For comparison, the mean percentage inhibition of autologous proliferative responses of patients (n = 13) and controls (n = 10) from panel E is shown. The P values reflect the difference between the suppression of proliferation of CD4+CD25− responder T cells from healthy donors using CD4+CD25hi from healthy controls versus CD4+CD25hi from patients. (G) Responder cells from patients with chronic ITP (n = 3) were cocultured with CD4+CD25hi from healthy controls (n = 3) as in panel E, and mean percentage inhibition was calculated (◇). For comparison, the mean percentage inhibition of autologous proliferative responses from panel E is shown. The P values reflect the difference between the suppression of proliferation of CD4+CD25− responder T cells from patients with chronic ITP using CD4+CD25hi from healthy controls versus autologous patient CD4+CD25hi.