Defective Circulating CD25 Regulatory T Cells in Patients with Chronic Immune Thrombocytopenic Purpura.

Chronic Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by increased destruction of platelets due to the production of anti-platelet autoantibodies. Regulatory CD4⁺ T cell (Treg) populations characterized by co-expression of CD25^hi and the forkhead/winged helix transcription factor (Foxp3) play an important role in the maintenance of peripheral tolerance. Reduced levels of Foxp3 mRNA in circulating mononuclear cells (Sakakura et al., 2007) and abnormal Treg function in spleen biopsies (Liu et al., 2007) in patients with ITP have been reported, indicating that deficiency in generation and/or defective functions of Tregs contribute to loss of immunologic self-tolerance in patients with ITP. To test the hypothesis that the pathogenesis of chronic ITP may be directly related to the levels or function of circulating Tregs, we first compared the frequency of Tregs in peripheral blood of patients with chronic ITP (n=16) and controls (n=26). We found similar frequency of CD4⁺CD25^bright T cells characteristic of Treg phenotype in patients and controls (6.2% ±0.6% vs. 5.6% ±0.3%, p=0.31). In addition, we found no significant differences in the level of FoxP3-expressing CD4⁺CD25^hi Tregs (patients 78.9% ±2.6% vs. controls 85.7% ±2.4%, p=0.06) and in the frequency of FoxP3⁺CD25^bright population (patients 4.9% ±0.5% vs. controls 5.1% ±0.3%, p=0.7) as measured by intracellular staining of CD4+ T cells. To assess the regulatory function of Tregs in control versus patient samples, sorted CD4⁺CD25^hi T cells were cultured alone or in combination with autologous responder CD4⁺CD25⁻ T cells at different ratios (responder/suppressor ratios: 1:1, 1:4, and 1:16) using two different T cell receptor signal strengths. We found that when cultured alone, CD4⁺CD25^hi Tregs derived from the circulation of patients with chronic ITP (n=13) had an anergic phenotype characteristic of normal Tregs whereas CD4⁺CD25⁻ cells from both patients with chronic ITP and healthy individuals responded similarly to stimulation (p≥0.7). In contrast, in cocultures, the patient Tregs had an overall twofold lower ability to suppress the proliferation of responder CD4⁺CD25⁻ T cells as compared with CD4⁺CD25^hi cells from healthy subjects (p ≤0.02). To determine whether the decrease in Treg function was due to a defect in the CD4⁺CD25^hi Tregs or whether the effector CD4⁺CD25⁻ T cells from chronic ITP patients were resistant to suppression, we performed co-mixing experiments, in which Tregs from either healthy controls or patients with chronic ITP were cocultured with the autologous and the converse responder patient and control CD4⁺CD25⁻ cells. We found that patient CD4⁺CD25^hi Tregs (n=5) had a decreased suppressive effect on proliferation of CD4⁺CD25⁻ cells from either patients or healthy controls (suppression =44%). In contrast, CD4⁺CD25^hi Tregs from controls (n=3) suppressed the proliferation of responder cells from both patients and controls to a similar degree (suppression =74%). We therefore demonstrate that CD4⁺CD25^hi T cells are functionally defective in patients with chronic ITP and that the lack of suppression is not due to increased resistance to suppression by the responder cells. In conclusion, these data support a role for Tregs in loss of tolerance in chronic ITP, opening up the possibility of using Tregs as a therapeutic target in the treatment of chronic ITP by transferring fully functional Tregs or by restoration of Treg function.