Foxp3+ regulatory T cells (Tregs) are crucial for the maintenance of immunological self-tolerance. Indeed, defective Treg compartments have been described in several autoimmune diseases, including in patients with ITP. We recently reported that ITP patients on treatment with thrombopoietic receptor (TPO-R) agonists, known to improve platelet counts, have increased Treg suppressive activity. We now find in a cohort of 31 ITP patients (median platelet count 66 ×109/L, range 5–326 ×109/L) who were tested at least at 2 different time points either before, during and after their treatment with TPO-R agents, a positive correlation between platelet counts and circulating Treg frequency (Foxp3+CD25hi, r=0.388, p=0.005) as well as in vitro suppressive activity (r=-0.556, p<0.0001), suggesting that patients with elevated platelet counts have an expanded Treg compartment, possibly due to generation of newly-formed (inducible) Tregs and/or proliferation of pre-existing Tregs. Interesting, patients' monocyte frequency were inversely correlated with platelet numbers (r=-0.467, p=0.006).and patients with platelet counts of >50×109/L had reduced frequency of CD14hiCD16neg classic monocytes compared to those with <50×109/L (5.3±0.5% versus 8± 1.2%, p=0.03) whereas both the Tregs frequency (3.4±0.1% versus 2.8±0.2%, p=0.04) and activity (p=0.003) were significantly elevated, raising the possibility that monocytes, which as mature antigen presenting state can affect Treg development, may be inhibiting Foxp3+ Treg frequency/activity in patients with ITP. As a first step to address the mechanisms of Treg expansion in patients with elevated platelet counts and to determine the role of moncytes, we investigated Foxp3 expression in ex vivo activated CD4+ cells from normal donors (n=7) and ITP patients (n=8) using flow cytometric methodology. Following 3 day stimulation of PBMCs with anti-CD3, a similar increase (p=0.9) in CD4+Foxp3+ T cell population was observed in cultures of normal (from 3.6±0.7% to 13.6±1.1%) and patients (from 2.9±1.4% to 12.6±2.1%), suggesting that mechanisms of Foxp3 upregulation after TCR triggering are intact in ITP patients. To determine whether the increase in CD4+Foxp3+ cell population was due to de novo expression of Foxp3 in non-Foxp3 CD4+ cells and/or selective outgrowth of pre-existing Foxp3+ cell population as has been reported for healthy controls, PBMCs were CFSE-labeled before stimulation with anti-CD3 and the proliferative responses and Foxp3 expression levels were analyzed after 3 days. Reports indicate that in stimulated PBMC cultures of normal volunteers, Foxp3high expression in dividing CFSElow CD4+ cells represent proliferation of pre-existing Foxp3+ cell populations whereas Foxp3 upregulation in non-dividing CFSEhigh CD4+ cells is representative of induced Foxp3+ cells and that both mechanisms account for Foxp3+Treg expansion in activated CD4+ cells. In controls and ITP patients, no significant differences were noted in the frequency of Foxp3+ cells in non-proliferating CFSEhigh (7.0±1.0% versus 9.2±1.7%, p=0.9) and dividing Foxp3highCFSElow (13.1±1.5% versus 10.5±1.2%, p=0.3) CD4+ cells, indicating that in ITP patients the proliferative responses of pre-existing Foxp3 cells as well as the ability to generate inducible Foxp3 cells are similar to those of healthy controls. To assess the role of monocytes in Foxp3 expansion, PBMCs were depleted of CD14+ fraction before stimulation with anti-CD3. Removal of CD14+ monocytes resulted in a 25% decrease in the frequency of proliferating CD4+ cells expressing Foxp3high in controls whereas it caused a 22% increase in patients (patients versus controls, p=0.02), but had a similar effect in the levels of Foxp3 expression in non-proliferating CD4+ cells in patients and controls (p=0.7), indicating that monocytes may have an inhibitory effect exclusively on expansion of pre-existing Tregs in patients with ITP. Altogether, our findings support the possibility that in ITP patients with low platelet counts, monocytes may inhibit Treg expansion and activity in response to T cell stimulation, but as platelet counts rise following treatment with TPO-R agonists, the inhibition is removed, enabling proliferation of preexisting CD4+Foxp3high T cells. Studies to characterize the “inhibitory” monocyte populations and the nature of the signals that drive Treg expansion in ITP patients are ongoing.
Bussel:Portola: Consultancy; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai, Inc.: Membership on an entity's Board of Directors or advisory committees; Cangene: Research Funding; Genzyme: Research Funding.
Asterisk with author names denotes non-ASH members.