Tregs are subpopulations of CD4⁺ T cells specialized in immune suppression and include a variety of subsets, such as Tregs expressing transcription factor forkhead box P3 (Foxp3), T-regulatory 1 (Tr1) cells, and Th3 cells. The negative selection process in the thymus deletes T cells reactive with autoantigenic peptides presented by thymic epithelial cells in high affinity and plays a critical role in the maintenance of central immune tolerance. However, a subset of autoreactive T cells, including those reactive with autoantigenic “cryptic” peptides that are not efficiently expressed in the thymus, escape from the negative selection process and are delivered to periphery. These autoreactive T cells are subsequently suppressed or deleted in periphery through Treg-mediated mechanisms, but Treg deficiency potentially leads to onset of harmful autoimmune conditions [17]. In fact, a reduced frequency of Tregs and defective function of Tregs have been reported in patients with various autoimmune diseases, including type 1 diabetes, multiple sclerosis, and systemic lupus erythematosus [18].

Given a critical role of Tregs in preventing the autoimmune response, dysregulation of Tregs could also be associated with pathophysiology of ITP. Multiple research groups have examined quantity of Foxp3 Tregs in patients with ITP. The majority of studies demonstrated a decreased frequency of Foxp3 Tregs in peripheral blood CD4⁺ T cells in ITP patients, compared with healthy controls [8]. Some studies failed to represent a significant difference in Treg frequencies between ITP patients and healthy controls, but these inconsistent results might be explained by use of different phenotypes for identification of Foxp3 Tregs, i.e., CD4⁺CD25^high, CD4⁺Foxp3⁺, CD4⁺CD25^highFoxp3⁺, and CD4⁺CD25⁺CD127^low. The lowest Foxp3 Treg frequency is detected in patients with acute phase of the disease and/or in those with low platelet count, and Foxp3 Treg count is recovered in remission. Foxp3 Treg frequency is also decreased in the bone marrow and spleen from patients with ITP [19–21]. A recent histologic analysis of ITP spleens has found reduced Foxp3 Tregs within the germinal center and proliferative lymphoid nodule, which accommodate B cells proliferating upon antigenic stimulation. In addition, Treg’s immunosuppressive function assessed using a principle of allogeneic T-cell response is shown to be inferior in ITP patients than in controls [22–25]. Finally, several studies have evaluated serial changes in frequency and function of Foxp3 Tregs before and after the treatment. High-dose dexamethasone and rituximab increase the proportion of Foxp3 Tregs in responders [24, 26], whereas TPO receptor agonists fail to increase Treg frequency, but improve Treg function [22].

The reduced number and impaired function of Foxp3 Tregs in ITP patients suggest that dysregulation of the peripheral immune tolerance may contribute to development of ITP. This hypothesis has been tested using Foxp3 Treg-deficient mice, which are generated by transferring Foxp3 Treg-depleted CD4⁺CD25⁻ T cells isolated from BALB/c mice into syngeneic nude mice [27]. Three weeks after transfer, approximately one third of Treg-deficient mice spontaneously develop bruises in association with thrombocytopenia, which sustains for at least 12 weeks. IgG antibodies capable of binding to platelets are detected in platelet eluates and in splenocyte culture supernatants from thrombocytopenic Foxp3 Treg-deficient mice. The primary target of antiplatelet autoantibodies produced in these mice was identified as GPIb/IX [28]. These findings indicate that thrombocytopenia observed in Foxp3 Treg-deficient mice is mediated through production of IgG antiplatelet autoantibodies, which is analogous to human ITP. In addition, Foxp3 Tregs can suppress effector T-cell responses through various mechanisms, and we have found that Foxp3 Treg’s immunosuppressive property is primarily mediated through engaging cytotoxic T lymphocyte antigen 4 (CTLA4), which suppresses T-cell activation by inhibiting a critical co-stimulatory interaction between CD28 and CD80/CD86. Interestingly, we have happened to find short-term treatment with recombinant TPO could promote the peripheral induction of Foxp3 Tregs and suppress specific T-cell responses to platelet GPs in Foxp3 Treg-deficient mice [29]. This may explain why some patients treated with TPO receptor antagonists maintain their platelet count after cessation of the drug [30]. In another ITP model mouse, in which GPIIIa knockout mice are immunized with wild-type platelets and their splenocytes are transferred into severe combined immunodeficiency mice, severe fatal thrombocytopenia occurs through production of IgG antiplatelet antibodies and induction of platelet-reactive cytotoxic T cells [31]. Interestingly, in this model, Foxp3 Tregs are increased in the thymus, but are decreased in the spleen, in comparison with control mice, proposing an intriguing theory that peripheral Foxp3 Treg deficiency is caused by thymic retention [32]. Therefore, Foxp3 Treg deficiency in secondary lymphoid organs is one of the critical mechanisms that develop and maintain the pathogenic loop of ITP (Fig. 1).

Tfh cells are a specialized CD4⁺ T subset that supports B-cell maturation and differentiation within the germinal center [33]. They can be identified by a combination of markers, including high expression of chemokine C-X-C motif receptor 5 (CXCR5), inducible co-stimulator (ICOS), programmed cell death 1 (PD-1), and the transcription repressor B cell lymphoma 6. In the germinal center, Tfh cells interact with antigen-specific B cells through various interactions, such as ICOS-ICOS ligand, PD-1-PD ligand-1, CD40-CD154, and IL-21 receptor-IL-21 in an antigen-dependent and/or -independent manner, resulting in maturation into specialized memory B cells and plasma cells. Increased numbers of Tfh cells and/or dysregulated Tfh function contribute to the development of autoimmune phenotype in mouse models, and expansion of Tfh cells has been reported in peripheral blood from patients with various autoimmune diseases, including systemic lupus erythematosus, Sjögren’s syndrome, rheumatoid arthritis, myasthenia gravis, and autoimmune thyroid disease [33–35]. With respect to ITP, some studies have reported an increased proportion of circulating Tfh cells in ITP patients compared to healthy controls and a positive correlation between the percentages of Tfh cells and anti-GPIIb/IIIa or anti-GPIb/IX antibodies [36, 37]. Interestingly, an increased frequency of Tfh cells was also observed in the spleen of ITP patients, and the Tfh proportion was correlated with the expansion of the germinal center structure [4]. In addition, CXCR5 is a B-cell zone homing chemokine receptor and is required for T-cell migration into the follicles for their co-localization with B cells, in response to the specific ligand CXCL13, which is elevated in circulation of ITP patients [38, 39]. Taken together, expansion of Tfh cells in the germinal center of the spleen from ITP patients may promote generation and maintenance of B cells and plasma cells that produce high-affinity IgG antiplatelet autoantibodies, although whether this process is antigen-specific is still controversial.

T-cell-mediated cytotoxicity is an alternative mechanism for platelet destruction in patients with ITP. The involvement of CD8⁺ CTLs in pathogenic process of ITP was first shown by increased expression of genes involved in cell-mediated cytotoxicity in circulating CD3⁺ T cells derived from ITP patients [6]. In this landmark study, the authors have successfully demonstrated that CD8⁺ CTLs from ITP patients are able to induce direct platelet apoptosis or lysis in vitro. In addition, CD8⁺ T cells in the bone marrow of ITP patients are shown to suppress thrombocytopoiesis by triggering apoptosis and inhibition of maturation in megakaryocytes, leading to a decrease in platelet production, which could be successfully corrected by high-dose dexamethasone [40]. In the ITP mouse model generated by induction of the isoimmune response to GPIIIa followed by transplantation of immune repertoire into severe combined immunodeficiency mice, CD8⁺ CTLs attack megakaryocytes in the bone marrow and almost completely inhibit platelet production [31]. Using this mouse model, it has been shown that B-cell depletion by treatment with anti-CD20 monoclonal antibody results in suppression of splenic CD8⁺ CTL proliferation together with normalization of platelet counts [41]. Recently, anti-GPIbα antibodies, but not anti-GPIIb/IIIa antibodies, were shown to induce Fc-independent platelet activation, sialidase neuraminidase-1 translocation, and desialylation, leading to platelet clearance in the liver [42]. This Fc-independent platelet clearance mechanism is fundamentally different from the classical Fc-Fcγ receptor-dependent mechanism, which is mediated through macrophage phagocytosis in the reticuloendothelial system. Moreover, this analogous mechanism is mediated by CD8⁺ T cells, which induce platelet desialylation and subsequently promote platelet clearance in the liver [43]. Thus, IL-27 may be a promising therapeutic targeting CD8⁺ T cells in ITP patients because of its ability to inhibit CTL-mediated platelet destruction in vitro [44].

Several lines of recent studies indicate there is a subset of CD8⁺ cells with regulatory function called CD8⁺ Tregs [45, 46]. These Tregs exert their immunosuppressive property against effector CD4⁺ cells and B cells via a cell-cell contact through direct lysis and/or secretion of soluble negative regulates, such as IL-10 and TGF-β. In vitro co-culture studies have revealed that CD8⁺ Tregs effectively inhibited proliferation of CD4⁺ T cells and B cells, resulting in suppression of antiplatelet-autoantibody production in the murine model of ITP [7]. In addition, corticosteroid therapy decreased effector CD4⁺ T cells and B cells, but expanded CD8⁺ Tregs.

A recent accumulating evidence in ITP patients and ITP mouse models has shown that a variety of T cell subsets are involved in pathogenic process of ITP, by promoting IgG antiplatelet autoantibody production and inducing CTLs with capacity to target platelets and megakaryocytes. The elucidation of mechanisms underlying the updated “pathogenic loop” model for the pathophysiology of ITP (Fig. 1) would be useful in developing novel therapeutic strategies.