In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP.

No abstract

Our previous clinical study demonstrated that low-dose decitabine showed sustained responses in nearly half of refractory immune thrombocytopenia (ITP) patients. The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells, and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient (SCID) mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA sequencing and cytokine analysis showed that low-dose decitabine rebalanced T cell homeostasis, decreased proinflammatory cytokines, and down-regulated phosphorylated STAT3 in ITP patients. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicated that the immunomodulatory effect of decitabine provided one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

Increased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi’s) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi’s attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi’s could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to two patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.