Although impaired myeloid-derived suppressor cells (MDSCs) recently have been studied in immune thrombocytopenia (ITP), another myeloid-derived cell population signified as M2 macrophages has not been investigated properly in ITP patients. In the present study, we intended to determine the features of circulating M2-like macrophages, to examine its relationship with MDSCs, and to explore their prognostic values in ITP. Peripheral blood mononuclear cells from healthy controls and primary ITP patients were isolated to test the circulating M2-like macrophages and MDSCs. The circulating M2-like macrophage population defined as CD68+CD163+ and circulating MDSC population as CD11b+CD33+HLA-DR− were determined by flow cytometry. Plasma inflammatory cytokines were measured by multiplex ELISA. The percentages of MDSCs were found to be expanded in newly diagnosed patients of ITP, especially among those of the complete response (CR) group (p < 0.0001). Positive linear correlation was verified between percentages of M2-like macrophages and MDSCs. The same correlation was also determined in the CR group. After treatment, the percentages of M2-like macrophages and MDSCs were both increased significantly in CR group, while those patients among the PR + NR group manifested a significant numeric decrease of MDSCs but only a moderate decrease in M2-like macrophages. MIP-1α/CCL3 was negatively correlated with M2-like macrophages while MCP-1 possessed a positive correlation with M2-like macrophages, eotaxin-1/CCL11 was negatively correlated with MDSCs and interleukin-1β (IL-1β) was found to be negatively correlated with both M2-like macrophages and MDSCs. The present findings indicated critical roles of both circulating M2-like macrophages and MDSCs in ITP. The positive correlation between them might be related to inflammatory factors-mediated bidirectional interactions or partially due to their similar background patterns during differentiation. MIP-1α/CCL3, MCP-1, eotaxin-1/CCL11 and IL-1β might play a critical role in the expansion of both M2 macrophages and MDSCs population in ITP patients, which deserves further investigation.

Background: Primary immune thrombocytopenia (ITP) is an autoimmune disease. Some ITP patients are associated with pathogen infection undetected with conventional technologies. Investigating the changes of T cells and potential metabolic mechanism are important for better understanding of ITP. Methods: The study enrolled 75 newly diagnosed ITP patients. The pathogens of patients were detected by metagenomic next-generation sequencing (mNGS). Plasma lipids were measured by liquid chromatography-mass spectrometry (LC–MS). CD4 T cell and CD8 T cell were analyzed using flow cytometry. Mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential were measured by flow cytometry. Seahorse XF real-time ATP rate assay was used to investigate the change of cellular metabolism. Results: Positive plasma pathogens were detected in seven ITP patients. Of them, 5 (71.4%) positive pathogen-ITP patients were no response (NR) after first-line treatment with corticosteroids. Regulatory T cells (Tregs) increased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and healthy controls (HC). Mitochondrial membrane potential of Th17 and Tregs were decreased in positive pathogen-ITP and negative pathogen-ITP patients, compared to HC (all p < 0.05). The overall metabolism flux of positive pathogen-ITP patients was decreased, as compared to HC (p = 0.004), of them a higher proportion of glycolysis-derived ATP and a smaller proportion of oxidative phosphorylation (OXPHOS)-derived ATP were found in Tregs. The ATP rate index of Tregs was decreased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and HC (p < 0.05). Conclusions: Impaired mitochondria function of Tregs in positive pathogen-ITP patients caused a decrease of OXPHOS-derived ATP and overall metabolism flux that might be the cause of steroid resistance in ITP patients.

Background.Th17/Treg balance skews towards Th17 in ITP patient. IRF4 has been highlighted for its close relationship to the immunosuppressive function of Treg cells and the IL-17 synthesis in CD4⁺T cells. This study was aimed at examining the effects of IRF4 to the Th17/Treg cells in patients with ITP.Methods.Treg and Teff cells were isolated from PBMCs of newly diagnosed ITP patients. The percentages of CD4⁺CD25hiFoxp3⁺Treg cells and the CD3⁺CD4⁺IL-17⁺Th17 cells were detected by flow cytometry. After being cultured, the supernatants of Tregs were collected for IL-10 concentration test. The IRF4 levels of Tregs were measured. Teffs were cultured alone or with Tregs for 24 hours. Then the supernatants were collected for IL-17 concentration test. The binding intensity of IRF4 to the gene IL-10 in Treg cells was detected by ChIP-qPCR. Metabolic assays for Teffs and Tregs were performed with Agilent Seahorse XF96 Analyzer.Results.The secretion of IL-10 by Tregs was decreased in ITP patients. The intensity of IRF4 binding to IL-10 DNA of Tregs in patients was higher than that of normal controls and Teffs in ITP patients. The expressions of IRF4 of Tregs in ITP patients were remarkably lower than that of healthy controls. The percentage of Th17 cells in healthy controls was significantly increased after IRF4 mRNA silencing. Abnormal metabolism of Treg and Teff cells was found in ITP patients.Conclusion.The skewed ratio of Th17/Treg cells and dysfunction of Treg cells in newly diagnosed ITP patients was at least partly caused by IRF4 dysfunction. The underlying mechanism might be the impact of IRF4 on the metabolism of Treg and Teff cells.

Background: Primary immune thrombocytopenia (ITP) is an autoimmune disorder. The existence of autoreactive T cells has long been proposed in ITP. Yet the identification of autoreactive T cells has not been achieved, which is an important step to elucidate the pathogenesis of ITP. Methods: ITP patients’ peripheral blood was collected prior to the treatment and one month after initiating dexamethasone treatment per related therapeutic guideline. Serum cytokines were profiled to examine T cell subtypes imbalance using a protein chip. TCR Vβ analysis in CD8⁺T cells of ITP patients, and TCR CDR3 DNA sequencing of CD4⁺T and CD8⁺T cells were performed to determine the autoreactive T cells’ clones. Results: Cytokine profiling revealed imbalanced distribution of T cells subtypes, which was confirmed by CD4⁺T and CD8⁺T cells’ oligoclonal expansion of TCR Vβ analysis and TCR CDR3 DNA sequencing. VDJ segments were found to be more frequently presented in ITP patients, when compared with health controls. There was an individualized CD4⁺T cell or CD8+T cell CDR3 sequence in each ITP patient. Conclusions: The present study revealed that T cell clones expanded in ITP patients’ peripheral blood, and each clone had an individualized TCR CDR3 sequence. The expanded T cell clones preferred to use some specific VDJ segment. Further studies are warranted to get access to individualized treatment such as Car-T in patients with ITP.