Abstract
IntroductionPrimary immune thrombocytopenia (ITP) is an autoimmune disease characterized by excessive platelet destruction and decreased platelet production. Although T cells are known to contribute to the pathophysiology of ITP, the details of how CD4+ T cells mediate the loss of immune tolerance in this disease remain elusive. Tacrolimus, an immunosuppressive agent, was demonstrated to be a promising therapeutic option for ITP patients in our previous reports (ASH, 2021; Br J Haematol, 2023), but its mechanism of action is not well understood. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from 14 ITP patients before and after tacrolimus treatment and from 6 healthy control (HC) individuals. We employed single-cell mass cytometry (cytometry by time-of-flight, CyTOF) with a predefined panel of 42 markers characterizing lineage, metabolism, and signaling pathways. Additionally, a 10x Genomics platform was used to perform single-cell RNA sequencing (scRNA-seq). Flow cytometry was further used to confirm the single-cell analysis. ResultsTo identify distinct and potentially targetable CD4+ T-cell subsets in ITP, unsupervised hierarchical clustering of single-cell RNA-seq data identified 15 discrete clusters. The subset among all the subgroups referred to herein as ‘cluster 15‘ was the most distinct and was significantly greater in the ITP group than in the HC group. Within cluster 15, CD4+ T cells from ITP patients expressed significantly increased levels of MKI67, CDK1, CDC6, TYMS, MTHFD2, CXCR3 and IFNG. Through pathway enrichment analysis, our data revealed that the upregulated differentially expressed genes (DEGs) in ITP patients were most prominently enriched in the cell cycle, DNA replication, one-carbon pool by folate, oxidative phosphorylation, and the Th1-, Th2-, and Th17-cell differentiation pathways. Thus, cluster 15 in ITP patients likely represents a Th1-like CD4+ T-cell subset characterized by elevated proliferation, metabolic activation, and proinflammatory features. Given the heterogeneity of T-cell activation states across clusters, pseudotime analysis was applied to deconvolve the developmental trajectory. The slingshot algorithm positioned cluster 15 in the terminal differentiation state, indicating a functionally mature effector subset marked by constitutive IFNγ secretion. The results were validated using the dynamical model of scVelo.We further investigated the heterogeneity of CD4+ T cells with CyTOF. Uniform manifold approximation and projection (UMAP) plot showed 20 clusters of CD4+ T cells. Intriguingly, CyTOF identified a similar Th1-like subset exhibiting a proinflammatory, metabolically active phenotype characterized by MTHFD2+CXCR3+CD4+ T cells. Notably, the Th1-like subset frequency was inversely correlated with the platelet count, suggesting that targeted depletion or functional inhibition of this population could ameliorate thrombocytopenia. Since the previously designed antibody panel used for mass spectrometry did not include MKI67, we were unable to detect the expression of proliferation markers in this subgroup using CyTOF. Flow cytometry analysis of PBMCs from an independent ITP patient cohort (n=15) confirmed that MTHFD2+MKI67+CXCR3+CD4+ T cells were indeed significantly expanded in ITP patients. These data suggest that we have identified a functionally distinct and potentially targetable T-cell subpopulation.Finally, we explored the effects of tacrolimus on immune cell populations in ITP patients treated with tacrolimus. As expected, patients who recovered from tacrolimus treatment had a lower proportion of the Th1-like T-cell subset. The dysregulated T-cell subset exhibited HC-like expression after treatment with tacrolimus. ConclusionsIn summary, this study uncovers substantial T-cell heterogeneity and defines a novel pathogenic Th1-like subset of PBMCs that exhibits aberrant expansion and dysfunction in ITP patients. This population is potentially amenable to therapeutic intervention to prevent pathogenic inflammatory factor production. Our data dissect the tacrolimus-driven remodeling of this pathogenic T-cell landscape, providing insights for precision ITP management.
