Low expressed SRSF1 promotes lck exon 8 skipping to attenuate TCR signaling and treg induction in immune thrombocytopenia

Abstract Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by platelet destruction and impaired production. A key feature involves dysfunctional immune regulation, particularly impaired regulatory T cell (Treg) frequency and function. While Treg deficiencies in ITP patients are well-documented, their molecular mechanisms remain unclear. Emerging evidence suggests that post-transcriptional regulation, particularly alternative splicing, plays a crucial role in immune dysfunction. However, the potential involvement of splicing dysregulation in ITP pathogenesis remains unexplored. The LCK gene encodes a lymphocyte-specific tyrosine kinase essential for TCR signaling and contains alternatively spliced exons influencing T cell activation. Given TCR signaling's importance in Treg development, we hypothesized that aberrant LCK splicing contributes to Treg impairment in ITP. Methods: To investigate this hypothesis, we performed RNA sequencing analysis of purified CD4+ naive T cells isolated from peripheral blood samples of treatment-naive ITP patients and age/sex-matched healthy controls. Bioinformatics pipelines were employed to identify differentially spliced transcripts and evaluate global alternative splicing patterns. For mechanistic studies, we constructed an LCK minigene system to specifically examine the regulation of exon 8 splicing. Potential splicing factors interacting with LCK were systematically screened using RNA pull-down assays coupled with mass spectrometry analysis. Candidate factor binding was subsequently validated through RNA chromatin immunoprecipitation (RNA-ChIP) experiments. Functional consequences were assessed through multiple approaches: TCR activation status was measured by phosphorylation of ZAP70 and CD3ζ using western blot; in vitro Treg induction capacity was evaluated using standard differentiation assays; and in vivo relevance was tested through adoptive transfer experiments using CD61 knockout mice as an established ITP model system. Molecular interactions between LCK isoforms and downstream signaling components were examined by co-immunoprecipitation followed by immunoblot analysis. Results: CD4+ naive T cells from ITP patients exhibited attenuated TCR signaling strength and significantly impaired capacity for in vitro Treg induction compared to healthy controls. Transcriptome analysis further revealed widespread dysregulation of alternative splicing patterns in CD4+ naive T cells from ITP patients, with particularly prominent skipping of exon 8 in the LCK transcript. Through systematic screening, we identified the splicing factor SRSF1 as a key regulator of LCK exon 8 inclusion, with RNA-ChIP assays confirming direct binding of SRSF1 to this specific exon. Notably, SRSF1 expression was significantly reduced in CD4+ naive T cells from ITP patients. Functional validation using antisense oligonucleotides (ASOs) designed to target the SRSF1 binding site effectively enhanced exon 8 skipping and, importantly, led to reduced TCR activation in Jurkat cells. While sequence analysis revealed that murine LCK lacks two human-specific recursive splicing sites, these two recursive splicing sites were found to be essential for exon 8 skipping in human LCK. In our murine ITP model, adoptive transfer experiments demonstrated that CD4+ naive T cells overexpressing the exon 8-skipped murine LCK variant showed significantly impaired Treg induction capacity compared to cells expressing full-length murine LCK. At the molecular level, co-immunoprecipitation experiments revealed that the exon 8-skipped LCK isoform exhibited significantly reduced binding affinity for ZAP70, providing a plausible mechanistic explanation for the observed attenuation in TCR signaling strength. Conclusions: Our findings establish a novel SRSF1-LCK splicing regulatory axis as a critical determinant of Treg biology in ITP. The downregulation of SRSF1 in ITP patients promotes preferential skipping of LCK exon 8, resulting in impaired interaction with ZAP70 and consequent attenuation of TCR signaling, which ultimately compromises proper Treg induction and function. These results provide important new insights into ITP pathogenesis and suggest potential therapeutic opportunities through splicing modulation. The identification of splicing dysregulation in immune tolerance breakdown may have broader implications for understanding and treating other Treg-related autoimmune disorders.