The novel role of signal transducer and activator of transcription 5B (STAT5B) in mediating T cell differentiation and regulatory functions

Abstract

Signal transducer and activator of transcription 5B (STAT5B) is an essential transcription factor of growth hormone and common γ-chain family cytokines including interleukin (IL)-2 IL-7 and IL-15 IL-2-STAT5B pathway plays an important role in differentiation of induced regulatory T (iTreg) cells The iTreg cells are crucial for maintaining immune tolerance and controlling inflammatory diseases In recent years the clinical biological and genetic characteristics of patients with STAT5B mutations have rapidly expanded Human STAT5B mutations can cause “non-classical” growth hormone insensitivity syndrome (GHIS) immune dysregulation autoimmunity and chronic lung disease However the mechanisms of how STAT5B mutations lead to the immune dysregulation and autoimmune syndrome are still unclear In addition therapeutic options to reverse both post-natal growth retardation and immunodeficiency at present are lacking at present We recently encountered a male patient (designated patient C) who suffered from post-natal growth retardation severe skin inflammation hypergammaglobulinemia and lymphoproliferative syndrome A novel heterozygous STAT5B missense mutation (c 1281G to A；p Ala371Thr) was identified in this patient through whole exome sequencing (WES) However the relationship between the mutation and immune dysregulation syndrome is still unknown Therefore we investigated the role of heterozygous STAT5BA371T variant in the pathogenesis of immune dysregulation syndrome in this study We confirmed that the mutant STAT5B protein is expressed and tyrosine phosphorylation in response to IL-2 stimulation is normal We found normal proportion of CD4+CD25+ T cells in the peripheral blood mononuclear cell (PBMC) of the patient before T cell receptor (TCR) stimulation Furthermore significant decrease in CD4+CD25high T cells was found in the patient’s PBMC after T cell activation Given that CD25 is one of the STAT5B target genes in T cells lower CD25 expression implicates that novel STAT5BA371T variant is a hypomorphic mutation Lower CD25 expression in turn leads to lower sensitivity to IL-2 hence ineffective induction of iTreg cells To investigate the role of this STAT5BA371T mutation in T cell differentiation and gene regulation we have established a human T cell lines model which was transfected with wild type (WT) or mutant STAT5B genes Dual luciferase reporter assay indicated the STAT5BA371T mutation showed dominate negative effect and significantly diminish transcription activity of CD25 after TCR activation The data indicated that the missense mutation partially compromised STAT5B in its downstream transcriptional function with 70% of the transcription activation activity on CD25 promoter conserved We then performed RNA sequencing (RNA-seq) to identify the changes in expression of STAT5BA371T target genes after TCR stimulation Gene set enrichment analysis (GSEA) showed that genes associated with tissue resident Treg were enriched in the STAT5BA371T compared to WT cell line In conclusion our data showed that a dominate-negative heterozygous STAT5BA371T variant may cause the immune dysregulation syndrome by deterring iTreg differentiation suggesting a critical role of STAT5B in iTreg differentiation This novel mutation may provide a direction into a better understanding of the complicated roles of STAT5B in regulating the human immune system

Date of Award	2020
Original language	English
Supervisor	Chi-Chang Shieh (Supervisor)