A BACH2-STAT5 transcriptional module licenses a short intracellular C3 proteoform in activated CD4+ T cells 2308647

Abstract

IL-2-STAT5 signaling is vital for CD4+ T cell function, whereas BACH2 restrains activation. How these transcription factors co-operate at the genome remains unclear. Intracellular C3 supports T cell survival and effector programming, but how it is transcriptionally controlled and whether it matches serum C3 is unknown. We hypothesized that a BACH2-STAT5 module coordinates C3 expression and licenses a distinct C3 proteoform in activated CD4+ T cells. We profiled wild-type and Bach2—/— CD4+ T cells by RNA-seq and mapped STAT5 and BACH2 binding using ChIP-seq and H3K27ac Hi-ChIP. STAT5 and BACH2 were re-expressed in Stat5—/— T cells, and shared elements at the C3 locus were deleted by CRISPR. IL-2/STAT5 inhibition, C3 overexpression or knockdown, and complement activation assays defined C3 expression and function. AlphaFold2 modeling and domain analyses characterized novel C3 proteoforms. IL-2/STAT5 target genes were enriched in Bach2—/— CD4+ T cells, with overlapping STAT5 and BACH2 binding at differentially expressed loci. Combined activating or repressive control was confirmed by re-expression in Stat5—/— cells. At the C3 locus, BACH2-STAT5 co-occupancy and IL-2/STAT5 signaling were required for C3 transcription; C3 was lost in Bach2—/— cells, after IL-2/STAT5 blockade, or upon CRISPR deletion of shared elements. Resting CD4+ T cells expressed canonical, signal peptide-containing C3, whereas activation induced a shorter, signal peptide-deficient transcript predicted to encode an intracellular proteoform. Purified short C3 failed to seed classical C3 convertase or support hemolysis, indicating altered function. We identify a BACH2—STAT5 transcriptional module that is essential for C3 expression in activated CD4+ T cells and redirects C3 output toward a short, intracellular proteoform with noncanonical complement activity. Proteoform-specific, T cell-intrinsic complement suggests new opportunities to target BACH2-STAT5 and C3 in autoimmunity and immunotherapy. NA Immune Response Regulation: Molecular Mechanisms (IRM)