Aiolos restrains acquisition of a cytotoxic program by CD4 +T cells

Abstract CD4 +cytotoxic T lymphocytes, or CD4-CTLs, comprise a CD4 +subset capable of performing functions normally observed in CD8 +T and Natural Killer cells. CD4-CTLs play critical roles in many immune contexts, including protective anti-viral responses to viruses such as SARS-CoV-2 and influenza. Despite their well-documented importance, the complete mechanisms that underlie their formation remain unclear. Here, we define the transcription factor Aiolos as a novel repressor of CD4-CTL differentiation. We find that Aiolos deficiency results in increased CD4-CTL responses in lungs of influenza-infected mice, as assessed by elevated expression of Granzyme B and Perforin, as well as the CTL marker NKG2A/C/E. We further find that Aiolos-deficient CD4-CTLs exhibit increased expression of transcription factors associated with cytotoxic programming, including Eomes and Blimp-1. Mechanistically, we find that Aiolos-deficient cells have a heightened sensitivity to IL-2/STAT5 signaling due to enhanced expression of IL-2 receptor subunits. This translates into increased chromatin accessibility and STAT5 association at regulatory regions of hallmark CD4-CTL genes in the absence of Aiolos. Consistent with these data, in silico analyses demonstrate that STAT5 DNA-binding motifs are significantly enriched at these same regions. Intriguingly, the STAT5 motif partially overlaps with that of the core Aiolos DNA binding motif, suggesting that Aiolos may function to broadly antagonize STAT5 activity throughout the genome. Collectively, this work establishes Aiolos as a novel repressor of cytotoxic programming in CD4 +T cells and highlights its potential as a therapeutic target for enhancing anti-viral immunity. This work was supported by grants from The National Institutes of Health AI134972 and AI127800 to K.J.O, AI156411 to P.L.C. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program. J.A.T. and S.P. are supported through funding from the Infectious Disease Institute T32 in Host-Microbe Interactions fellowship.