Whole-transcriptome quantitative analysis of WT versus Ikzf3-/- CD4+ T cells cultured in Th1-polarizing or Tfh-polarizing conditions

Effective immunity to viruses such as influenza and SARS-CoV-2 requires the generation of CD4+ T cell subsets that coordinate complementary aspects of the immune response. These include T follicular helper (TFH) and T helper 1 (TH1) cells, which promote humoral and cell-mediated helper responses, respectively. A third population, CD4+ cytotoxic T lymphocytes (CD4-CTLs) facilitates clearance of infection via mechanisms normally associated with CD8+ T cells, including perforin-mediated destruction of infected cells. Here, we identify the transcription factor Aiolos as a reciprocal regulator of TFH and CD4-CTL responses. We demonstrate that Aiolos deficiency compromises TFH differentiation and antibody production during influenza infection. Conversely, we find that CD4+ T cells acquire a cytotoxic-like program in the absence of Aiolos, including increased expression of the CTL-associated transcription factors Eomes and Blimp-1. We further show that while Aiolos positively regulates the TFH transcriptional regulators Zfp831, Tcf-1 and Bcl-6, it also directly represses expression of IL-2Ra and IL-2/STAT5-driven expression of the cytotoxic gene program. Thus, our findings identify Aiolos as a pivotal regulator of TFH and CD4-CTL differentiation and highlight its potential as a therapeutic target for the manipulation of CD4+ T cell humoral and cytotoxic responses. Naïve WT or Ikzf3-/- CD4+ T cells were cultured in complete IMDM ((IMDM [Life Technologies], 10% FBS [26140079, Life Technologies], 1% Penicillin-Streptomycin [Life Technologies], and 50uM 2-mercapto-ethanol [Sigma-Aldrich]) under Th1 (5ug/mL anti-IL-4, 5ng/mL IL-12, 50U/mL IL-2) or Tfh (5ug/mL anti-IL-4, 100ng/mL IL-6) polarizing conditions on plate-bound anti-CD3 (5ug/mL) and anti-CD28 (2ug/mL) for 3 days (72 hrs). Total RNA was isolated and provided to Azenta Life Sciences for library preparation and downstream analysis.