A network of bZIP transcription factors and inhibitors enforces differentially proliferative and cytotoxic states in T cells

The bZIP family of transcription factors (TFs) plays a central role in regulating T-cell exhaustion. Their complex dimeric interactions obscure the roles of individual members, which genetic knockout or overexpression alone poorly resolve. Here, we use overexpression of full-length bZIP family TFs alongside genetically encoded, protein-based inhibitors to probe the effect of increased or decreased bZIP TF activity on CAR T-cell expansion under exhausting conditions. Enrichment of inhibitors of pro-exhaustion TFs (CREM, NFIL3) illustrated the utility of this approach. The screen enriched full-length TFs of established anti-exhaustion relevance and identified underappreciated bZIP TFs such as BATF2. Detailed comparison of several bZIP TFs and inhibitors revealed distinct effects on proliferation, persistence, and cytotoxicity. We investigated combinations of pro-cytotoxic JUN, pro-proliferative BATF2, and a novel inhibitor (AEL-CREM) of pro-dysfunction CREM. Distinct pairs were more advantageous for maintenance of cytotoxicity or proliferation, revealing relationships between BATF2, JUN-, and CREB1-family bZIPs. Using a combination of epistasis, transcriptome, proteome, and AP-MS interactome analyses of BATF2 and AEL-CREM, we discovered functional overlap in their inhibition of pro-dysfunction CREB1-family activity, potentially driven by unexpected binding of CREB1-family members by overexpressed BATF2. Their function diverged in BATF2's early promotion of pro-proliferation AP-1 activity and later physical and functional interactions with BACH2, contributing to persistence. These findings were mirrored in murine chronic viral infection and syngeneic tumor models, where BATF2 or AEL-CREM overexpression were notably pro-proliferative and anti-exhaustion. Together, these results illuminate new connections and functional roles enabled by bZIP family perturbations, with implications for modulating T-cell state.