Tu160 - Chimeric Antigen Receptor Induced Signaling Imbalance Compromises Human Regulatory T Cell Function

Abstract Text: Regulatory T cells (Tregs) hold tremendous promise as living therapeutics against autoimmunity and transplant rejection. Antigen specificity is key for Treg efficacy. Yet, while chimeric antigen receptor (CAR) engineering enables new antigen specificities, CAR Tregs have shown limited efficacy in solid organ transplant rejection and autoimmune disease models, even when combined with immunosuppressive regimens. In contrast, antigen-specific T cell receptor (TCR) transgenic Tregs alone reverse autoimmune disease. We hypothesized that CAR signaling fails to fully recapitulate TCR/CD28 signaling in Tregs. Single-cell RNA-seq of Tregs and effector T (Teff) cells activated via CAR or TCR/CD28 identified a pro-inflammatory FOXP3+HELIOS+ Treg subset uniquely induced by CAR signaling. These cells were characterized by high expression of inflammatory cytokines and chemokines, such as IFNy, granzyme B, and CD40L at the mRNA and protein levels. Gene pathway analysis revealed that CAR, but not TCR/CD28, uniquely induces noncanonical NF-kB pathway activity in Tregs to levels comparable to those in activated Teff cells. Consistently, NF-kB inhibition reduced IFNy by CAR Tregs. Interestingly, reducing CAR affinity by 40-fold rescued noncanonical NF-kB pathway activity to the level found in TCR/CD28 activated Tregs. Transcription factor enrichment analysis revealed reduced BATF expression specifically in high affinity CAR Tregs. Ectopically expressing BATF in CAR Tregs curbed IFNy production and enhanced suppressive function. These findings shed light on key signaling pathways controlling CAR Treg biology. Ongoing studies are dissecting the impact of NF-kB and BATF on CAR Treg function in humanized mice towards designing the next generation of CAR Treg therapies.