Optimizing chimeric antigen receptors to minimize pro-inflammatory programs in human regulatory T cells 3265

Abstract Description Regulatory T cells (Tregs) are essential for immune tolerance and hold therapeutic promise in autoimmunity and transplant rejection. 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 in conjunction with non-specific immunosuppression. In contrast, antigen-specific Tregs alone reverse autoimmune disease. We hypothesized that CAR signaling leads to diminished Treg activity compared to endogenous TCR. We found that CAR-activated Tregs remained stable (FOXP3+HELIOS+) but exhibited reduced suppression of antigen-presenting cells and effector T cells. Surprisingly, CAR Tregs secreted high levels of pro-inflammatory mediators, such as IFNγ, and acquired CD40L expression. Single-cell RNA-seq identified a pro-inflammatory FOXP3+HELIOS+ Treg subset uniquely induced by CAR signaling. Transcription factor enrichment analysis revealed elevated IER3 and reduced BATF expression in this subset. Ectopically expressing IER3 and BATF skewed CAR Tregs towards a more pro-inflammatory and anti-inflammatory phenotype, respectively. Moreover, reducing CAR affinity 40-fold mitigated pro-inflammatory activity by CAR Tregs. These findings underscore receptor affinity as a critical modulator of CAR Treg biology. Ongoing studies are elucidating the signaling cascades triggered by high-affinity CAR binding in Tregs and dissect their impact on CAR Treg function. Funding Sources Human Islet Research Network (HIRN, NIDDK) U24DK104162-07; American Cancer Society (ACS) IRG-19-137-20; Diabetes Research Connection (DRC) IPF 22-1224; Swim Across America (SAA) grant 23-1579; 5T32GM132055; Hollings Cancer Center (HCC) Lowvelo Graduate Fellowship. Topic Categories Therapeutic Approaches to Autoimmunity (THER)