Enhancing specific thymic Treg activation and function with 4-1BB monomeric streptavidin-based CARs

Hyperinflammatory diseases arise from excessive immune activation, causing tissue damage and systemic inflammation. Regulatory T (Treg) cells play a key role in maintaining immune homeostasis, but their function or numbers may be impaired in pathological conditions. Conventional immunosuppressive therapies often fail to restore immune balance and are associated with significant adverse effects. An emerging therapeutic strategy involves the use of chimeric antigen receptor (CAR)-engineered Tregs to suppress aberrant immune responses. However, antigen-specific CAR-Tregs may be insufficient due to the heterogeneity of inflammatory diseases. Universal CAR-Tregs (UniCAR-Tregs), which may recognize broad immune markers, represent a more flexible and potentially effective alternative. In this study, second- and third-generation UniCAR constructs containing a monomeric streptavidin extracellular domain were introduced into human thymus-derived Tregs (thyTregs) via lentiviral transduction. The phenotype and transcriptomic profile of engineered thyTregs were characterized and compared to unmodified controls. Their suppressive capacity was assessed in vitro using a mixed lymphocyte reaction with a biotinylated intermediary, followed by evaluation in a preclinical graft-versus-host disease (GvHD) mouse model. CARs incorporating CD28 co-stimulation resulted in non-specific activation or failed to enhance suppression. In contrast, the UniCAR41BB construct more specifically activated thyTregs and augmented their suppressive function. In vivo, UniCAR41BB thyTregs delayed GvHD onset and improved survival. This study demonstrates, for the first time, that thyTregs can be effectively transduced without compromising their regulatory phenotype. Furthermore, second-generation UniCAR41BB construct enhances antigen-dependent suppressive function, highlighting its potential as a versatile therapeutic platform for GvHD and other inflammatory disorders.