CD28 costimulatory domain protects against tonic signaling-induced functional impairment in CAR-Tregs

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance in transplantation. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling resulting from ligand-independent baseline activation. Tonic signaling may cause CAR-T cell dysfunction, especially when the CAR structure incorporates the CD28 costimulatory domain (CSD) rather than the 4-1BB CSD. Here, we explored the impact of tonic signaling on human CAR-Tregs according to the type of CSD. Compared to CD28-CAR-Tregs, 4-1BB-CAR-Tregs showed enhanced proliferation and greater activation of MAP kinase and mTOR pathways but exhibited decreased lineage stability and reduced abilities to produce IL-10 and be restimulated through the CAR. Although both CAR-Treg populations were suppressive in vivo, cell tracking with bioluminescence imaging found longer persistence for CD28-CAR-Tregs than for 4-1BB-CAR-Tregs. This study demonstrates that CD28-CAR best preserves Treg function and survival in the context of tonic signaling, in contrast with previous findings for Tconvs. SUMMARY Lamarthée et al investigated the impact of chimeric antigen receptor (CAR) tonic signaling on CAR-engineered Tregs according to the incorporated costimulatory domain (either CD28 or 4-1BB). CD28 ameliorated Treg stability, long-term survival and function compared to 4-1BB. ### Competing Interest Statement The authors have declared no competing interest. * CAR : Chimeric Antigen Receptor CD : Cluster of differentiation CSD : Costimulatory domains CTLA4 : Cytotoxic T-lymphocyte antigen 4 EC : Endothelial Cells EFs : Elongation Factor 1-α shortened EGFRt : truncated Epidermal Growth Factor Receptor FOXP3 : Forkhead-box protein 3 FVD : Fixable Viability Dye GMP : Good Manufacturing Practice GVHD : Graft-versus-host disease MFI : Mean Fluorescence Intensity MOI : Multiplicity of Infection mTOR : mechanistic Target Of Rapamycin mTORC : mTOR complex PBMCs : Peripheral Blood Mononuclear Cells PGE2 : Prostaglandin E2 qPCR : quantitative Polymerase Chain Reaction RFI : Ratio of fluorescence intensity scFv : Single-chain variable fragment T2A : Thosea asigna virus 2A Tconvs : Conventional T cells TNFRSF : TNF Receptor Superfamily Tregs : T regulatory cells TSDR : Treg-Specific Demethylated Region VCN : Vector Copy Number