Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection

Abstract

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2–specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti–HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

CAR Tregs mediate linked suppression and infectious tolerance in islet transplantation

“…Linked and bystander suppression are thought to be mediated at least in part through Treg-mediated induction of tolerogenic APCs, via removal of co-stimulatory molecules and MHC molecules ( 29 ). To test if NOD A2-CAR Tregs influenced APC activity, the former were co-cultured with HLA-A2 negative or HLA-A2 + NOD splenic dendritic cells (DCs) ( 16 ). After 48 hours, DCs were analyzed for expression of the co-stimulatory molecules CD80 and CD86, and the NOD MHC class II molecule, I-A g7 .…”

“…Our findings raise the question of where A2-CAR Tregs mediated their effects. Although A2-CAR Tregs suppress DC function ( 16 ), NSG mice have dysfunctional DCs and small, hypocellular lymphoid organs ( 42 ), so in this model, A2-CAR Tregs most likely suppress BDC2.5 T cells at the site of transplantation, resulting in a systemic effect. This possibility is supported by intravital imaging studies of mice with ACE islet transplants and injected with BDC Teff and Tregs which showed that Tregs create long-lasting bonds with BDC2.5 T cells, more so than with DCs, in a partially CTLA-4-dependent manner ( 33 ).…”

“…This assay has been previously described ( 16 ). Spleens were collected from adult, sex-matched NOD/ShiLtJ or NOD-HLA-A2 mice and enzymatically digested (STEMCELL Technologies).…”

“…Focusing on CARs, we and others developed CARs which recognize HLA-A2 (A2-CAR), a commonly mismatched protein in organ transplantation. Tregs engineered to express an A2-CAR suppress anti-HLA-A2-directed alloimmunity significantly better than polyclonal Tregs in multiple humanized and mouse transplant models ( 9–16 ). This pre-clinical work led to rapid clinical translation and two ongoing clinical trials of A2-CAR Tregs in kidney and liver transplantation (( 17 ) NCT04817774 for kidney, NCT05234190 for liver).…”

Cellular Strategies for Separating GvHD from GvL in Haploidentical Transplantation

“…According to a study conducted by Boroughs and colleagues, using a 4-1BB-based CAR in Tregs had a detrimental impact on their ability to carry out their regulatory functions [99]. Another study by Dawson et al conducted an extensive investigation into how various co-stimulatory domains influence the function of an anti-HLA-A2 CAR in an allotransplantation model [100,101]. Their data revealed that the CAR encoding CD28 was more effective both in vitro and in vivo concerning proliferation, suppression, and the delay of GvHD symptoms, while the presence of 4-1BB-CAR had a negative impact on Treg function and stability [100].…”