Functional effects of chimeric antigen receptor co-receptor signaling domains in human Tregs

Abstract

Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptor (CARs) are a potent immunosuppressive cellular therapy in multiple disease models. To date the majority of CAR Treg studies employed second generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3z, but it was not known if this CAR design was optimal for Tregs.Using an HLA-A2-specific CAR platform and human Tregs, we compared ten CARs with different co-receptor signaling domains and systematically tested their function. Tregs expressing a CAR encoding wild-type CD28 were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and ability to suppress CD80 expression on DCs as key in vitro predictors of in vivo function.This comprehensive study of CAR signaling-domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.

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

“…In line with a recent report (Dawson et al, 2020), we observed that the proliferative capacity of CAR-Tregs greatly varied between CD28 CAR-Tregs and 4-1BB CAR-Tregs. Indeed, 4-1BB CAR-Tregs had achieved 20-fold greater expansion than their CD28 counterparts at day 21.…”

“…These results agreed with those for human chimerism in the blood, which was strongly inhibited by cotransfer of CAR-Tregs ( Figure 6D). Strikingly, circulating CAR-Tregs were readily detected on day 24 ( Figure 6D), far later than in previous reports (Dawson et al, 2020;Nowak et al, 2018). In our hands, a sizeable population of CAR-Tregs could be detected in the blood (median: 4.4 [0.2-9.1]% of circulating cells) as late as day 60-62 for CD28 CAR-Tregs, which was when mice were sacrificed ( Figure Supp 3A).…”

“…Adoptive regulatory cell therapy represents a promising strategy to promote operational tolerance in solid organ transplantation. Although substantial efforts have been made to understand the effects of the CSD in CAR-T cells, this important issue has only recently started to be investigated in Tregs (Dawson et al, 2020;Nowak et al, 2018). In this study, we used HLA-A2-targeted CARs in a transplant model to demonstrate that the CD28 CSD more strongly abates the negative impact of CAR tonic signaling on Treg lineage stability and function than does the 4-1BB CSD.…”

“…A few pioneering studies have provided proof-of-concept evidence that the Treg response can be redirected toward a donor mismatched antigen. More specifically, Human Leukocyte Antigen (HLA) A2-targeted CAR-Tregs efficiently prevent graft-versus-host disease (GVHD) and skin graft rejection in an HLA-A2-specific manner (Boardman et al, 2017;Boroughs et al, 2019;MacDonald et al, 2016;Noyan et al, 2017;Pierini et al, 2017;Dawson et al, 2020). Most of these pilot studies have used HLA-A2-targeted CARs incorporating the CD28 endodomain based on evidence that the CD28 signal is critical for Treg ontogeny (Salomon et al, 2000;Watanabe et al, 2005), survival (Zhang et al, 2013) and epigenetic stability (DuPage et al, 2015).…”

“…On the other hand, 4-1BB expression is a hallmark marker of activated human Tregs (Bacher et al, 2016;Schoenbrunn et al, 2012), and TNFRSF9 (Tumor Necrosis Factor Receptor SuperFamily 9, encoding 4-1BB) belongs to a handful of genes whose epigenetic marks are highly conserved in activated Tregs across species (Arvey et al, 2015). Emerging data suggest that the 4-1BB CSD could support greater proliferation than its CD28 counterparts at the expense of Treg suppressive function (Boroughs et al, 2019;Dawson et al, 2020;Nowak et al, 2018) .…”