Abstract
Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.
Highlights
Regulatory T cells (Tregs) are a small subset of CD4+ T cells that are key for maintaining self-tolerance and preventing autoimmune disease [1]
(CDRs) of the heavy and light chains of the SN607D8 scFv into the framework regions of an scFv derived from the anti-HER2 antibody Herceptin, which is known to be compatible with chimeric antigen receptor (CAR) surface expression [42]
In human leukocyte antigen (HLA)-A2 transgenic mice reconstituted with HLA-A2+ Peripheral blood mononuclear cells (PBMCs), we found that the circulating HLA-A2– cells, i.e. the engineered A2 CAR (A2-CAR)+TCRdeficient Tregs, remained FOXP3+ (Figure 6A), albeit with limitations in the number of acquired events due to the marked decrease in the number of CD4+ and CD4+HLA-A2- cells over time (Figure 6B)
Summary
Regulatory T cells (Tregs) are a small subset of CD4+ T cells that are key for maintaining self-tolerance and preventing autoimmune disease [1]. A plethora of preclinical models have shown that the infusion of Tregs can suppress graft rejection and promote transplant tolerance [2]. Several phase I/II clinical studies using Tregs have been reported [3, 4]. The ONE Study is the largest coordinated international study of regulatory cell therapies in kidney transplantation. The study includes 28 patients who received Treg therapy in 4 nonrandomized single-arm phase I/IIa trials. While a significant fraction of Tregs in the polyclonal pool can react to allogeneic donor antigens, data from preclinical models show that donor-reactive Tregs are more effective than polyclonal Tregs in promoting transplant tolerance [6]. Donor alloantigen-reactive Tregs may be functionally altered or induced to migrate out of the peripheral blood following transplantation, limiting the frequency of alloantigen-reactive clones within polyclonal Treg products and thereby posing challenges for consistent expansion of donorreactive Tregs [2]
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