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Expression of a chimeric antigen receptor specific for donor HLA class I enhances the potency of human regulatory T cells human skin transplant rejection ABSTRACT Regulatory T cell (Treg) therapy using recipient-derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically-translatable synthetic fusion proteins which can redirect the specificity of T cells towards designated antigens. We used CAR technology to redirect human polyclonal Tregs towards donor-MHC class I molecules which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3 ζ signalling domain (CAR) and one lacking an intracellular signalling domain ( Δ CAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or Δ CAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and Δ CAR Tregs preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune-mediated skin injury caused by transferring allogeneic PBMCs more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation. Cells were stained in phosphate buffered saline (PBS) supplemented with 1% heat-inactivated foetal calf serum (FCS) and 5 mM EDTA (all from Thermo Fisher Scientific) using fluorescently-conjugated antibodies specific for HLA-A2 (BB7.2), CD4 (OKT4), FOXP3 (PCH101), CD39 (eBioA1), CD69 (H1.2F3) (all from eBioscience), CD25 (4E3 or 2A3), CTLA-4 (BNI3) (all from BD Biosciences) CD127 (A019D5), CCR4 (L291H4), CCR9 (L053E8), CCR10 (5688-5), CD62L (DREG-56), integrin β 7 (FIB504), CLA (HECA-452), HLA-DR (all from BioLegend), HLA-A2 (REA142) (Miltenyi-Biotec) and HLA class I (Tu149) (Thermo Fisher Scientific). Cells were stained with PE-conjugated HLA-A*0201/CINGVCWTV and HLA-B*0702/DPRRRSRNL dextramers (Immudex) provided by Dr. Marc Martinez-Llordella; KCL, London, UK. Dead cells were excluded using live/dead near-IR staining (Thermo Fisher Scientific). Intracellular staining was performed using the eBioscience Fix/Perm kit. Cells were acquired and sorted using an LSRFortessa II and FACSAria II (BD), respectively. Data was analysed using FlowJo 7 or 10 (Tree Star). Animal models of transplantation have demonstrated that allospecific Tregs are superior to polyclonal Tregs at protecting from allograft rejection (4, 17, 19, 31-34). Here, we isolated human Tregs using GMP-compatible protocols and used CAR technology to generate donor-MHC class I-allospecific CAR Tregs which were functionally superior to polyclonal Tregs in vitro and in a human skin xenograft transplant model. These results demonstrated a promising new direction for clinical trials which are currently assessing the safety and efficacy of polyclonal Treg therapy in kidney (the ONE Study: NCT02129881) and liver (ThRIL: NCT02166177) transplant recipients (29, 30). Tregs which recognise allo-MHC-peptide complexes (direct allorecognition) and/or allopeptides presented in the context of recipient MHC (indirect allospecificity) have been shown to suppress alloimmune responses more effectively than polyclonal Tregs (4, 17-19, 31-34). However, the potential of CD4 + Tregs is limited as they are MHC class II-restricted thus activated primarily by professional APCs. CARs recognise their target antigen in an MHC-independent manner and as such, can confer specificity for donor MHC class I, an alloantigen ubiquitously expressed on tissue parenchyma throughout an allograft.