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• W2563656670 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 that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR 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 peripheral blood mononuclear cells 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. 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 that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR 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 peripheral blood mononuclear cells 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. The long-term benefits of organ transplantation are hindered by graft rejection (1Booth AJ Grabauskiene S Wood SC Lu G Burrell BE Bishop DK IL-6 promotes cardiac graft rejection mediated by CD4 + cells.J Immunol. 2011; 187: 5764-5771Crossref PubMed Scopus (49) Google Scholar, 2Pasquet L Douet JY Sparwasser T Romagnoli P van Meerwijk JP Long-term prevention of chronic allograft rejection by regulatory T-cell immunotherapy involves host Foxp3-expressing T cells.Blood. 2013; 121: 4303-4310Crossref PubMed Scopus (27) Google Scholar, 3Sagoo P Lombardi G Lechler RI Relevance of regulatory T cell promotion of donor-specific tolerance in solid organ transplantation.Front Immunol. 2012; 3: 184Crossref PubMed Scopus (46) Google Scholar, 4Tsang JY Tanriver Y Jiang S et al.Indefinite mouse heart allograft survival in recipient treated with CD4(+)CD25(+) regulatory T cells with indirect allospecificity and short term immunosuppression.Transpl Immunol. 2009; 21: 203-209Crossref PubMed Scopus (59) Google Scholar, 5Rogers NJ Lechler RI Allorecognition.Am J Transplant. 2001; 1: 97-102Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), a detrimental process driven by alloreactive T cells that recognize donor MHC antigens via the direct, indirect, and semidirect pathways of allorecognition (3Sagoo P Lombardi G Lechler RI Relevance of regulatory T cell promotion of donor-specific tolerance in solid organ transplantation.Front Immunol. 2012; 3: 184Crossref PubMed Scopus (46) Google Scholar,6Game DS Lechler RI Pathways of allorecognition: Implications for transplantation tolerance.Transpl Immunol. 2002; 10: 101-108Crossref PubMed Scopus (173) Google Scholar). Current immunosuppressive regimens target the direct pathway and reduce acute allograft rejection (7Safinia N Leech J Hernandez-Fuentes M Lechler R Lombardi G Promoting transplantation tolerance; adoptive regulatory T cell therapy.Clin Exp Immunol. 2013; 172: 158-168Crossref PubMed Scopus (51) Google Scholar) but are associated with severe side-effects (8Meier-Kriesche HU Kaplan B The search for CNI-free immunosuppression: No free lunch.Am J Transplant. 2011; 11: 1355-1356Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) and do not effectively prevent chronic rejection (2Pasquet L Douet JY Sparwasser T Romagnoli P van Meerwijk JP Long-term prevention of chronic allograft rejection by regulatory T-cell immunotherapy involves host Foxp3-expressing T cells.Blood. 2013; 121: 4303-4310Crossref PubMed Scopus (27) Google Scholar,9Wojciechowski D Vincenti F Tofacitinib in kidney transplantation.Expert Opin Investig Drugs. 2013; 22: 1193-1199Crossref PubMed Scopus (18) Google Scholar); thus, the half-life of allografts remains limited to 10–15 years (10Burgos D Gonzalez-Molina M Ruiz-Esteban P et al.Rate of long-term graft loss has fallen among kidney transplants from cadaveric donors.Transplant Proc. 2012; 44: 2558-2560Crossref PubMed Scopus (2) Google Scholar,11Gruessner RW Gruessner AC The current state of pancreas transplantation.Nat Rev Endocrinol. 2013; 9: 555-562Crossref PubMed Scopus (159) Google Scholar). Thymus-derived regulatory T cells (tTregs) are immunosuppressive T cells with a fundamental role in the maintenance of tolerance in vivo (12Sakaguchi S Wing K Onishi Y Prieto-Martin P Yamaguchi T Regulatory T cells: How do they suppress immune responses?.Int Immunol. 2009; 21: 1105-1111Crossref PubMed Scopus (659) Google Scholar, 13Barzaghi F Passerini L Bacchetta R Immune dysregulation, polyendocrinopathy, enteropathy, x-linked syndrome: A paradigm of immunodeficiency with autoimmunity.Front Immunol. 2012; 3: 211Crossref PubMed Scopus (247) Google Scholar, 14Katoh H Zheng P Liu Y FOXP3: Genetic and epigenetic implications for autoimmunity.J Autoimmun. 2013; 41: 72-78Crossref PubMed Scopus (50) Google Scholar, 15Miyara M Gorochov G Ehrenstein M Musset L Sakaguchi S Amoura Z Human FoxP3 + regulatory T cells in systemic autoimmune diseases.Autoimmun Rev. 2011; 10: 744-755Crossref PubMed Scopus (265) Google Scholar). These cells are characterized in humans as CD4+CD25hiCD127lo and constitutively express the transcription factor forkhead-box protein 3 (FOXP3) (7Safinia N Leech J Hernandez-Fuentes M Lechler R Lombardi G Promoting transplantation tolerance; adoptive regulatory T cell therapy.Clin Exp Immunol. 2013; 172: 158-168Crossref PubMed Scopus (51) Google Scholar,12Sakaguchi S Wing K Onishi Y Prieto-Martin P Yamaguchi T Regulatory T cells: How do they suppress immune responses?.Int Immunol. 2009; 21: 1105-1111Crossref PubMed Scopus (659) Google Scholar,16Elinav E Waks T Eshhar Z Redirection of regulatory T cells with predetermined specificity for the treatment of experimental colitis in mice.Gastroenterology. 2008; 134: 2014-2024Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In rodent models, Tregs significantly prolong the survival of skin (17Tsang JY Tanriver Y Jiang S et al.Conferring indirect allospecificity on CD4 + CD25 + Tregs by TCR gene transfer favors transplantation tolerance in mice.J Clin Invest. 2008; 118: 3619-3628Crossref PubMed Scopus (213) Google Scholar, 18Golshayan D Jiang S Tsang J Garin MI Mottet C Lechler RI In vitro-expanded donor alloantigen-specific CD4 + CD25 + regulatory T cells promote experimental transplantation tolerance.Blood. 2007; 109: 827-835Crossref PubMed Scopus (262) Google Scholar, 19Sagoo P Ali N Garg G Nestle FO Lechler RI Lombardi G Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells.Sci Transl Med. 2011; 3: 83ra42Crossref PubMed Scopus (261) Google Scholar) and heart (4Tsang JY Tanriver Y Jiang S et al.Indefinite mouse heart allograft survival in recipient treated with CD4(+)CD25(+) regulatory T cells with indirect allospecificity and short term immunosuppression.Transpl Immunol. 2009; 21: 203-209Crossref PubMed Scopus (59) Google Scholar) allografts and in humans, correlations between the proportion of Tregs within allografts and graft survival have been observed (20Krustrup D Madsen CB Iversen M Engelholm L Ryder LP Andersen CB The number of regulatory T cells in transbronchial lung allograft biopsies is related to FoxP3 mRNA levels in bronchoalveolar lavage fluid and to the degree of acute cellular rejection.Transpl Immunol. 2013; 29: 71-75Crossref PubMed Scopus (14) Google Scholar, 21Krystufkova E Sekerkova A Striz I Brabcova I Girmanova E Viklicky O Regulatory T cells in kidney transplant recipients: The effect of induction immunosuppression therapy.Nephrol Dial Transplant. 2012; 27: 2576-2582Crossref PubMed Scopus (46) Google Scholar, 22Louis S Braudeau C Giral M et al.Contrasting CD25hiCD4 + T cells/FOXP3 patterns in chronic rejection and operational drug-free tolerance.Transplantation. 2006; 81: 398-407Crossref PubMed Scopus (237) Google Scholar). With the safety of Treg therapy having been demonstrated in the contexts of graft-versus-host disease (GvHD) (23Di Ianni M Falzetti F Carotti A et al.Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.Blood. 2011; 117: 3921-3928Crossref PubMed Scopus (812) Google Scholar, 24Brunstein CG Miller JS Cao Q et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: Safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (815) Google Scholar, 25Trzonkowski P Bieniaszewska M Juscinska J et al.First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4 + CD25 + CD127- T regulatory cells.Clin Immunol. 2009; 133: 22-26Crossref PubMed Scopus (498) Google Scholar, 26Theil A Tuve S Oelschlagel U et al.Adoptive transfer of allogeneic regulatory T cells into patients with chronic graft-versus-host disease.Cytotherapy. 2015; 17: 473-486Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and type I diabetes (27Bluestone JA Buckner JH Fitch M et al.Type 1 diabetes immunotherapy using polyclonal regulatory T cells.Sci Transl Med. 2015; 7: 315ra189Crossref PubMed Scopus (612) Google Scholar,28Marek-Trzonkowska N Mysliwiec M Dobyszuk A et al.Therapy of type 1 diabetes with CD4(+)CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets – results of one year follow-up.Clin Immunol. 2014; 153: 23-30Crossref PubMed Scopus (259) Google Scholar), we are conducting Phase I/II clinical trials to investigate the therapeutic potential of adoptive polyclonal Treg therapy to promote tolerance in kidney (The ONE Study: NCT02129881) and liver (ThRIL: NCT02166177) transplant recipients (29Safinia N Vaikunthanathan T Fraser H et al.Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation.Oncotarget. 2016; 7: 7563-7577Crossref PubMed Scopus (104) Google Scholar,30Afzali B Edozie FC Fazekasova H et al.Comparison of regulatory T cells in hemodialysis patients and healthy controls: Implications for cell therapy in transplantation.Clin J Am Soc Nephrol. 2013; 8: 1396-1405Crossref PubMed Scopus (63) Google Scholar). However, we (4Tsang JY Tanriver Y Jiang S et al.Indefinite mouse heart allograft survival in recipient treated with CD4(+)CD25(+) regulatory T cells with indirect allospecificity and short term immunosuppression.Transpl Immunol. 2009; 21: 203-209Crossref PubMed Scopus (59) Google Scholar,17Tsang JY Tanriver Y Jiang S et al.Conferring indirect allospecificity on CD4 + CD25 + Tregs by TCR gene transfer favors transplantation tolerance in mice.J Clin Invest. 2008; 118: 3619-3628Crossref PubMed Scopus (213) Google Scholar,19Sagoo P Ali N Garg G Nestle FO Lechler RI Lombardi G Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells.Sci Transl Med. 2011; 3: 83ra42Crossref PubMed Scopus (261) Google Scholar,31Putnam AL Safinia N Medvec A et al.Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.Am J Transplant. 2013; 13: 3010-3020Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and others (32Joffre O Santolaria T Calise D et al.Prevention of acute and chronic allograft rejection with CD4 + CD25 + Foxp3 + regulatory T lymphocytes.Nat Med. 2008; 14: 88-92Crossref PubMed Scopus (441) Google Scholar,33Lee K Nguyen V Lee KM Kang SM Tang Q Attenuation of donor-reactive T cells allows effective control of allograft rejection using regulatory T cell therapy.Am J Transplant. 2014; 14: 27-38Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) have demonstrated in animal models that allospecific Tregs are superior to polyclonal Tregs at reducing graft rejection, upon adoptive transfer. Murine Tregs expanded with allogeneic antigen-presenting cells (APC) (direct allospecificity) and/or transduced to express an allospecific TCR (indirect allospecificity) prolonged graft survival significantly more effectively than polyclonal Tregs. These findings were confirmed using human Tregs expanded with allogeneic dendritic cells (19Sagoo P Ali N Garg G Nestle FO Lechler RI Lombardi G Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells.Sci Transl Med. 2011; 3: 83ra42Crossref PubMed Scopus (261) Google Scholar) and B cells (31Putnam AL Safinia N Medvec A et al.Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.Am J Transplant. 2013; 13: 3010-3020Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar,34Landwehr-Kenzel S Issa F Luu SH et al.Novel GMP-compatible protocol employing an allogeneic B cell bank for clonal expansion of allospecific natural regulatory T cells.Am J Transplant. 2014; 14: 594-606Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) in human skin xenograft transplant models. As such, clinical trials are assessing the safety and efficacy of Tregs with direct allospecificity in kidney (DART: NCT02188719) and liver (deLTa: NCT02188719 and NCT01624077) transplant recipients. Here, we investigated whether allospecificity may be conferred onto Tregs using chimeric antigen receptors (CAR) (35Boardman D Maher J Lechler R Smyth L Lombardi G Antigen-specificity using chimeric antigen receptors: The future of regulatory T-cell therapy?.Biochem Soc Trans. 2016; 44: 342-348Crossref PubMed Scopus (33) Google Scholar). CARs are synthetic fusion proteins capable of redirecting the specificity of T cells toward desired antigens. Classical CARs comprise an extracellular antigen-targeting moiety that binds a specific antigen in an MHC-independent manner and a series of customized intracellular TCR and costimulatory signaling domains. As such, CARs translate the binding of specific antigens into the activation of desired signaling cascades (36Maher J Immunotherapy of malignant disease using chimeric antigen receptor engrafted T cells.ISRN Oncol. 2012; 2012: 278093Crossref PubMed Google Scholar). CARs are primarily used clinically to generate tumor antigen–specific T cells (37Porter DL Levine BL Kalos M Bagg A June CH Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med. 2011; 365: 725-733Crossref PubMed Scopus (2622) Google Scholar, 38Kalos M Levine BL Porter DL et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med. 2011; 3: 95ra73Crossref PubMed Scopus (1795) Google Scholar, 39Grupp SA Kalos M Barrett D et al.Chimeric antigen receptor-modified t cells for acute lymphoid leukemia.N Engl J Med. 2013; 368: 1509-1518Crossref PubMed Scopus (2495) Google Scholar, 40Brentjens RJ Davila ML Riviere I et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med. 2013; 5: 177ra38Crossref PubMed Scopus (1499) Google Scholar, 41Till BG Jensen MC Wang J et al.CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: Pilot clinical trial results.Blood. 2012; 119: 3940-3950Crossref PubMed Scopus (405) Google Scholar, 42Kochenderfer JN Wilson WH Janik JE et al.Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19.Blood. 2010; 116: 4099-4102Crossref PubMed Scopus (984) Google Scholar, 43Kohn DB Dotti G Brentjens R et al.CARs on track in the clinic.Mol Ther. 2011; 19: 432-438Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). However, preclinical studies have shown the efficacy of CAR-modified Tregs for the treatment of colitis (16Elinav E Waks T Eshhar Z Redirection of regulatory T cells with predetermined specificity for the treatment of experimental colitis in mice.Gastroenterology. 2008; 134: 2014-2024Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar,44Elinav E Adam N Waks T Eshhar Z Amelioration of colitis by genetically engineered murine regulatory T cells redirected by antigen-specific chimeric receptor.Gastroenterology. 2009; 136: 1721-1731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar,45Blat D Zigmond E Alteber Z Waks T Eshhar Z Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells.Mol Ther. 2014; 22: 1018-1028Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and multiple sclerosis (46Fransson M Piras E Burman J et al.CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery.J Neuroinflammation. 2012; 9: 112Crossref PubMed Scopus (189) Google Scholar). The functionality of CARs has also been demonstrated in human Tregs (47Lee JC Hayman E Pegram HJ et al.In vivo inhibition of human CD19-targeted effector T cells by natural T regulatory cells in a xenotransplant murine model of B cell malignancy.Cancer Res. 2011; 71: 2871-2881Crossref PubMed Scopus (76) Google Scholar,48Jethwa H Adami AA Maher J Use of gene-modified regulatory T-cells to control autoimmune and alloimmune pathology: Is now the right time?.Clin Immunol. 2014; 150: 51-63Crossref PubMed Scopus (45) Google Scholar), most recently as a means of protecting against xeno-GvHD (49MacDonald KG Hoeppli RE Huang Q et al.Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.J Clin Invest. 2016; 126: 1413-1424Crossref PubMed Scopus (283) Google Scholar). However, to date, no one has demonstrated the capacity of CAR Tregs to protect from solid transplant rejection. In this study, we applied CAR technology to generate allospecific Tregs with the aim of promoting organ transplant tolerance. We redirected human CD4+CD25+CD127loFOXP3+ Tregs toward an MHC class I molecule (HLA-A2), an alloantigen that is ubiquitously expressed in an allograft, with the hypothesis that they would be more potent than polyclonal Tregs at protecting from graft rejection, upon adoptive transfer. A previously described (50Watkins NA Brown C Hurd C Navarrete C Ouwehand WH The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library.Tissue Antigens. 2000; 55: 219-228Crossref PubMed Scopus (19) Google Scholar) HLA-A2-specific single-chain variable fragment (scFv) sequence (3PB2 VH and DPK1 VL) was validated by immunoprecipitation (data not shown) and cloned upstream of (i) a 9E10 cMyc epitope; (ii) a human CD28 hinge/transmembrane domain; (iii) a human CD28-CD3ζ signaling domain; and (iv) an enhanced green fluorescent protein (eGFP) open reading frame in a second-generation pLNT/SFFV lentiviral plasmid (provided by Prof. Adrian Thrasher; UCL, London, UK). A truncated CAR (ΔCAR) was generated by removing the CD28-CD3ζ signaling domain from the full-length CAR-eGFP fusion. All plasmids were verified by sequencing (Beckman Coulter Genomics, Takeley, Essex, UK). Anonymized healthy donor peripheral blood was obtained from the National Blood Service (NHS Blood and Transplantation, Tooting, London, UK) with informed consent and ethical approval (Institutional Review Board of Guy’s Hospital; reference 09/H0707/86). CD4+ T cells were enriched using RosetteSep™ (StemCell Technologies, Waterbeach, Cambridgeshire, UK), after which CD4+CD25− Tregs and CD4+CD25− Teffs were separated using CD25 MicroBeads II (Miltenyi Biotec, Bisley, Surrey, UK). Tregs/effector T cells (Teffs) were activated with anti-CD3/CD28 beads (1:1 bead:cell ratio; Thermo Fisher Scientific, Paisley, Renfrewshire, UK) and cultured in X-vivo™ 15 (Lonza, Slough, Berkshire, UK) supplemented with 5% human AB serum (BioSera, Uckfield East, Sussex, UK). Treg were cultured with 100 nM rapamycin (LC-Laboratories, Woburn, MA) and 1000 U/mL recombinant IL-2 (Proleukin-Novartis, Camberley, Surrey, UK), whereas Teffs were cultured with 100 U/mL IL-2 only. Cells were stained in phosphate-buffered saline (PBS) supplemented with 1% heat-inactivated fetal 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, Hatfield, Hertfordshire, UK), CD25 (4E3 or 2A3), CTLA-4 (BNI3) (all from BD Biosciences, Oxford, Oxfordshire, UK) CD127 (A019D5), CCR4 (L291H4), CCR9 (L053E8), CCR10 (5688-5), CD62L (DREG-56), integrin β7 (FIB504), CLA (HECA-452), HLA-DR (all from BioLegend, London, UK), 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, Copenhagen, Denmark) 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 were analyzed using FlowJo 7 or 10 (Tree Star, Ashland, OR). HEK293T, MCF-7, and T-47D epithelial cells were grown in DMEM-based media. B-Lymphoblastoid cell lines (B-LCL) and K562s (donated by Dr. Marc Martinez-Llordella) were grown in RPMI-based media. All culture media were supplemented with 10% heat-inactivated FCS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine (all from Thermo Fisher Scientific). HLA-transfected K562 cultures were supplemented with 5 μg/mL neomycin (Thermo Fisher Scientific). All cells were grown at 37°C in the presence of 5% (vol/vol) CO2. HEK293T cells were cotransfected with pLNT/SFFV-CAR-eGFP or pLNT/SFFV-ΔCAR-eGFP (Figure 1A), pΔ8.91 and pCMV-VSV-G plasmids at a mass ratio of 4:3:1 using polyethylenimine (3:1 PEI:DNA wt/wt; Sigma-Aldrich, Gillingham, Dorset, UK). Viral supernatant was harvested 48–56 h posttransfection and lentiviral particles were concentrated using PEG-it™ (System Biosciences, Bar Hill, Cambridgeshire, UK). Tregs/Teffs were transduced in RetroNectin®-treated plates (50 μg/μL; Takara Bio Inc., Saint-Germain-en-Laye, Yvelines, France) 3 days postisolation using fourfold concentrated viral supernatant. eGFP+ cells were purified by fluorescence-assisted cell sorting (FACS) 7 days posttransduction. Teffs/Tregs were cocultured overnight with confluent MCF-7 or T-47D cell monolayers. Culture supernatants were collected to measure IL-2, interferon (IFN)γ, and IL-10 production by cytokine-specific enzyme-linked immunosorbent assays (ELISA) (eBioscience). Monolayers were washed and the viability of the monolayer cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Absorbance was measured at 560 nm. Results are shown as percent viability relative to monolayers cultured alone. Tregs were cocultured with autologous CD4+CD25−‒ Teff responders that were labeled with CellTrace Violet (CTV; Thermo Fisher Scientific) and activated with anti-CD3/CD28 beads (1:40 bead:cell ratio) or irradiated (12 000 cGy) allogeneic B-LCLs (3:1 T:B-LCL ratio); DBB (HLA-A2+DR7+), MOU (HLA-A2−DR7+), SPO (HLA-A2+DR11+), and BM21 (HLA-A2−DR11+). Teff CTV dilution was measured after 5 days using flow cytometry. Results are shown as percent suppression (inverse of percent Teff proliferation) relative to Teffs cultured alone. Primary human umbilical vein endothelial cells (HUVECs) were isolated by collagenase digestion using ethically approved protocols (East London & The City Local Research Ethics Committee reference 05/Q0603/34 ELCHA). HUVECs were stimulated with 15 ng/mL IFNγ (R&D Systems, Abingdon, Oxfordshire, UK) for 72 h prior to experimentation and seeded in μ-Slides VI 0.4 (Ibidi, Planegg/Martinsried, Germany) coated with 0.5% bovine gelatin. Tregs were suspended at 1 × 106 cells/mL in PBS (with Ca2+ and Mg2+) and flowed across HUVEC layers using a shear stress of 1 dyn/cm2. The number of Tregs that migrated in 10-s frames was assessed. BALB/c recombination activating gene (RAG)2−/−γc−/− (BRG) mice were maintained under sterile conditions (Biological Services Unit, New Hunt’s House, King’s College London). All procedures were performed in accordance with all legal, ethical, and institutional requirements (PPL70/7302). Human skin was obtained from routine surgical procedures with informed consent and ethical approval (Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London; reference 06/Q0704/18). Donor HLA-A2 expression was determined by flow cytometry, staining skin-derived cells obtained by collagenase digestion (100 μg/mL; Sigma-Aldrich). Split-thickness skin grafts (1.5 cm2) were transplanted onto 10–11-week-old recipient BRG mice as previously described (19Sagoo P Ali N Garg G Nestle FO Lechler RI Lombardi G Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells.Sci Transl Med. 2011; 3: 83ra42Crossref PubMed Scopus (261) Google Scholar) and mice were administered 100 μg anti-mouse Gr-1 (BioXCell, Upper Heyford, Oxfordshire, UK) intraperitoneally every 3–4 days. After 5–6 weeks (Figure S4), mice were injected intravenously with 5 × 106 peripheral blood mononuclear cells (PBMCs) ± 1 × 106 Tregs. Skin grafts were harvested for histological analysis 5 weeks following PBMCs/Tregs transfer. Skin grafts were frozen in optimal cutting temperature (OCT) (Thermo Fisher Scientific). Eight- or 16-μm-thick sections were fixed in 4% paraformaldehyde, blocked with a mixture of 10% donkey serum, 0.1% fish skin gelatin, 0.1% Triton X-100, and 0.5% Tween-20 (all from Sigma-Aldrich) in PBS and stained with the following antibodies: anti-human CD3 (polyclonal rabbit; DAKO, Stockport, Cheshire, UK), anti-FOXP3 (236A/E7; Abcam, Milton, Cambridgeshire, UK), anti-CD45 (HI30, eBioscience), anti-involucrin (CY5; Sigma-Aldrich), anti-human CD31, and Ki67 (both polyclonal rabbit; Abcam). Sections were stained with secondary donkey anti-mouse Alexa Fluor®555 and anti-rabbit Alexa Fluor®488 or Alexa Fluor®647 antibodies with 4′,6-diamidino-2-phenylindole (DAPI) (all from Thermo Fisher Scientific) and mounted with Fluorescence Mounting Medium (DAKO). Maximum intensity projection images consisting of 10 z-stacks (1.1 μm apart) were acquired at ×20 magnification using a C2+ point scanning confocal microscope (Nikon, Kingston Upon Thames, Surrey, UK) and analyzed/quantified with NIS Elements and FIJI imaging software (51Schindelin J Arganda-Carreras I Frise E et al.Fiji: An open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (30116) Google Scholar). Data shown are mean ± SEM or mean ± SD. Statistical significance was determined using two-tailed paired Student’s t-tests or analysis of variance (ANOVA) with the Tukey multiple comparison post-hoc test. p < 0.05, p < 0.01, p < 0.001, and p < 0.0001. A HLA-A2-specific CAR incorporating a human CD28-CD3ζ signaling domain was generated using a patient-derived HLA-A2-specific scFv sequence (50Watkins NA Brown C Hurd C Navarrete C Ouwehand WH The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library.Tissue Antigens. 2000; 55: 219-228Crossref PubMed Scopus (19) Google Scholar) (Figure 1A and B). A second-generation CAR was selected based on the superior function of these CARs relative to first-generation CARs (52Krause A Guo HF Latouche JB Tan C Cheung NK Sadelain M Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes.J Exp Med. 1998; 188: 619-626Crossref PubMed Scopus (208) Google Scholar, 53Finney HM Lawson AD Bebbington CR Weir AN Chimeric receptors providing both prima" @default.
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• W2563656670 date "2017-04-01" @default.
• W2563656670 modified "2023-10-10" @default.
• W2563656670 title "Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection" @default.
• W2563656670 cites W1509896974 @default.
• W2563656670 cites W1511438228 @default.
• W2563656670 cites W1531095893 @default.
• W2563656670 cites W1541810537 @default.
• W2563656670 cites W1574443686 @default.
• W2563656670 cites W1593835418 @default.
• W2563656670 cites W1598242657 @default.
• W2563656670 cites W1963811203 @default.
• W2563656670 cites W1966542058 @default.
• W2563656670 cites W1967278561 @default.
• W2563656670 cites W1967521828 @default.
• W2563656670 cites W1974039913 @default.
• W2563656670 cites W1981690705 @default.
• W2563656670 cites W1988181665 @default.
• W2563656670 cites W1991344211 @default.
• W2563656670 cites W1991890073 @default.
• W2563656670 cites W1994454155 @default.
• W2563656670 cites W1997688914 @default.
• W2563656670 cites W1998157143 @default.
• W2563656670 cites W2005354199 @default.
• W2563656670 cites W2005451808 @default.
• W2563656670 cites W2013274514 @default.
• W2563656670 cites W2017529374 @default.
• W2563656670 cites W2018715802 @default.
• W2563656670 cites W2030058137 @default.
• W2563656670 cites W2035346630 @default.
• W2563656670 cites W2054901613 @default.
• W2563656670 cites W2054951100 @default.
• W2563656670 cites W2063820155 @default.
• W2563656670 cites W2067270731 @default.
• W2563656670 cites W2067687448 @default.
• W2563656670 cites W2068392572 @default.
• W2563656670 cites W2073045132 @default.
• W2563656670 cites W2075080208 @default.
• W2563656670 cites W2076991609 @default.
• W2563656670 cites W2087637747 @default.
• W2563656670 cites W2100495173 @default.
• W2563656670 cites W2102490214 @default.
• W2563656670 cites W2105400883 @default.
• W2563656670 cites W2105484520 @default.
• W2563656670 cites W2113692100 @default.
• W2563656670 cites W2113802879 @default.
• W2563656670 cites W2118836795 @default.
• W2563656670 cites W2119860862 @default.
• W2563656670 cites W2121987769 @default.
• W2563656670 cites W2126072190 @default.
• W2563656670 cites W2130011407 @default.
• W2563656670 cites W2130338247 @default.
• W2563656670 cites W2132337808 @default.
• W2563656670 cites W2132475439 @default.
• W2563656670 cites W2136365031 @default.
• W2563656670 cites W2137768092 @default.
• W2563656670 cites W2147266645 @default.
• W2563656670 cites W2149184543 @default.
• W2563656670 cites W2150966665 @default.
• W2563656670 cites W2167279371 @default.
• W2563656670 cites W2167550136 @default.
• W2563656670 cites W2173141813 @default.
• W2563656670 cites W2261516588 @default.
• W2563656670 cites W2303852857 @default.
• W2563656670 cites W2315461301 @default.
• W2563656670 cites W2582906781 @default.
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