Anti-HLA-A2-CAR Tregs prolong vascularized mouse heterotopic heart allograft survival

Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99 days, whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors, A2-CAR Tregs slightly increased median graft survival from 11 to 14 days, which was further extended to >100 days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients. Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99 days, whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors, A2-CAR Tregs slightly increased median graft survival from 11 to 14 days, which was further extended to >100 days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients. Solid organ transplantation is a life-changing, and often lifesaving, therapy option for patients with end-stage organ diseases. However, life-long immunosuppression is needed to prevent allograft rejection, which can be toxic to both host and transplanted organs, and increase patient susceptibilities to cancer development and infections. Thus, strategies to induce persistent immune tolerance have the potential to reduce the burden of immunosuppression and alleviate these side effects. Many studies across various preclinical transplantation models have demonstrated therapeutic potential of adoptively transferred regulatory T cells (Tregs) to promote allograft survival.1Tang Q Bluestone JA Regulatory T-cell therapy in transplantation: moving to the clinic.Cold Spring Harbor Perspect Med. 2013; 3: a015552Crossref PubMed Scopus (0) Google Scholar, 2Brennan TV Tang Q Liu F-C et al.Requirements for prolongation of allograft survival with regulatory T cell infusion in lymphosufficient hosts.J Surg Res. 2011; 169: e69-e75Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 3Tsang JY-S Tanriver Y Jiang S et al.Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice.J Clin Investig. 2008; 118: 3619-3628Crossref PubMed Scopus (0) Google Scholar, 4Joffre 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 (438) Google Scholar, 5Lee K Nguyen V Lee K-M Kang S-M 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 These studies also highlighted the heightened potency of alloantigen-reactive Tregs over polyclonal Tregs,2Brennan TV Tang Q Liu F-C et al.Requirements for prolongation of allograft survival with regulatory T cell infusion in lymphosufficient hosts.J Surg Res. 2011; 169: e69-e75Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 3Tsang JY-S Tanriver Y Jiang S et al.Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice.J Clin Investig. 2008; 118: 3619-3628Crossref PubMed Scopus (0) Google Scholar, 4Joffre 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 (438) Google Scholar,6Sagoo P Ali N Garg G et al.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 (260) Google Scholar as Treg-mediated immune tolerance requires local Treg activation in the graft tissue and graft-draining lymph nodes (LN).7Samy ET Parker LA Sharp CP Tung KSK Continuous control of autoimmune disease by antigen-dependent polyclonal CD4+CD25+ regulatory T cells in the regional lymph node.J Exp Med. 2005; 202: 771-781Crossref PubMed Scopus (150) Google Scholar Although alloantigen-reactive Tregs can be selectively expanded ex vivo via stimulation with donor-derived antigen-presenting cells (APCs),8Alzhrani A Bottomley M Wood K Hester J Issa F Identification, selection, and expansion of non-gene modified alloantigen-reactive Tregs for clinical therapeutic use.Cell Immunol. 2020; 357: 104214Crossref PubMed Scopus (11) Google Scholar this approach is complicated by challenges in donor-cell collection under Good Manufacturing Process-compliant conditions. Alternatively, Tregs can be genetically engineered to express a synthetic chimeric antigen receptor (CAR) targeting donor alloantigen, thus redirecting the Tregs to the graft. Since donor and recipient major histocompatibility complex (MHC) mismatches are the main trigger of allograft rejection, donor-specific MHCs are attractive targets for CAR Treg therapy. Several groups have developed CARs targeting HLA-A2 (A2-CARs),9MacDonald KG Hoeppli RE Huang Q et al.Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.J Clin Investig. 2016; 126: 1413-1424Crossref PubMed Scopus (0) Google Scholar, 10Boardman DA Philippeos C Fruhwirth GO et al.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.Am J Transplant. 2017; 17: 931-943Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 11Noyan F Zimmermann K Hardtke-Wolenski M et al.Prevention of allograft rejection by use of regulatory T cells with an MHC-specific chimeric antigen receptor.Am J Transplant. 2017; 17: 917-930Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 12Dawson NA Lamarche C Hoeppli RE et al.Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells.JCI Insight. 2019; 4: e123672PubMed Google Scholar, 13Bézie S Charreau B Vimond N et al.Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice.Blood Adv. 2019; 3: 3522-3538Crossref PubMed Scopus (35) Google Scholar, 14Muller YD Ferreira LMR Ronin E et al.Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance.Front Immunol. 2021; 12: 686439Crossref PubMed Scopus (0) Google Scholar because up to 25% of transplantations are characterized by an HLA-A2–positive donor and an HLA-A2–negative recipient. Most published studies evaluated A2-CAR Treg function in humanized NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice for protection of HLA-A2–positive skin grafts or in the context of xenogenic GvHD (graft versus host disease).9MacDonald KG Hoeppli RE Huang Q et al.Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.J Clin Investig. 2016; 126: 1413-1424Crossref PubMed Scopus (0) Google Scholar, 10Boardman DA Philippeos C Fruhwirth GO et al.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.Am J Transplant. 2017; 17: 931-943Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 11Noyan F Zimmermann K Hardtke-Wolenski M et al.Prevention of allograft rejection by use of regulatory T cells with an MHC-specific chimeric antigen receptor.Am J Transplant. 2017; 17: 917-930Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 12Dawson NA Lamarche C Hoeppli RE et al.Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells.JCI Insight. 2019; 4: e123672PubMed Google Scholar, 13Bézie S Charreau B Vimond N et al.Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice.Blood Adv. 2019; 3: 3522-3538Crossref PubMed Scopus (35) Google Scholar, 14Muller YD Ferreira LMR Ronin E et al.Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance.Front Immunol. 2021; 12: 686439Crossref PubMed Scopus (0) Google Scholar However, NSG mice are severely immunodeficient due to the absence of many immune cell types and incompatibilities between ligands and receptors from human T cells and mouse hosts. A fraction of human T cells is also xenoreactive against mouse antigens, leading to systemic inflammation and limiting experimental duration while confounding the anti-graft immune response. One recent study reported prolongation of HLA-A2–positive skin graft survival following administration of mouse A2-CAR Tregs in immunocompetent mice.15Sicard A Lamarche C Speck M et al.Donor-specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients.Am J Transplant. 2020; 20: 1562-1573Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar All prior studies have focused on models of non-vascularized grafts, whereas most clinical grafts are vascularized. This is especially important in the context of Tregs, which naturally recognize antigens presented by MHC class II expressed on APCs. Redirecting Tregs to HLA-A2, a class I antigen, allows Tregs to recognize all cells in an A2-positive graft and, particularly, the vascular endothelial cells lining the donor-derived blood vessels. Furthermore, while prior studies examined CAR Treg efficacy in single A2-mismatch models, clinical grafts are often characterized by multiple MHC mismatches and greater immunogenicity. Thus, it remains unknown whether CAR Treg infusions would be sufficient to prolong graft survival, or if adjunct immunosuppressive drugs would be necessary. Here, we demonstrate the therapeutic potential of mouse A2-CAR Tregs in vascularized heart transplant models with either a single A2- or haplo-MHC-mismatch. C57BL/6J (Jackson, stock no. 000664), BALB/cJ (Jackson, stock no. 000651), B6. Cg-Immp2lTg(HLA−A/H2−D)2Enge/J (Jackson, stock no. 004191; B6.A2 hereafter), B6.A2XBALB/c F1, and TCR75 Tg mice16Brennan TV Jaigirdar A Hoang V et al.Preferential priming of alloreactive T cells with indirect reactivity.Am J Transplant. 2009; 9: 709-718Crossref PubMed Scopus (40) Google Scholar,17Honjo K Xy XU Bucy RP CD4+ T-cell receptor transgenic T cells alone can reject vascularized heart transplants through the indirect pathway of alloantigen recognition.Transplantation. 2004; 77: 452-455Crossref PubMed Scopus (45) Google Scholar were housed and bred under specific pathogen-free conditions at the UCSF Animal Barrier Facility. All mouse experiments were performed according to a UCSF Institutional Animal Care and Use Committee-approved protocol. The following antibodies were used for phenotyping and proliferation assays: anti-CD4 BUV496 (clone GK1.5, BD), anti-CD25 PE (clone PC61, Biolegend), anti-CD62L APC (clone MEL-14, BD), anti-CD69 PE-Cy7 (clone H1.2F3, BD), anti-Foxp3 AF488 (clone FJK-16s, Invitrogen), anti-myc tag AF488/AF647 (clone 9B11, CellSignaling), anti-EGFRt PE/BV711 (clone AY13, Biolegend), anti-Thy1.1 PE-Cy7 (clone HIS51, eBioscience), anti-CD45.1 PE-Cy7 (clone A20, Biolegend), CellTrace Violet (CTV, ThermoFisher), and Fixable Viability Dye eFluor780 (ThermoFisher). CD4+ T cells were enriched from pooled LNs and spleens via the EasySepTM Mouse CD4+ T Cell Isolation Kit (STEMCELL, Vancouver, Canada, Cat.#19852), according to manufacturer instructions. Cells were stained with anti-CD4-APC, anti-CD25-PE, and anti-CD62L-FITC, and Tregs (CD4+CD25+CD62Lhi) and Tconv (CD4+CD25−CD62Lhi) were sorted on a FACS Aria II cytometer to >98% purity. Flow cytometric analyses were performed on a Fortessa or LSRII flow cytometer and FlowJo software (TreeStar). The complementarity determining regions of the anti-HLA-A2 scFv from a human B-cell-derived hybridoma (clone SN607D8) were grafted onto the framework of Herceptin/trastuzumab (clone 4DG) as previously described.14Muller YD Ferreira LMR Ronin E et al.Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance.Front Immunol. 2021; 12: 686439Crossref PubMed Scopus (0) Google Scholar To generate an anti-HLA-A2 murine CAR, the grafted scFv was cloned in-frame to the murine CD8 hinge and transmembrane domains followed by CD28 and CD3ζ intracellular domains via isothermal DNA assembly. The A2-CAR was cloned into a murine stem cell virus-based retroviral vector with an N-terminal myc tag and in-frame to a truncated EGFR (EGFRt), separated by a P2A peptide. Viral particles were produced using the Platinum-E Retroviral Packaging Cell Line according to manufacturer recommendations (CellBiolabs, San Diego). FACS-purified Tconv and Tregs were stimulated and expanded as previously described,18Tang Q Henriksen KJ Bi M et al.In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.J Exp Med. 2004; 199: 1455-1465Crossref PubMed Scopus (978) Google Scholar undergoing retroviral spinfection on day 1 or 3 of the expansion culture, respectively. First, retroviral particles were centrifuged on plates pre-coated with RetroNectin (Takara Bio Inc., Kusatsu, Japan) for 2 h at 2000 × g. T cells were subsequently loaded at 1 × 106/ml and spinfected for 15 min at 2000 × g. EGFRt-positive cells were FACS-purified on day 8 and rested overnight in complete medium (Tconv) or in medium supplemented with 200 IU/ml recombinant human IL-2 (rhIL-2, Tregs), prior to use in all subsequent experiments. A2-CAR T cells were co-cultured with irradiated HLA-A2–expressing K562, parental (HLA-A2–negative) K562, or splenocytes from B6 or B6.A2 mice for 2–3 days prior to evaluation of activation and proliferation (CTV-dilution) via flow cytometry. Tconv cultures were maintained in medium without rhIL-2, while Treg cultures were supplemented with 200 or 2000 IU/ml rhIL-2 for activation and proliferation assays, respectively. For in vitro suppression assays, 25,000 CTV-labeled B6.TCR75 Tg+ CD4+ responder cells were co-cultured with polyclonally expanded Tregs (plus anti-CD3) or A2-CAR Tregs at the indicated ratios, without rhIL-2 and in the presence of 175,000 irradiated splenocytes from B6.A2XBALB/c F1 or B6XBALB/c F1. On day 4, cells were harvested, stained and analyzed using flow cytometry. Heterotopic, intra-abdominal heart transplantation was performed by transplanting hearts from B6.A2 (single A2-mismatch), B6.A2XBALB/c F1 (haplo-mismatch), or B6 (syngeneic) donor mice into B6 recipient mice, as previously described.19Plenter RJ Grazia TJ Murine heterotopic heart transplant technique.J Visual Exper. 2014; 89: 51511Google Scholar Polyclonal or A2-CAR Tregs were administered intravenously at the indicated doses, on day 1 or 2 post-heart transplantation for the haplo-mismatch and single A2-mismatch model, respectively. For haplo-mismatch experiments, rapamycin-treated mice received a therapeutic regimen of 1mg/kg i.p. daily from days 0 to 9, as previously described,20Raimondi G Sumpter TL Matta BM et al.Mammalian target of rapamycin inhibition and alloantigen-specific regulatory T cells synergize to promote long-term graft survival in immunocompetent recipients.J Immunol. 2010; 184: 624-636Crossref PubMed Scopus (81) Google Scholar and A2-CAR Tregs were injected intravenously on day 7 post-transplantation. Transplant survival was assessed by transabdominal palpation and scored on a scale of 0 to 4 (0 = non-beating, rejected graft; 1 = contraction barely palpable, muscle fibrillation only; 2 = obvious decrease in contraction strength, both ventricles still contracting in a coordinated manner; 3 = strong, coordinated contraction of both ventricles, with noticeable decrease in strength or rate; 4 = normal amplitude and frequency). Palpation scores of 0 and 1 were considered as rejection, as confirmed histologically by H.E. stain. For lymphocyte isolation, mice were euthanized according to IACUC standard operating procedure and perfused with PBS. Lymphoid tissues (spleen, LNs) were mechanically disintegrated to generate single-cell suspensions for antibody staining. Non-lymphoid tissues (lung, liver, transplanted and endogenous heart) were digested in RPMI1640 (ThermoFischer) supplemented with 100 µg/ml DNase I (Sigma-Aldrich) and 1 mg/ml collagenase type IV (Sigma-Aldrich) for 30 min at 37°C followed by mechanical disintegration. Single cell suspensions were washed, resuspended in 40% Percoll (Sigma-Aldrich), overlaid on 80% Percoll, and centrifuged for 20 min at 2000 rpm at 20°C. Mononuclear cells were collected from the interface for antibody staining. Analyses were performed using the GraphPad Prism 9 software. Specific statistical tests used for different experiments are indicated in the figure legends. We designed a murine anti-HLA-A2 CAR with a myc tag and EGFRt transduction reporter (Figure 1A). On day 8, expanded, retrovirally transduced Tconvs and Tregs were sorted for CAR-positive cells based on EGFRt-expression (Figure 1B). The %EGFRt+ and myc MFI (mean fluorescent intensity) indicated comparable transduction efficiencies and CAR surface expression levels between Tconvs and Tregs (Figure 1C). Transduced and sorted A2-CAR Tregs remained Foxp3-positive (Figure 1C, right panel). A2-CAR—expressing Tconvs and Tregs upregulated activation markers CD25 and CD69 upon co-incubation with A2-expressing but not parental K562 cells (Figure 2A). Moreover, in the presence of rhIL-2, A2-CAR Tregs proliferated significantly more in response to irradiated B6.A2 splenocytes than B6 splenocytes (Figure 2B). There was also a positive correlation between the degree of CTV-dilution and CAR surface expression levels (Figure 2B). Next, we evaluated the ability of A2-CAR Tregs to suppress the proliferation of CD4+ cells from B6.TCR75 transgenic mice in vitro. The TCR75 TCR is specific for a H2-Kd-derived peptide presented by H2-Iab,17Honjo K Xy XU Bucy RP CD4+ T-cell receptor transgenic T cells alone can reject vascularized heart transplants through the indirect pathway of alloantigen recognition.Transplantation. 2004; 77: 452-455Crossref PubMed Scopus (45) Google Scholar and thus recognizes BALB/c-derived alloantigens presented by B6 APCs. In the absence of Tregs, CTV-labeled TCR75 Tconvs proliferated robustly in response to stimulation with irradiated splenocytes from BALB/cXB6.HLA-A2 F1 (Figure 2C). The addition of A2-CAR Tregs strongly inhibited the proliferation of TCR75 Tconvs, exhibiting 8- to 16-fold greater suppressive activity than untransduced (UT), polyclonally expanded Tregs (p < .0001, Figure 2C). Importantly, Treg stimulation via anti-CD3 elicited comparable suppressive capacities between A2-CAR—expressing and untransduced Tregs (p = .6506, Figure 2D), indicating that enhancement of Treg suppressive capacity is driven specifically by A2-CAR activation. Together, these results demonstrate A2-CAR-induced Treg activation and suppressive function in response to HLA-A2 antigen. To assess the in vivo function of A2-CAR Tregs, we used a single A2-mismatch transplantation model in which hearts from B6.A2 mice were transplanted heterotopically into B6 recipients (Figure 3A). Single A2-mismatch hearts had a median survival of 23 days (n = 7). To evaluate the ability of A2-CAR Tregs to prolong graft survival, we infused Tregs on day 2 post-transplantation, an infusion time chosen to distinguish between technical failure and potential acute A2-CAR Treg toxicity. Injection of 1 × 106 ex vivo-expanded, untransduced polyclonal Tregs failed to significantly influence graft survival (median survival 35 days, n = 4, p = .0754, Figure 3B). In contrast, 1 × 106 A2-CAR Tregs prolonged median graft survival to 99 days (n = 4, p = .0144, Figure 3B). Interestingly, while we noted a progressive decline of graft function via palpation score across all recipients (Figure 3C), histological analysis of hearts surviving to 100 days revealed reduced lymphocyte infiltration specifically in mice treated with CAR Tregs (Figure 3D). Since 1 × 106 A2-CAR Tregs did not completely prevent graft rejection, we increased the A2-CAR Treg dose to 2 × 106 and 4 × 106 but did not see further improvements in graft survival (p = .6617) nor palpitation score (Figure 3E,F). Moreover, while flow cytometric analysis of surviving day 100 grafts revealed elevated T cell infiltration relative to transplanted hearts in syngeneic recipients, the percentages of infiltrating CD4+ and CD8+ T cells were comparable between mice that received different doses of A2-CAR Tregs (Figure 3G). We also analyzed recipients of different A2-CAR Treg doses at the 100-day endpoint for the presence of infused Tregs, carrying the congenic markers Thy1.1 or CD45.1. Unexpectedly, we did not detect Thy1.1+ or CD45.1+ cells in the graft-draining para-aortic LN, nor any of the non-draining lymphoid or non-lymphoid tissues, including the graft tissue (Figure 3H). Although undetectable by the 100-day endpoint, day 8 analysis of a 4 × 106 A2-CAR Treg recipient showed infused Tregs were present at this early timepoint, with the highest frequencies in the para-aortic LN, cutaneous LN, and the transplanted heart (0.11–0.13%, Figure 3H). Having established 1 × 106 A2-CAR Tregs as an effective dose in a single A2-mismatch model, we then assessed A2-CAR Treg-mediated protection of grafts harboring multiple MHC mismatches, a scenario more consistent with clinical settings. We transplanted hearts from BALB/cxB6.HLA-A2 F1 mice into B6 recipients (Figure 4A). While A2.F1 grafts were rapidly rejected with a median survival of 11 days (n = 4), injection of 1 × 106 A2-CAR Tregs significantly prolonged median graft survival, albeit only to day 14 (n = 4, p = .0207, Figure 4B). We next explored rapamycin treatment for potential synergy with A2-CAR Tregs. Postoperative treatment for 9 days with rapamycin prolonged median graft survival to 27.5 days (n = 4, p = .01, Figure 4B). We combined the 9-day rapamycin treatment with infusion of 1 × 106 A2-CAR Tregs on day 7, a regimen adapted from a previous report demonstrating synergy between rapamycin and alloantigen-specific Tregs in a mouse heart allograft model.20Raimondi G Sumpter TL Matta BM et al.Mammalian target of rapamycin inhibition and alloantigen-specific regulatory T cells synergize to promote long-term graft survival in immunocompetent recipients.J Immunol. 2010; 184: 624-636Crossref PubMed Scopus (81) Google Scholar This regimen also models established protocols of immunosuppression withdrawal after Treg infusion, as used in several clinical trials of Treg cell therapy (NCT02474199, NCT03654040). Although we again observed a gradual decline of graft function (Figure 4C) and mild cell infiltrates (Figure 4D), combination therapy dramatically prolonged graft survival (n = 4, p = .0266, Figure 4B), with several grafts persisting to the 100-day endpoint. Natural alloantigen-reactive Tregs recognize class II MHCs, via direct, semi-direct, or indirect pathways.21Wagner JC Tang Q CAR-tregs as a strategy for inducing graft tolerance.Curr Transplant Rep. 2020; 7: 205-214Crossref PubMed Scopus (5) Google Scholar Engineering Tregs to recognize class I MHCs thus expands the antigen pool that can be targeted by adoptive Treg therapy. Consistent with prior reports, our results show that re-directing Tregs to allogeneic class I MHC on parenchymal cells can effectively protect the targeted tissue. To date, studies characterizing the impact of A2-CAR Tregs in transplantation have focused on the prevention of skin graft rejection or systemic GvHD in mouse models. Although the therapeutic potential of antigen-specific CAR Tregs is evident within these contexts,9MacDonald KG Hoeppli RE Huang Q et al.Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.J Clin Investig. 2016; 126: 1413-1424Crossref PubMed Scopus (0) Google Scholar,10Boardman DA Philippeos C Fruhwirth GO et al.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.Am J Transplant. 2017; 17: 931-943Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar,12Dawson NA Lamarche C Hoeppli RE et al.Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells.JCI Insight. 2019; 4: e123672PubMed Google Scholar,13Bézie S Charreau B Vimond N et al.Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice.Blood Adv. 2019; 3: 3522-3538Crossref PubMed Scopus (35) Google Scholar,15Sicard A Lamarche C Speck M et al.Donor-specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients.Am J Transplant. 2020; 20: 1562-1573Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar evaluations of the CAR-Treg function within immunocompetent models of vascularized transplant and haplo-MHC-mismatch have yet to be reported. In this study, we provide a proof-of-concept demonstration that adoptive transfer of A2-CAR Tregs can significantly prolong the survival and function of a vascularized allograft harboring either a single A2-mismatch or multiple MHC-mismatches in immunocompetent mice. In the single-A2 mismatch model, the administration of 1 × 106 A2-CAR-expressing Tregs was sufficient to extend graft survival to a median of 99 days, demonstrating a strong therapeutic effect. However, multiple HLA mismatches are commonplace in clinical transplant scenarios, and greater immunogenicity often correlates with stronger and faster rejection. To better evaluate the A2-CAR Treg function in a multi-MHC-mismatch setting, we used a heterotopic heart transplant model in which hearts from BALB/cXB6.HLA-A2 F1 donors are transplanted into B6 mice. In this more immunogenic model, while treatment with A2-CAR Tregs again statistically outperformed treatment with untransduced controls, we found combination therapy with rapamycin necessary to induce clinically meaningful prolongation of graft survival. The inability of A2-CAR Treg monotherapy to prevent gradual decline of graft function in both models may be due to several contributing factors. Prior reports suggest that Tregs alone may be insufficient to attenuate runaway alloimmune responses.5Lee K Nguyen V Lee K-M Kang S-M 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 Moreover, direct CAR-mediated recognition of graft tissues may also risk unintended toxicities. For example, Tregs utilize the perforin and granzyme B pathway to trigger direct cytotoxicity against APCs upon TCR-mediated recognition of class II MHC.22Schmidt A Oberle N Krammer PH Molecular mechanisms of treg-mediated T cell suppression.Front Immunol. 2012; 3 (https://doi.org/10.3389/fimmu.2012.00051)Crossref Scopus (432) Google Scholar By redirecting Treg specificity to target parenchymal cells in the transplanted tissue, A2-CAR expression may reprogram Tregs to exhibit direct cytotoxicity against the same cells the Tregs are engineered to protect. It is important to point out that the slow rejection kinetics in the single A2-mismatch model provided an opportunity to evaluate potential toxicities of CAR Tregs toward the allograft. However, very few A2-CAR Tregs could be detected in the grafts beyond the early post-infusion period, suggesting that the gradual loss of graft protection is unlikely to have been due to direct toxicity by the CAR Tregs. Furthermore, in the single A2-mismatch model, we did not accelerate graft rejection nor decline graft function even when administering double or quadruple the CAR Treg dose. Rather, the lack of A2-CAR Treg persistence and the emergence of cell infiltrates within the graft tissue both point toward a gradual loss of immunosuppressive control. Thus, one potential approach toward extending the durability of CAR Treg-mediated protection could be repeated administration of CAR Tregs. Alternatively, designing CAR Tregs with indirect allospecificity could confer graft tolerance as it has been shown that Treg-mediated promotion of long-term graft survival is strongly dependent upon indirect allospecificity.3Tsang JY-S Tanriver Y Jiang S et al.Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice.J Clin Investig. 2008; 118: 3619-3628Crossref PubMed Scopus (0) Google Scholar Indeed, donor alloantigen-specific TCR-transgenic Tregs can also induce graft tolerance by inducing a quiescent state that allows endogenous, indirect Tregs to expand and propagate the immunosuppressive effect.5Lee K Nguyen V Lee K-M Kang S-M 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 Therefore, it would be interesting to directly compare the efficacy of MHC class I- and class II-targeting CAR Tregs acting with direct and indirect allospecificity, respectively. Unfortunately, a robust strategy to synthetically transfer indirect allospecificity remains to be developed, thus preventing us from evaluating the two strategies in this work. In summary, we show that adoptively transferred A2-CAR Tregs can significantly prolong graft survival in both single A2-mismatch and haplo-mismatch models of heterotopic heart transplantation. While further studies are required to characterize the site(s) of suppressive activity and further optimize treatment strategies to better preserve graft function, our study offers proof-of-concept demonstration that CAR-Tregs mediate long-term graft survival in a fully immunocompetent vascularized transplant model. This project was funded in part by grants from the NIDDK (UC4 DK116264 and P30 DK063720), Juno Therapeutics, and JDRF (SRA-2019-776-S-B). JCW was funded by the IZKF Wuerzburg (Z2-CSP5) and received a travel grant from BaCaTec (5 [2018-2]). The authors thank Juan Du for technical support and lab management. We would like to thank the Flow Cytometry Core and the Histology Core at UCSF for supporting this study. The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. QT received grant support from Juno Therapeutics for this project. QT is a co-founder, share holder, and scientific advisor of Sonoma Biotherapeutics. JCW, ER, YP, and QT are inventors of a provisional patent application on A2-CAR Treg. PH has no relevant competing interests to disclose. The data that support the findings of this study are available from the corresponding author upon reasonable request. All DNA constructs used in this study will be made available upon request under a material transfer agreement with the Office of Technology Management of the University of California, San Francisco.