Preclinical assessment of antigen-specific chimeric antigen receptor regulatory T cells for use in solid organ transplantation.

A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scid gamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.

PTEN is required for human Treg suppression of costimulation in vitro.

Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

Donor-specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients.

Cell therapy with autologous donor-specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor-specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR-Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor-specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti-HLA-A2-specific CAR were administered to Bl/6 mice at the time of transplanting an HLA-A2+ Bl/6 skin graft. Donor-specific CAR-Tregs, but not irrelevant-CAR Tregs, significantly delayed skin rejection and diminished donor-specific antibodies (DSAs) and frequencies of DSA-secreting B cells. Donor-specific CAR-Treg-treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen-specific suppression. When donor-specific CAR Tregs were tested in HLA-A2-sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor-specific CAR-Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.

Insulin-producing intestinal K cells protect nonobese diabetic mice from autoimmune diabetes.

BACKGROUND & AIMS: Type 1 diabetes is caused by an aberrant response against pancreatic ß cells. Intestinal K cells are glucose-responsive endocrine cells that might be engineered to secrete insulin. We generated diabetes-prone non-obese diabetic (NOD) mice that express insulin, via a transgene, in K cells. We assessed the effects on immunogenicity and diabetes development. METHODS: Diabetes incidence and glucose homeostasis were assessed in NOD mice that expressed mouse preproinsulin II from a transgene in K cells and nontransgenic NOD mice (controls); pancreas and duodenum tissues were collected and analyzed by histology. We evaluated T cell responses to insulin, levels of circulating autoantibodies against insulin, and the percentage of circulating antigen-specific T cells. Inflammation of mesenteric and pancreatic lymph node cells was also evaluated. RESULTS: The transgenic mice tended to have lower blood levels of glucose than control mice, associated with increased plasma levels of immunoreactive insulin and proinsulin. Fewer transgenic mice developed diabetes than controls. In analyses of pancreas and intestine tissues from the transgenic mice, insulin-producing K cells were not affected by the immune response and the mice had reduced destruction of endogenous ß cells. Fewer transgenic mice were positive for insulin autoantibodies compared with controls. Cells isolated from mesenteric lymph nodes of the transgenic mice had significantly lower rates of proliferation and T cells from transgenic mice tended to secrete lower levels of inflammatory cytokines than from controls. At 15 weeks, transgenic mice had fewer peripheral CD8(+) T cells specific for NRP-V7 than control mice. CONCLUSIONS: NOD mice with intestinal K cells engineered to express insulin have reduced blood levels of glucose, are less likely to develop diabetes, and have reduced immunity against pancreatic ß cells compared with control NOD mice. This approach might be developed to treat patients with type 1 diabetes.

Short Report: CAR Tregs mediate linked suppression and infectious tolerance in islet transplantation.

Regulatory T cells (Tregs) have potential as a cell-based therapy to prevent or treat transplant rejection and autoimmunity. Using an HLA-A2-specific chimeric antigen receptor (A2-CAR), we previously showed that adoptive transfer of A2-CAR Tregs limited anti-HLA-A2 alloimmunity. However, it was unknown if A2-CAR Tregs could also limit immunity to autoantigens. Using a model of HLA-A2+ islet transplantation into immunodeficient non-obese diabetic mice, we investigated if A2-CAR Tregs could control diabetes induced by islet-autoreactive (BDC2.5) T cells. In mice transplanted with HLA-A2+ islets, A2-CAR Tregs reduced BDC2.5 T cell engraftment, proliferation and cytokine production, and protected mice from diabetes. Tolerance to islets was systemic, including protection of the HLA-A2negative endogenous pancreas. In tolerant mice, a significant proportion of BDC2.5 T cells gained FOXP3 expression suggesting that long-term tolerance is maintained by de novo Treg generation. Thus, A2-CAR Tregs mediate linked suppression and infectious tolerance and have potential therapeutic use to simultaneously control both allo- and autoimmunity in islet transplantation.

Implanted islets in the anterior chamber of the eye are prone to autoimmune attack in a mouse model of diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes is an autoimmune disease resulting from the destruction of insulin-producing beta cells. Along with advances in generating replacement beta cells for treating diabetes, there is also increasing demand for non-invasive tools to evaluate the recurrence of autoimmune attack on transplanted tissue. Here, we examined the anterior chamber of the eye as a potential islet transplant site, and also evaluated whether in vivo imaging of the islets transplanted in the eye could enable real-time visualisation of autoimmune processes underway in the pancreas. METHODS: Syngeneic islet equivalents were transplanted into the eye or kidney capsule of streptozotocin-induced diabetic C57BL/6 mice to compare islet dose (25-125 islet equivalents) and function across transplant sites. Autoimmune attack of syngeneic islets was evaluated in the pancreas and eye tissues of NOD and NOD-severe combined immunodeficient (SCID) mice given diabetogenic splenocytes. RESULTS: Islet transplantation in the eye decreased fasting plasma glucose levels and increased weight gain and survival in an islet-dose-dependent manner. Even 50 islets in the eye reduced blood glucose levels, whereas ≥ 200 islets were required in the kidney for a similar effect. Autoimmune destruction of pancreatic islets in the eye mirrored that in the pancreas and could be visualised in real time by non-invasive imaging. CONCLUSIONS/INTERPRETATION: We found that far fewer islets were required to restore normoglycaemia when transplanted into the anterior chamber of the eye vs the kidney capsule. However, our results suggest that islets are not protected against autoimmune attack in the eye, making this a suitable site for visualising autoimmune processes against transplanted tissue.

Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection.

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease.

Background: Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD). Methods: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. Results: The speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. Conclusions: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.

Insulin B peptide-MHC class II-specific chimeric antigen receptor-Tregs prevent autoimmune diabetes.

Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IA g7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs stably expressed Foxp3 and prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes. Brief Summary: Chimeric antigen receptor Tregs specific for an insulin B-chain peptide presented by MHC class II prevent autoimmune diabetes.

Human A2-CAR T Cells Reject HLA-A2 + Human Islets Transplanted Into Mice Without Inducing Graft-versus-host Disease.

BACKGROUND: Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). METHODS: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4 + and CD8 + T cells and tested their ability to reject HLA-A2 + islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T-cell engraftment, islet function, and xGVHD were assessed longitudinally. RESULTS: The speed and consistency of A2-CAR T-cell-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of coinjected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, coinjection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2 + human islets within 1 wk and without xGVHD for 12 wk. CONCLUSIONS: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of islet-replacement therapies.

Tregs with an MHC class II peptide-specific chimeric antigen receptor prevent autoimmune diabetes in mice.

Adoptive immunotherapy with Tregs is a promising approach for preventing or treating type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR redirected NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Cotransfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In WT NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising therapeutic approach for the prevention of autoimmune diabetes.

Insulin Null ß-cells Have a Prohormone Processing Defect That Is Not Reversed by AAV Rescue of Proinsulin Expression.

Up to 6% of diabetes has a monogenic cause including mutations in the insulin gene, and patients are candidates for a gene therapy. Using a mouse model of permanent neonatal diabetes, we assessed the efficacy of an adeno-associated virus (AAV)-mediated gene therapy. We used AAVs with a rat insulin 1 promoter (Ins1) regulating a human insulin gene (INS; AAV Ins1-INS) or native mouse insulin 1 (Ins1; AAV Ins-Ins1) to deliver an insulin gene to ß-cells of constitutive insulin null mice (Ins1-/-Ins2-/-) and adult inducible insulin-deficient mice [Ins1-/-Ins2f/f PdxCreER and Ins1-/-Ins2f/f mice administered AAV Ins1-Cre)]. Although AAV Ins1-INS could successfully infect and confer insulin expression to ß-cells, insulin null ß-cells had a prohormone processing defect. Secretion of abundant proinsulin transiently reversed diabetes. We reattempted therapy with AAV Ins1-Ins1, but Ins1-/-Ins2-/- ß-cells still had a processing defect of both replaced Ins1 and pro-islet amyloid polypeptide (proIAPP). In adult inducible models, ß-cells that lost insulin expression developed a processing defect that resulted in impaired proIAPP processing and elevated circulating proIAPP, and cells infected with AAV Ins1-Ins1 to rescue insulin expression secreted proinsulin. We assessed the subcellular localization of prohormone convertase 1/3 (PC1/3) and detected defective sorting of PC1/3 to glycogen-containing vacuoles and retention in the endoplasmic reticulum as a potential mechanism underlying defective processing. We provide evidence that persistent production of endogenous proinsulin within ß-cells is necessary for ß-cells to be able to properly store and process proinsulin.

Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells.

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. This protocol was validated in: Sci Transl Med (2020), DOI: 10.1126/scitranslmed.aaz3866 Graphical abstract.

Antibodies to the wheat storage globulin Glo-3A in children before and at diagnosis of celiac disease.

OBJECTIVE: Celiac disease (CD) is an autoimmune disease triggered by exposure to gluten-containing foods. IgA autoantibodies to tissue transglutaminase (TTG) are elevated in CD, but little is known about the gastrointestinal state before the appearance of TTG. Antibodies to wheat storage globulin Glo-3A have been studied in type 1 diabetes, and may be a marker of altered mucosal barrier and/or immune function. In the present study, we investigated antibody responses to Glo-3A in CD. PATIENTS AND METHODS: In the Diabetes Autoimmunity Study in the Young, children were studied prospectively from birth for the appearance of TTG and CD. Fifty cases of CD were frequency matched with 50 controls on age (of TTG seroconversion in the case), sex, ethnicity, presence of a first-degree relative with type 1 diabetes mellitus, and human leukocyte antigen -DR3 genotype. In cases and controls, IgG antibodies to Glo-3A were analyzed in a blinded manner in the sample collected at the time of seroconversion to TTG positivity (or the matched sample in controls) and in all of the previous samples since birth (mean 4.5 samples). The association between Glo-3A antibody levels and CD case status was explored using t tests at the TTG-positive visit and when Glo-3A levels were highest, and mixed modeling to describe Glo-3A over time. RESULTS: At the time of first elevated TTG (mean 4.9 years), patients with CD had higher Glo-3A antibody levels than controls (13.3 ± 17.2 vs 7.6 ± 11.7, P = 0.005). In both cases and controls, Glo-3A antibodies appear to peak at a mean age of 2.9 years, before mean age of initial TTG seroconversion. The peak Glo-3A antibody levels were higher in cases than controls (25.5 ± 21.8 vs 14.9 ± 18.3 P = 0.0007). Using mixed modeling to account for multiple visits per person, cases had higher levels of Glo-3A antibodies than controls at all ages from birth to TTG seroconversion (ß = 0.53, P = 0.002). CONCLUSIONS: Compared with controls, CD cases have higher Glo-3A antibody responses in the beginning years, before initial detection of TTG.

Antibodies from a patient with type 1 diabetes and celiac disease bind to macrophages that express the scavenger receptor CD163.

Antibodies against the wheat storage globulin Glo-3A from a patient with both type 1 diabetes (T1D) and celiac disease were enriched to identify potential molecular mimicry between wheat antigens and T1D target tissues. Recombinant Glo-3A was used to enrich anti-Glo-3A immunoglobulin G antibodies from plasma by batch affinity chromatography. Rat jejunum and pancreas, as well as human duodenum and monocytes were probed, and binding was evaluated by immunohistochemistry and confocal microscopy. Glo-3A-enriched antibodies bound to a specific subset of cells in the lamina propria of rat jejunum that co-localized mostly with a marker of resident, alternatively activated CD163-positive (CD163⁺) macrophages. Blood monocytes and macrophage-like cells in human duodenum were also labelled with the enriched antibodies. Blocking studies revealed that binding to CD163⁺ macrophages was not due to cross-reactivity with anti-Glo-3A antibodies, but rather to non-Glo-3A antibodies co-purified during antibody enrichment. The novel finding of putative autoantibodies against tolerogenic intestinal CD163⁺ macrophages suggests that regulatory macrophages were targeted in this patient with celiac disease and T1D.

Developmental Timing of High-Fat Diet Exposure Impacts Glucose Homeostasis in Mice in a Sex-Specific Manner.

We previously demonstrated that male, but not female, Swiss Webster mice are susceptible to diabetes, with incidence increased by early overnutrition and high-fat diet (HFD). In this study, we investigated how HFD in Swiss Webster males and females during preweaning, peripubertal, and postpubertal periods alters glucose homeostasis and diabetes susceptibility. In males, HFD throughout life resulted in the highest diabetes incidence. Notably, switching to chow postpuberty was protective against diabetes relative to switching to chow at weaning, despite the longer period of HFD exposure. Similarly, HFD throughout life in males resulted in less liver steatosis relative to mice with shorter duration of postpubertal HFD. Thus, HFD timing relative to weaning and puberty, not simply exposure length, contributes to metabolic outcomes. Females were protected from hyperglycemia regardless of length or timing of HFD. However, postpubertal HFD resulted in a high degree of hepatic steatosis and adipose fibrosis, but glucose regulation and insulin sensitivity remained unchanged. Interestingly, peri-insulitis was observed in the majority of females but was not correlated with impaired glucose regulation. Our findings reveal critical periods of HFD-induced glucose dysregulation with striking sex differences in Swiss Webster mice, highlighting the importance of careful consideration of HFD timing relative to critical developmental periods.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.

Peripherin-reactive antibodies in mouse, rabbit, and human blood.

Type 1 diabetes (T1D) is an autoimmune disorder that results from the destruction of insulin-producing beta-cells in the islets of Langerhans. To date, autoimmune T-cell response and antibody reactivity to more than 20 autoantigens have been linked to this disease. Some studies have described the intermediate filament protein peripherin (PRPH) as an autoantigen associated with T1D in non-obese diabetic (NOD) mice. We evaluated immune reactivity of mouse and rabbit sera and human plasma to a 58 kDa protein expressed in RIN-m5F rat insulinoma cells. The protein was isolated using 2-DE and identified by mass spectrometry as PRPH. Antibodies from healthy humans and T1D patients, CD-1 mice, C57BL/6 mice, NOR (non-obese diabetes resistant) mice, and NOD mice reacted with PRPH on Western blots. However, antibody response to PRPH was stronger in NOD than non-autoimmune prone C57BL/6 mice. We conclude that immune reactivity to PRPH is not exclusively associated with NOD mice or human patients with T1D. Furthermore, the frequent occurrence of PRPH-reactive antibodies in mouse and human blood suggests that binding may be non-specific or could reflect the presence of natural autoantibodies against PRPH. These findings point to the need for a re-evaluation of PRPH as a T1D autoantigen in NOD mice and raise the question of the physiological relevance of such widespread immune reactivity against this peripheral nervous system protein.

Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells.

Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs To date, most of the CAR Treg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs Using a human leukocyte antigen-A2-specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs expressing a CAR encoding CD28wt 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 an ability to suppress CD80 expression on dendritic cells 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.

T reg-specific insulin receptor deletion prevents diet-induced and age-associated metabolic syndrome.

Adipose tissue (AT) regulatory T cells (T regs) control inflammation and metabolism. Diet-induced obesity causes hyperinsulinemia and diminishes visceral AT (VAT) T reg number and function, but whether these two phenomena were mechanistically linked was unknown. Using a T reg-specific insulin receptor (Insr) deletion model, we found that diet-induced T reg dysfunction is driven by T reg-intrinsic insulin signaling. Compared with Foxp3cre mice, after 13 wk of high-fat diet, Foxp3creInsrfl/fl mice exhibited improved glucose tolerance and insulin sensitivity, effects associated with lower AT inflammation and increased numbers of ST2+ T regs in brown AT, but not VAT. Similarly, Foxp3creInsrfl/fl mice were protected from the metabolic effects of aging, but surprisingly had reduced VAT T regs and increased VAT inflammation compared with Foxp3cre mice. Thus, in both diet- and aging-associated hyperinsulinemia, excessive Insr signaling in T regs leads to undesirable metabolic outcomes. Ablation of Insr signaling in T regs represents a novel approach to mitigate the detrimental effects of hyperinsulinemia on immunoregulation of metabolic syndrome.