Helical TomoTherapy Total Lymphoid Irradiation and Hematopoietic Cell Transplantation for Kidney Transplant Tolerance in Rhesus Macaques.

Development of a post-transplant kidney transplant tolerance induction protocol involving a novel total lymphoid irradiation (TLI) conditioning method in a rhesus macaque model is described. We examined the feasibility of acheiving tolerance to MHC 1-haplotype matched kidney transplants by establishing a mixed chimeric state with infusion of donor hematopoietic cells (HC) using TomoTherapy TLI. The chimeric state was hypothesized to permit the elimination of all immunosuppressive (IS) medications while preserving allograft function long-term without development of graft-versus-host-disease (GVHD) or rejection. An experimental group of 11 renal transplant recipients received the tolerance induction protocol and outcomes were compared to a control group (n = 7) that received the same conditioning but without donor HC infusion. Development of mixed chimerism and operational tolerance was accomplished in two recipients in the experimental group. Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD. None of the animals in the control group achieved tolerance when IS was eliminated. This novel experimental model demonstrated the feasibility for inducing of long-term operational tolerance when mixed chimerism is achieved using a TLI post-transplant conditioning protocol in 1-haplotype matched non-human primate recipients of combined kidney and HC transplantation.

Extracellular Vesicle-associated GARP/TGFß:LAP Mediates "Infectious" Allo-tolerance.

Here we test the hypothesis that, like CD81-associated "latent" IL35, the transforming growth factor (TGF)ß:latency-associated peptide (LAP)/glycoprotein A repetitions predominant (GARP) complex was also tethered to small extracellular vesicles (sEVs), aka exosomes, produced by lymphocytes from allo-tolerized mice. Once these sEVs are taken up by conventional T cells, we also test whether TGFß could be activated suppressing the local immune response. Methods: C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes followed by anti-CD40L/CD154 antibody treatment on days 0, 2, and 4. On day 35, spleen and lymph nodes were extracted and isolated lymphocytes were restimulated with sonicates of CBA splenocytes overnight. sEVs were extracted from culture supernatants by ultracentrifugation (100 000g) and assayed for (a) the presence of TGFß:LAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFß:LAP and to activation from its latent form, as well as various TGFß receptors; and (c) TGFß-dependent function in 1° and 2° immunosuppression of tetanus toxoid-immunized B6 splenocytes using trans-vivo delayed-type hypersensitivity assay. Results: After tolerization, CBA-restimulated lymphocytes secreted GARP/TGFß:LAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFß:LAP was mainly associated with CD81+ exosomes. sEV-bound GARP/TGFß:LAP became active in both 1° and 2° immunosuppression, the latter requiring sEV uptake by "bystander" T cells and reexpression on the cell surface. Conclusions: Like other immune-suppressive components of the Treg exosome, which are produced in a latent form, exosomal GARP/TGFß:LAP produced by allo-specific regulatory T cells undergoes either immediate activation (1° suppression) or internalization by naive T cells, followed by surface reexpression and subsequent activation (2°), to become suppressive. Our results imply a membrane-associated form of TGFß:LAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFß:LAP along with Treg-derived GARP as part of the infectious tolerance network.

Cross-decoration of dendritic cells by non-inherited maternal antigen-containing extracellular vesicles: Potential mechanism for PD-L1-based tolerance in cord blood and organ transplantation.

Exposure to non-inherited maternal antigens (NIMA) during the fetal period induces lifelong split tolerance to grafts expressing these allo-antigens. In adult mice, the production of extracellular vesicles (EVs) from maternal microchimeric cells causes cross-decoration (XD) of offspring dendritic cells (DC) with NIMA and upregulation of PD-L1, contributing to NIMA tolerance. To see how this may apply to humans, we tested NIMA acquisition by fetal DCS in human cord blood. The average percentage of NIMA-XD among total DCs was 2.6% for myeloid and 4.5% for Plasmacytoid DC. These cells showed higher PD-L1 expression than their non-XD counterparts (mDC: p = .0016; pDC: p = .024). We detected CD9+ EVs bearing NIMA and PD-L1 in cord blood. To determine if this immune regulatory mechanism persists beyond the pregnancy, we analyzed NIMA-expressing kidney and liver transplant recipients. We found donor antigen XD DCs in peripheral blood and graft-infiltrating DCs. As in cord blood, the pattern of donor antigen expression was punctate, and PD-L1 expression was upregulated, likely due to both protein and miRNA acquired from EV. Our findings support a mechanism for split tolerance to NIMAs that develops during pregnancy and is recapitulated in adult transplant recipients.

Tomotherapy Applied Total Lymphoid Irradiation and Allogeneic Hematopoietic Cell Transplantation Generates Mixed Chimerism in the Rhesus Macaque Model.

Development of a new methodology to induce immunological chimerism after allogeneic hematopoietic cell (HC) transplantation in a rhesus macaque model is described. The chimeric state was achieved using a non-myeloablative, helical tomotherapy-based total lymphoid irradiation (TomoTLI) conditioning regimen followed by donor HC infusions between 1-haplotype matched donor/recipient pairs. The technique was tested as a feasibility study in an experimental group of seven rhesus macaques that received the novel TomoTLI tolerance protocol and HC allo-transplants. Two tomotherapy protocols were compared: TomoTLI (n = 5) and TomoTLI/total-body irradiation (TBI) (n = 2). Five of seven animals developed mixed chimerism. Three of five animals given the TomoTLI protocol generated transient mixed chimerism with no graft-versus-host disease (GVHD) with survival of 33, 152 and >180 days. However, the inclusion of belatacept in addition to a single fraction of TBI resulted in total chimerism and fatal GVHD in both animals, indicating an unacceptable conditioning regimen.

Donor HLA-DR Drives the Development of De Novo Autoimmunity Following Lung and Heart Transplantation.

Individuals harbor preexisting HLA-DR/DQ-restricted responses to collagen type V (ColV) mediated by Th17 cells under Treg control, both specific to peptides that bind to inherited HLA class II antigens. Yet after transplant, the donor-DR type somehow influences graft outcome. We hypothesized that, long after a lung or heart allograft, the particular HLA-DR type of the mismatched transplant donor transforms the specificity of the "anti-self" response. This could explain why, over long term, certain donor DRs could be more immunogenic than others. METHODS: We analyzed 7 HLA-DR15neg patients who had received a lung allograft from a DR15+ donor. To determine the mechanism of acquired specificity in self-reactivity, we analyzed the kinetics of DR1 (host) and DR15 (donor) peptide restriction in a heart transplant model using DR-transgenic mice. RESULTS: Beyond 1.5 years post-lung transplant, all patients tested had acquired DR15-restricted immune responses to ColV peptides. These responses were either unrestrained Th17 type (n = 4) or Th17 controlled by Treg arising early (<5 y) or late (>7 y) after transplant (n = 4). Treg suppression via conventional (transforming growth factor-ß [TGF-ß]) and extracellular vesicle-associated (IL-35) cytokines correlated with superior outcomes. Naïve DR1 and DR15 transgenic mice had preexisting DR-restricted responses, exclusively to ColV fragments containing DR1- or DR15-binding peptides. When HLA-DR1 transgenic recipients of a HLA-DR15 heart developed ColV reactivity post-transplant, mice that acutely rejected (20-25 d) responded only to the DR1-restricted ColV peptide epitope. In animals whose grafts survived long term, we could detect acquisition of DR from the transplant donor onto the surface of recipient dendritic cells, and immune responses against a donor DR15-restricted ColV peptide. CONCLUSIONS: These results might explain how certain donor HLA-DR types redirect host immune responses to novel peptides of critical self-antigens. Unless regulated, such responses may predispose the allograft to chronic rejection.

Infectious Tolerance as Seen With 2020 Vision: The Role of IL-35 and Extracellular Vesicles.

Originally identified as lymphocyte regulation of fellow lymphocytes, our understanding of infectious tolerance has undergone significant evolutions in understanding since being proposed in the early 1970s by Gershon and Kondo and expanded upon by Herman Waldman two decades later. The evolution of our understanding of infectious tolerance has coincided with significant cellular and humoral discoveries. The early studies leading to the isolation and identification of Regulatory T cells (Tregs) and cytokines including TGFß and IL-10 in the control of peripheral tolerance was a paradigm shift in our understanding of infectious tolerance. More recently, another potential, paradigm shift in our understanding of the "infectious" aspect of infectious tolerance was proposed, identifying extracellular vesicles (EVs) as a mechanism for propagating infectious tolerance. In this review, we will outline the history of infectious tolerance, focusing on a potential EV mechanism for infectious tolerance and a novel, EV-associated form for the cytokine IL-35, ideally suited to the task of propagating tolerance by "infecting" other lymphocytes.

NFATc3 regulation of collagen V expression contributes to cellular immunity to collagen type V and hypoxic pulmonary hypertension.

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

Mycophenolate Monotherapy in HLA-Matched Kidney Transplant Recipients: A Case Series of 20 Patients.

The ideal minimizing strategy for maintenance immunosuppression in HLA-matched kidney transplant recipients (KTR) is unknown. We hypothesized that mycophenolate (MPA) monotherapy is a safe and effective approach for maintenance therapy in this group of KTR. METHODS: Data were abstracted for 6-antigen HLA-matched KTR between 1994 and 2013. Twenty recipients receiving MPA monotherapy secondary to infection, cancer, calcineurin inhibitor (CNI) side effects, or immunosuppression minimization strategies were evaluated in this case series. RESULTS: MPA monotherapy had a low incidence of death-censored graft failure (3.19/100 person-y), rejection (0/100 person-y), hospitalization (1.62/100 person-y), malignancy (3.61/100 person-y), and infection (1.75/100 person-y). Further, 12-month mean or median serum creatinine (1.29 mg/dL), estimated glomerular filtration rate (64.3 mL/min/1.73 m2), urine protein creatinine ratio (143.2 mg/g), hemoglobin (13.9 g/dL), platelets (237.8 K/uL), and white blood cell count (9.04 K/uL) were favorable. There was a successful conversion rate of 90% (18 of 20) with 2 patients converting back to CNI-based regimens secondary to recurrence of membranous nephropathy and post-transplant lymphoproliferative disorder. CONCLUSIONS: Our findings indicate that MPA monotherapy may be a promising immunosuppression minimization strategy for HLA-matched KTR.

Treg-Cell-Derived IL-35-Coated Extracellular Vesicles Promote Infectious Tolerance.

Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in TregEbi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3+ and Foxp3neg (iTr35) T cells. Furthermore, IL-35 producers, although rare, secrete Ebi3 and p35 on extracellular vesicles (EVs) targeting a 25- to 100-fold higher number of T and B lymphocytes, causing them to acquire surface IL-35. This surface IL-35 is absent when EV production is inhibited or if Ebi3 is genetically deleted in Treg cells. The unique ability of EVs to coat bystander lymphocytes with IL-35, promoting exhaustion in, and secondary suppression by, non-Treg cells identifies a novel mechanism of infectious tolerance.

Oxidized-ATP Attenuates Kidney Allograft Rejection By Inhibiting T-Cell, B-Cell, and Macrophage Activity.

Background: Extracellular ATP binds to purinergic receptors and promotes inflammatory responses. We tested whether oxidized ATP (oATP), P2X7 receptor antagonist can attenuate acute kidney allograft rejection. Methods: Brown Norway kidney allografts were transplanted into Lewis recipients. Three groups were defined: oATP (n=8), cyclosporine A (n=6), and no treatment (n=8). On day 7, we assessed kidney allograft survival, function, and rejection characteristics. We further determined T-cell, B-cell, and macrophage response to oATP in vivo and in vitro and examined intragraft inflammatory gene transcripts. Results: Kaplan-Meier survival analyses demonstrated significantly better graft survival rates in oATP and CsA groups compared with no treatment (P<0.05). Similarly, serum creatinine (Scr) and BUN levels were significantly lower in oATP and CsA groups (P<0.05). oATP reduced both T cell-mediated rejection and antibody-mediated rejection, inhibited B-cell and T-cell activation, and downregulated intragraft IL-6 mRNA levels (P<0.0001). In vitro, oATP prevented proliferation in mixed lymphocyte reaction assays, and inhibited macrophage P2X7R activity in a dose-dependent manner. Conclusions: Our findings suggest that oATP mitigates kidney allograft rejection by inhibiting T-cell, B-cell, and macrophage activity and indicate a potential role for the purinergic system and oATP in solid organ transplantation.

Role of exosomes in tumour and transplant immune regulation.

Exosomes are a potent means for intercellular communication. However, exosomes have received intensive research focus in immunobiology only relatively recently. Because they transport proteins, lipids and genetic material between cells, they are especially suited to amplify their parental cell's message and overcome the physical constraints of cell-to-cell contact, that is exosome release gives cells the ability to alter distant, non-contiguous cells. As progress is made in this field, it has become increasingly obvious that exosomes are involved in most biological processes. In the immune system, exosomes are fundamental tools used by every immune cell type to fulfil its function and promote inflammation or tolerance. In this review, we first summarize key aspects of immune cell-specific exosomes and their functions. Then, we describe how exosomes have been shown to be indispensable orchestrators of the immune response in two immunological scenarios, namely transplant rejection or tolerance, and tumour evasion or initiation of anti-tumour immune responses.

A Human Pluripotent Stem Cell-Based Screen for Smooth Muscle Cell Differentiation and Maturation Identifies Inhibitors of Intimal Hyperplasia.

Contractile to synthetic phenotypic switching of smooth muscle cells (SMCs) contributes to stenosis in vascular disease and vascular transplants. To generate more contractile SMCs, we performed a high-throughput differentiation screen using a MYH11-NLuc-tdTomato human embryonic stem cell reporter cell line. We identified RepSox as a factor that promotes differentiation of MYH11-positive cells by promoting NOTCH signaling. RepSox induces SMCs to exhibit a more contractile phenotype than SMCs generated using PDGF-BB and TGF-ß1, two factors previously used for SMC differentiation but which also cause intimal hyperplasia. In addition, RepSox inhibited intimal hyperplasia caused by contractile to synthetic phenotypic switching of SMCs in a rat balloon injury model. Thus, in addition to providing more contractile SMCs that could prove useful for constructing artificial blood vessels, this study suggests a strategy for identifying drugs for inhibiting intimal hyperplasia that act by driving contractile differentiation rather than inhibiting proliferation non-specifically.

Epigenetic Priming of Human Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Accelerates Cardiomyocyte Maturation.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

AIMS/HYPOTHESIS: Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. METHODS: We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. RESULTS: Results in mice showed >300 days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400 days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. CONCLUSIONS/INTERPRETATION: We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas.

Extracellular matrix (ECM) plays an important developmental role by regulating cell behaviour through structural and biochemical stimulation. Tissue-specific ECM, attained through decellularization, has been proposed in several strategies for tissue and organ replacement. Decellularization of animal pancreata has been reported, but the same methods applied to human pancreas are less effective due to higher lipid content. Moreover, ECM-derived hydrogels can be obtained from many decellularized tissues, but methods have not been reported to obtain human pancreas-derived hydrogel. Using novel decellularization methods with human pancreas we produced an acellular, 3D biological scaffold (hP-ECM) and hydrogel (hP-HG) amenable to tissue culture, transplantation and proteomic applications. The inclusion of a homogenization step in the decellularization protocol significantly improved lipid removal and gelation capability of the resulting ECM, which was capable of gelation at 37 °C in vitro and in vivo, and is cytocompatible with a variety of cell types and islet-like tissues in vitro. Overall, this study demonstrates the characterisation of a novel protocol for the decellularization and delipidization of human pancreatic tissue for the production of acellular ECM and ECM hydrogel suitable for cell culture and transplantation applications. We also report a list of 120 proteins present within the human pancreatic matrisome.

Leukocyte-Associated Ig-like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses.

Leukocyte-associated Ig-like receptor 1 (LAIR1) is an ITIM-bearing collagen receptor expressed by leukocytes and is implicated in immune suppression. However, using a divalent soluble LAIR1/Fc recombinant protein to block interaction of cell surface LAIR1 with matrix collagen, we found that whereas Th1 responses were enhanced as predicted, Th17 responses were strongly inhibited. Indeed, LAIR1 on both T cells and monocytes was required for optimal Th17 responses to collagen type (Col)V. For pre-existing "natural" Th17 response to ColV, the LAIR1 requirement was absolute, whereas adaptive Th17 and Th1/17 immune responses in both mice and humans were profoundly reduced in the absence of LAIR1. Furthermore, the addition of C1q, a natural LAIR1 ligand, decreased Th1 responses in a dose-dependent manner, but it had no effect on Th17 responses. In IL-17-dependent murine organ transplant models of chronic rejection, LAIR1+/+ but not LAIR1-/- littermates mounted strong fibroproliferative responses. Surface LAIR1 expression was higher on human Th17 cells as compared with Th1 cells, ruling out a receptor deficiency that could account for the differences. We conclude that LAIR1 ligation by its natural ligands favors Th17 cell development, allowing for preferential activity of these cells in collagen-rich environments. The emergence of cryptic self-antigens such as the LAIR1 ligand ColV during ischemia/reperfusion injury and early acute rejection, as well as the tendency of macrophages/monocytes to accumulate in the allograft during chronic rejection, favors Th17 over Th1 development, posing a risk to long-term graft survival.

A Humanized Mouse Model Generated Using Surplus Neonatal Tissue.

Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs). Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue.

Specific Donor HLA-DR Types Correlate With Altered Susceptibility to Development of Chronic Lung Allograft Dysfunction.

BACKGROUND: The greatest challenge to long-term graft survival is the development of chronic lung allograft dysfunction. Th17 responses to collagen type V (colV) predispose lung transplant patients to the severe obstructive form of chronic lung allograft dysfunction, known as bronchiolitis obliterans syndrome (BOS). In a previous study cohort (n = 54), pretransplant colV responses were increased in recipients expressing HLA-DR15, consistent with the high binding avidity of colV (α1) peptides for HLA-DR15, whereas BOS incidence, which was known to be strongly associated with posttransplant autoimmunity to colV, was higher in patients who themselves lacked HLA-DR15, but whose lung donor expressed it. METHODS: To determine if this DR-restricted effect on BOS incidence could be validated in a larger cohort, we performed a retrospective analysis of outcomes for 351 lung transplant recipients transplanted between 1988 and 2008 at the University of Wisconsin. All subjects were followed until graft loss, death, loss to follow-up, or through 2014, with an average follow-up of 7 years. Comparisons were made between recipients who did or did not develop BOS. Grading of BOS followed the recommendations of the international society for heart and lung transplantation. RESULTS: Donor HLA-DR15 was indeed associated with increased susceptibility to severe BOS in this population. We also discovered that HLA-DR7 expression by the donor or HLA-DR17 expression by the recipient decreased susceptibility. CONCLUSIONS: We show in this retrospective study that specific donor HLA class II types are important in lung transplantation, because they are associated with either protection from or susceptibility to development of severe BOS.