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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 Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD.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.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Transplante de Rim , Radioterapia de Intensidade Modulada , Animais , Macaca mulatta , Irradiação Linfática , Tolerância Imunológica , Tolerância ao Transplante , Condicionamento Pré-Transplante/métodos , Rim , Quimeras de TransplanteRESUMO
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.
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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.
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Vesículas Extracelulares , Transplante de Órgãos , Animais , Antígenos , Antígeno B7-H1 , Células Dendríticas , Feminino , Sangue Fetal , Tolerância Imunológica , Camundongos , Gravidez , Linfócitos T Reguladores , Tolerância ao TransplanteRESUMO
BACKGROUND: Complement activation in kidney transplantation is implicated in the pathogenesis of delayed graft function (DGF). This study evaluated the therapeutic efficacy of high-dose recombinant human C1 esterase inhibitor (rhC1INH) to prevent DGF in a nonhuman primate model of kidney transplantation after brain death and prolonged cold ischemia. METHODS: Brain death donors underwent 20 h of conventional management. Procured kidneys were stored on ice for 44-48 h, then transplanted into ABO-compatible major histocompatibility complex-mismatched recipients. Recipients were treated with vehicle (n = 5) or rhC1INH 500 U/kg plus heparin 40 U/kg (n = 8) before reperfusion, 12 h, and 24 h posttransplant. Recipients were followed up for 120 d. RESULTS: Of vehicle-treated recipients, 80% (4 of 5) developed DGF versus 12.5% (1 of 8) rhC1INH-treated recipients (P = 0.015). rhC1INH-treated recipients had faster creatinine recovery, superior urinary output, and reduced urinary neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2-insulin-like growth factor-binding protein 7 throughout the first week, indicating reduced allograft injury. Treated recipients presented lower postreperfusion plasma interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, and IL-18, lower day 4 monocyte chemoattractant protein 1, and trended toward lower C5. Treated recipients exhibited less C3b/C5b-9 deposition on day 7 biopsies. rhC1INH-treated animals also trended toward prolonged mediated rejection-free survival. CONCLUSIONS: Our results recommend high-dose C1INH complement blockade in transplant recipients as an effective strategy to reduce kidney injury and inflammation, prevent DGF, delay antibody-mediated rejection development, and improve transplant outcomes.
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Transplante de Rim , Animais , Função Retardada do Enxerto/etiologia , Função Retardada do Enxerto/prevenção & controle , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Rim , Transplante de Rim/efeitos adversos , Primatas , Doadores de TecidosRESUMO
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.
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Quimerismo , Transplante de Células-Tronco Hematopoéticas , Tecido Linfoide/efeitos da radiação , Modelos Biológicos , Radioterapia de Intensidade Modulada/métodos , Animais , Doença Enxerto-Hospedeiro , Macaca mulatta , Modelos Animais , Transplante HomólogoRESUMO
Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor ß (TGF-ß) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGF-ß (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-ß treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-ß treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-ß.
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Transplante de Fígado , Traumatismo por Reperfusão , Animais , Citocinas , Interleucina-10 , Fígado , Preservação de Órgãos , Perfusão , Ratos , Fator de Crescimento Transformador beta , Fatores de Crescimento TransformadoresRESUMO
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.
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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.
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Alergia e Imunologia/história , Vesículas Extracelulares/imunologia , Tolerância Imunológica/imunologia , Interleucinas/imunologia , Animais , História do Século XX , História do Século XXI , HumanosRESUMO
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.
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Colágeno Tipo V/metabolismo , Hipertensão Pulmonar/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Núcleo Celular/metabolismo , Imunidade Celular/fisiologia , Transplante de Pulmão/métodosRESUMO
OBJECTIVES: Emerging evidence has shown a role for tumor antigen-specific regulation in cancer. Identifying individuals with pre-existing regulatory responses may be key to understand those who are more likely to respond to Programmed Death-1 (PD-1) or PD-1 Ligand 1 (PD-L1) checkpoint blockade. We hypothesized that a functional assay could identify the role of PD-1/PD-L1 interactions on tumor-specific immune cells in the peripheral blood in patients with advanced non-small-cell lung cancer (NSCLC). METHODS: We performed the trans vivo delayed-type hypersensitivity assay to identify the role of PD-1/PD-L1-mediated tumor-specific immune regulation in ten patients with advanced NSCLC. RESULTS: The majority of patients had PD-1-mediated anergic immune responses towards their tumor antigens. Eight out of nine of these patients did not respond to their own tumor antigens but responded in the presence of anti-PD-1 antibody ('PD-1 anergy' phenotype). A minority (3/9) also had 'active' PD-1-mediated immune suppressive regulatory responses. Our results suggest that PD-1-anergy is a common feature of NSCLC immune responses, whereas PD-1-mediated immune suppression is present only in a minority of patients. The latter was associated with poor clinical outcomes in our sample. CONCLUSIONS: Overall, our results indicate that bystander suppression or the 'anergy-only' phenomenon may be novel biomarkers in NSCLC and suggest prediction value based on these phenotypes.
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Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Receptor de Morte Celular Programada 1/imunologia , Idoso , Animais , Feminino , Humanos , Masculino , CamundongosRESUMO
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.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Rejeição de Enxerto/prevenção & controle , Terapia de Imunossupressão/métodos , Imunossupressores/uso terapêutico , Transplante de Rim , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Células Dendríticas/imunologia , Rejeição de Enxerto/imunologia , Humanos , Terapia de Imunossupressão/efeitos adversos , Macrófagos/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
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.
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Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement-driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement-blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43-48 hours prior to implantation into ABO-compatible, nonsensitized, MHC-mismatched recipients. Animals were divided into 3 donor-treatment groups: G1 - vehicle, G2 - rhC1INH+heparin, and G3 - heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b-9, and reduced urinary neutrophil gelatinase-associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor-management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD-induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation.
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Transplante de Rim , Animais , Morte Encefálica , Função Retardada do Enxerto/etiologia , Função Retardada do Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Transplante de Rim/efeitos adversos , Primatas , Fatores de Risco , Doadores de TecidosRESUMO
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.
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Vesículas Extracelulares/metabolismo , Tolerância Imunológica , Subunidade p35 da Interleucina-12/metabolismo , Interleucinas/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Citocinas/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Técnicas de Cocultura , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/ultraestrutura , Feminino , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Inativação de Genes , Transplante de Coração , Terapia de Imunossupressão , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Linfócitos T Reguladores/metabolismoRESUMO
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.
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Rejeição de Enxerto , Linfócitos T , Trifosfato de Adenosina/metabolismo , Aloenxertos/metabolismo , Rejeição de Enxerto/prevenção & controle , Rim/metabolismo , Macrófagos/metabolismo , Linfócitos T/metabolismoRESUMO
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.
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Exossomos/metabolismo , Rejeição de Enxerto/imunologia , Tolerância Imunológica/imunologia , Neoplasias/imunologia , Evasão Tumoral/imunologia , Linfócitos B/imunologia , Transporte Biológico/fisiologia , Comunicação Celular/fisiologia , Células Dendríticas/imunologia , Humanos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
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 (â¼90%). 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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Poli I-C/farmacologia , Receptores Notch/genética , Animais , Tamanho Celular , Histonas/genética , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Rim , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Notch/metabolismo , Sarcômeros/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Transplante de Células-Tronco/métodos , Transplante Heterotópico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.