RESUMO
Somatic mutations in T cells can cause cancer but also have implications for immunological diseases and cell therapies. The mutation spectrum in nonmalignant T cells is unclear. Here, we examined somatic mutations in CD4+ and CD8+ T cells from 90 patients with hematological and immunological disorders and used T cell receptor (TCR) and single-cell sequencing to link mutations with T cell expansions and phenotypes. CD8+ cells had a higher mutation burden than CD4+ cells. Notably, the biggest variant allele frequency (VAF) of non-synonymous variants was higher than synonymous variants in CD8+ T cells, indicating non-random occurrence. The non-synonymous VAF in CD8+ T cells strongly correlated with the TCR frequency, but not age. We identified mutations in pathways essential for T cell function and often affected lymphoid neoplasia. Single-cell sequencing revealed cytotoxic TEMRA phenotypes of mutated T cells. Our findings suggest that somatic mutations contribute to CD8+ T cell expansions without malignant transformation.
Assuntos
Linfócitos T CD8-Positivos , Mutação , Receptores de Antígenos de Linfócitos T , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Adulto , Análise de Célula Única , Masculino , Feminino , Pessoa de Meia-Idade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Frequência do Gene , Fenótipo , IdosoRESUMO
Despite recent progress in acute lymphoblastic leukemia (ALL) therapies, a significant subset of adult and pediatric ALL patients has a dismal prognosis. Better understanding of leukemogenesis and recognition of germline genetic changes may provide new tools for treating patients. Given that hematopoietic stem cell transplantation, often from a family member, is a major form of treatment in ALL, acknowledging the possibility of hereditary predisposition is of special importance. Reports of comprehensive germline analyses performed in adult ALL patients are scarce. Aiming at fulfilling this gap of knowledge, we investigated variants in 93 genes predisposing to hematologic malignancies and 70 other cancer-predisposing genes from exome data obtained from 61 adult and 87 pediatric ALL patients. Our results show that pathogenic (P) or likely pathogenic (LP) germline variants in genes associated with predisposition to ALL or other cancers are prevalent in ALL patients: 8% of adults and 11% of children. Comparison of P/LP germline variants in patients to population-matched controls (gnomAD Finns) revealed a 2.6-fold enrichment in ALL cases (CI 95% 1.5-4.2, p = 0.00071). Acknowledging inherited factors is crucial, especially when considering hematopoietic stem cell transplantation and planning post-therapy follow-up. Harmful germline variants may also predispose patients to excessive toxicity potentially compromising the outcome. We propose integrating germline genetics into precise ALL patient care and providing families genetic counseling.
Assuntos
Mutação em Linhagem Germinativa , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Criança , Exoma , Predisposição Genética para Doença , Células Germinativas , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMO
Gemtuzumab-ozogamicin (GO) is a humanized anti-CD33 antibody, which is conjugated to a cytotoxic calicheamicin. It is used to treat acute myeloid leukemia (AML) in combination with chemotherapy. We describe here two GO-treated acute myeloid leukemia (AML) cases: both patients suffered from a toxic syndrome, which manifested as impaired hemoglobin-haptoglobin scavenging and accumulation of hemolysis-related products. Our observations and earlier reports indicated that the reaction was caused by GO-targeted destruction of CD33 + CD163+ monocytes/macrophages, which are responsible for the clearance of hemoglobin-haptoglobin complexes. The rise of plasma lactate dehydrogenase was an early sign of the reaction, and both patients had high levels of free plasma hemoglobin, but plasma haptoglobin and bilirubin levels were paradoxically normal. Symptoms included septic fever and abnormalities in cardiac tests and in the case of the first patient, severe neurological symptoms which required intensive care unit admittance. Therapeutic plasma exchanges supported the patients until the recovery of normal hematopoiesis. The symptoms may be easily confounded with infectious complications-related organ damage. Regarding the increasing use of gemtuzumab-ozogamicin and other emerging CD33-targeted cell therapies, we want to highlight this mostly unknown and probably underdiagnosed toxicity.
RESUMO
The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.
Assuntos
Anemia Aplástica/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Mutação/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Common variable immunodeficiency and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is however implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in common variable immunodeficiency and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+ and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.
Assuntos
Síndromes de Imunodeficiência , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Regiões Determinantes de Complementaridade/genética , Humanos , Mutação , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-γ response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide/genética , Linfoma Difuso de Grandes Células B/genética , Doença Aguda , Epigênese Genética , Genômica/métodos , Antígenos HLA/genética , Humanos , Imunoterapia/métodos , Leucemia Mieloide/imunologia , Leucemia Mieloide/terapia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/terapia , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Mutação , Proteína Supressora de Tumor p53/genéticaRESUMO
Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.
Assuntos
Doença Enxerto-Hospedeiro/genética , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/genética , Western Blotting , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células HEK293 , Humanos , Imunidade Celular/genética , Imunidade Celular/fisiologia , Imunoprecipitação , Mutação/genética , Ligação Proteica/genética , Ligação Proteica/fisiologiaRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.
Assuntos
Citotoxicidade Imunológica/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Testes Imunológicos de Citotoxicidade/métodos , Humanos , Ativação Linfocitária/imunologia , Linfoma Difuso de Grandes Células B/imunologia , Receptores de Antígenos QuiméricosRESUMO
Hypoxia-inducible factors (HIFs) play a critical role in inflammatory properties of myeloid-derived cells. The effect of HIFs on myeloid-derived cell functions in organ transplantation remains unknown, however. We transplanted hearts into transgenic mice with myeloid cell-targeted deletions of HIF-1α or its negative regulator von Hippel-Lindau (VHL) to investigate the effects of HIF-1α inactivation or HIF pathway activation, respectively, on ischemia-reperfusion injury (IRI) and acute rejection. Deletion of VHL in myeloid cells enhanced mRNA expression of anti-inflammatory genes IDO, Arg-1, and HO-1 in vitro. In vivo, VHL-/- myeloid-derived cells of allograft recipients alleviated IRI and acute rejection, evidenced by reduced cardiomyocyte damage, decreased proinflammatory cytokine mRNA levels, and absence of inflammatory infiltrate at 5 days after transplantation. Ultimately, allograft survival was significantly prolonged. In vitro, VHL-/- myeloid-derived cells dose-dependently inhibited T-cell proliferation. Myeloid cells with HIF-1α-deletion retained proinflammatory qualities in vitro and in vivo. Deletion of VHL in myeloid cells of nonimmunosuppressed cardiac allograft recipients reduced myocardial injury and acute rejection. We suggest that HIF transcription factors induce a regulatory phenotype in myeloid-derived cells, which may be harnessed as a novel therapeutic strategy to regulate immune responses after heart transplantation.
Assuntos
Transplante de Coração , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Mieloides/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aloenxertos , Animais , Proliferação de Células , Feminino , Sobrevivência de Enxerto , Inflamação , Linfócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão , Linfócitos T/citologia , Transplante HomólogoAssuntos
Artrite Reumatoide/sangue , Evolução Clonal , Hematopoese , Artrite Reumatoide/genética , Biomarcadores , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mutação , Proteínas Proto-Oncogênicas/genéticaRESUMO
BACKGROUND: Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. METHODS AND RESULTS: We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-ß, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. CONCLUSIONS: Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.
Assuntos
Terapia Genética/métodos , Transplante de Coração/efeitos adversos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-sis/biossíntese , Adenoviridae/genética , Aloenxertos , Animais , Becaplermina , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Vetores Genéticos , Mesilato de Imatinib/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-sis/genética , Ratos Wistar , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de TempoRESUMO
Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Transplante de Coração , Coração/virologia , Animais , Dependovirus/classificação , Isoenxertos , Luciferases/genética , Masculino , Miocárdio/metabolismo , Ratos , Transgenes/genéticaRESUMO
BACKGROUND: Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. METHODS AND RESULTS: Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell-endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury-induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-ß1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-ß1-induced microvascular endothelial-to-mesenchymal transition. CONCLUSIONS: Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Microvasos/efeitos dos fármacos , Disfunção Primária do Enxerto/prevenção & controle , Traumatismo por Reperfusão/prevenção & controle , Sinvastatina/uso terapêutico , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotelina-1/biossíntese , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/enzimologia , Heme Oxigenase-1/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Masculino , Microvasos/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Fenômeno de não Refluxo/prevenção & controle , Fosfatos de Poli-Isoprenil/farmacologia , Disfunção Primária do Enxerto/enzimologia , Ratos , Ratos Endogâmicos WF , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
BACKGROUND: Preservation injury decreases patient survival and promotes the development of cardiac allograft vasculopathy. We investigated the sequential effects of hypothermic preservation on ischemia-reperfusion injury (IRI), subsequent innate immune activation, and adaptive immune response in rat cardiac allografts. METHODS: Allografts were transplanted from fully major histocompatibility complex-mismatched Dark Agouti to Wistar Furth rats without pre-operative hypothermia or after 4 hours of hypothermic preservation. Recipients received cyclosporine A immunosuppression. The allografts were recovered at 6 hours (n = 6, 7), 24 hours (n = 6), 10 days (n = 5), and 8 weeks (n = 5). Immunohistochemical, histologic, and reverse-transcription polymerase chain reaction analysis was performed. RESULTS: In IRI, significantly increased messenger RNA (mRNA) levels for Toll-like receptor 4, hyaluronan synthases (HAS)1-2 (p = 0.03), high-mobility group box 1 (p = 0.05), CD80/83 (p = 0.01, p = 0.048), and the cytokines tumor necrosis factor-alpha (p = 0.004), interferon-gamma (p = 0.012), and interleukin (IL)-6 (p = 0.019) were seen in allografts subjected to hypothermic preservation. During established alloimmune response, allografts subjected to hypothermic preservation expressed prominent infiltration of CD4+ T cells (p = 0.043) and dendritic cells (p = 0.029) and significantly up-regulated mRNA levels of CD80 (p = 0.036), chemokine (C-C motif) ligand 21 (p = 0.008), C-C chemokine receptor type 7 (p = 0.003), vascular endothelial growth factor-C (p = 0.016), and vascular endothelial growth factor receptor-3 (p = 0.02). These allografts also showed prominent mRNA upregulation of Foxp3 (p = 0.014), IL-17 (p = 0.038), and IL-23 (p = 0.043). Preservation significantly increased the incidence and intensity of allograft arteriosclerosis (p < 0.05) and cardiac fibrosis (p = 0.003) at 8 weeks. CONCLUSION: Our results demonstrate that preservation injury induced a cascade leading to an innate immune response that modulated the adaptive immune response towards Th17 rather than Th1 T-cell response in rat cardiac allografts and ultimately enhanced cardiac fibrosis and arterial occlusion. Our results also suggest that this immune response was not regulated by the calcineurin inhibitor cyclosporine A.
Assuntos
Transplante de Coração/imunologia , Células Th1/imunologia , Células Th17/imunologia , Doença Aguda , Animais , Antígenos CD/genética , Doença Crônica , Ciclosporina/uso terapêutico , Células Dendríticas/imunologia , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/imunologia , Transplante de Coração/patologia , Imunoglobulinas/genética , Imunossupressores/uso terapêutico , Glicoproteínas de Membrana/genética , Preservação de Órgãos/métodos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Ratos Endogâmicos WF , Reperfusão/métodos , Fatores de Tempo , Transplante Homólogo/imunologia , Antígeno CD83RESUMO
BACKGROUND: Hypoxia-inducible factor-1 (HIF-1), a key transcription factor in hypoxia, affects a wide range of adaptive cell functions. We examined the kinetics of endogenous HIF-1alpha during acute and chronic rejection, and the effect of exogenous HIF-1alpha in chronically rejecting rat cardiac allografts. METHODS: Heterotopic cardiac transplantations were performed between major MHC-mismatched Dark Agouti and Wistar-Furth rats. Cyclosporine A (CsA) was used to prevent acute rejection in the chronic rejection model. The effect of HIF-1alpha overexpression was investigated by adeno-assocated virus 2 (AAV2)-mediated gene transfer of a constitutively stabilized form of mouse HIF-1alpha (AAV-HIF-1alpha). The analysis of allografts was based on histology, immunohistochemistry and quantitative reverse transcript-polymerase chain reaction (RT-PCR). RESULTS: Acute and chronic rejection significantly induced HIF-1alpha mRNA in rat cardiac allografts when compared with syngeneic controls. Immunohistochemistry localized significantly increased HIF-1alpha immunoreactivity to vascular smooth muscle cells, vascular endothelial cells, post-capillary venules and graft-infiltrating mononuclear inflammatory cells of the allograft, whereas expression in cardiomyocytes remained unchanged. Regression analysis revealed a linear correlation between the progression of cardiac allograft vasculopathy (CAV) and HIF-1alpha immunoreactivity in post-capillary venules and graft-infiltrating mononuclear inflammatory cells in chronically rejecting rat cardiac allografts. AAV-HIF-1alpha enhanced cardiomyocyte HIF-1alpha production and significantly reduced cardiomyocyte apoptosis and the development of CAV in chronically rejecting rat cardiac allografts. CONCLUSIONS: We found that acute and chronic rejection increased HIF-1alpha mRNA and protein levels in rat cardiac allografts. On the other hand, cardiomyocyte-targeted HIF-1alpha gene transfer inhibited cardiomyocyte apoptosis and the development of CAV, suggesting a novel therapeutic strategy for HIF-1alpha in cardiac allografts.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Transplante de Coração/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Abdome , Animais , Apoptose , Anidrase Carbônica IX , Anidrases Carbônicas/metabolismo , Rejeição de Enxerto/patologia , Transplante de Coração/imunologia , Transplante de Coração/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Complicações Pós-Operatórias/patologia , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Ratos Endogâmicos WF , Transplante Heterotópico , Transplante Homólogo/imunologia , Transplante Homólogo/patologiaRESUMO
BACKGROUND: Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. METHODS AND RESULTS: Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C(+) inflammatory cell and hyaluronan receptor-1 (LYVE-1)(+) lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3(+), contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein-positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8(+) effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62(+) dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4(+) T cells in chronically rejecting mouse cardiac allografts. CONCLUSIONS: These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel-targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.
Assuntos
Arteriosclerose/prevenção & controle , Quimiocina CCL21/biossíntese , Transplante de Coração/imunologia , Imunomodulação/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Células Apresentadoras de Antígenos , Arteriosclerose/tratamento farmacológico , Movimento Celular/imunologia , Rejeição de Enxerto/imunologia , Camundongos , Camundongos Knockout , Ratos , Transdução de Sinais/imunologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
OBJECTIVE: Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A-D)--potent mesenchymal cell mitogens--in rat cardiac allografts. METHODS AND RESULTS: PDGFR-alpha mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac centhatn allografts. In acute rejection, PDGFR-alpha immunoreactivity increased in the media of arteries. In chronically-rejecting allografts, immunoreactivity of all PDGF ligands and receptors--except that of PDGF-B ligand--was found in the intima of arteries, and the expression of PDGF-A and PDGF-C was seen in cardiomyocytes. Intracoronary adeno-associated virus-2 (AAV2)-mediated PDGF-A and -D gene transfer enhanced cardiac allograft inflammation. AAV2-PDGF-A, AAV2-PDGF-C, and AAV2-PDGF-D significantly upregulated profibrotic TGF-beta1 mRNA and accelerated cardiac fibrosis and arteriosclerosis. In contrast, AAV2-PDGF-B did not aggravate chronic rejection. CONCLUSIONS: We found that alloimmune response induces PDGF-A, PDGF-C, and PDGF-D expression in the graft vasculature. PDGF-A, PDGF-C, and PDGF-D mediated profibrotic and proarteriosclerotic effects in transplanted hearts involving the TGF-beta1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.
Assuntos
Arteriosclerose/imunologia , Rejeição de Enxerto/metabolismo , Transplante de Coração/imunologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Modelos Animais de Doenças , Fibrose/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Transplante Homólogo , Regulação para CimaRESUMO
OBJECTIVE: Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. METHODS AND RESULTS: Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow- or non-bone marrow-derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis. CONCLUSIONS: These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.