RESUMO
Adoptive cell therapy (ACT), especially with CD4+ regulatory T cells (CD4+ Tregs), is an emerging therapeutic strategy to minimize immunosuppression and promote long-term allograft acceptance, although much research remains to realize its potential. In this study, we investigated the potency of novel Ab-suppressor CXCR5+CD8+ T cells (CD8+ TAb-supp) in comparison with conventional CD25highFoxp3+CD4+ Tregs for suppression of humoral alloimmunity in a murine kidney transplant (KTx) model of Ab-mediated rejection (AMR). We examined quantity of peripheral blood, splenic and graft-infiltrating CD8+ TAb-supp, and CD4+ Tregs in KTx recipients and found that high alloantibody-producing CCR5 knockout KTx recipients have significantly fewer post-transplant peripheral blood and splenic CD8+ TAb-supp, as well as fewer splenic and graft-infiltrating CD4+ Tregs compared with wild-type KTx recipients. ACT with alloprimed CXCR5+CD8+ T cells reduced alloantibody titer, splenic alloprimed germinal center (GC) B cell quantity, and improved AMR histology in CCR5 knockout KTx recipients. ACT with alloprimed CD4+ Treg cells improved AMR histology without significantly inhibiting alloantibody production or the quantity of splenic alloprimed GC B cells. Studies with TCR transgenic mice confirmed Ag specificity of CD8+ TAb-supp-mediated effector function. In wild-type recipients, CD8 depletion significantly increased alloantibody titer, GC B cells, and severity of AMR pathology compared with isotype-treated controls. Anti-CD25 mAb treatment also resulted in increased but less pronounced effect on alloantibody titer, quantity of GC B cells, and AMR pathology than CD8 depletion. To our knowledge, this is the first report that CD8+ TAb-supp cells are more potent regulators of humoral alloimmunity than CD4+ Treg cells.
Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Transplante de Rim , Linfócitos T Reguladores , Animais , Camundongos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Isoanticorpos , Transplante de Rim/efeitos adversos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores CXCR5/imunologia , Imunidade Humoral/imunologiaRESUMO
BACKGROUND: Adoptive cellular therapy (ACT) with antibody-suppressor CXCR5 + CD8 + T cells (CD8 + T Ab-supp ) inhibits alloantibody production, antibody-mediated rejection (AMR), and prolongs graft survival in multiple transplant mouse models. However, it is not known how conventional immunosuppressive agents impact the efficacy of CD8 + T Ab-supp ACT. METHODS: We investigated the efficacy of CD8 + T Ab-supp cell ACT when combined with calcineurin inhibitor (CNi) or mammalian target of rapamycin inhibitor (mTORi) in a murine model of kidney transplant. RESULTS: ACT-mediated decrease in germinal center B cells, posttransplant alloantibody titer, and amelioration of AMR in high alloantibody-producing CCR5 knockout kidney transplant recipients were impaired when ACT was combined with CNi and enhanced when combined with mTORi. CNi (but not mTORi) reduced ACT-mediated in vivo cytotoxicity of IgG + B cells and was associated with increased quantity of germinal center B cells. Neither CNi nor mTORi treatment impacted the expression of cytotoxic effector molecules (FasL, Lamp1, perforin, granzyme B) by CD8 + T Ab-supp after ACT. Concurrent treatment with CNi (but not mTORi) reduced in vivo proliferation of CD8 + T Ab-supp after ACT. The increase in quantity of splenic CD44 + CXCR5 + CD8 + T cells that occurs after ACT was reduced by concurrent treatment with CNi but not by concurrent treatment with mTORi (dose-dependent). CONCLUSIONS: Impaired efficacy of ACT by CNi is attributed to reduced persistence and/or expansion of CD8 + T Ab-supp cells after ACT. In contrast, concurrent immunosuppression with mTORi preserves CD8 + T Ab-supp cells quantity, in vivo proliferation, and in vivo cytotoxic effector function after ACT and enhances suppression of humoral alloimmunity and AMR.
Assuntos
Calcineurina , Imunossupressores , Animais , Camundongos , Imunossupressores/farmacologia , Inibidores de Calcineurina , Linfócitos T CD8-Positivos , Serina-Treonina Quinases TOR/metabolismo , Isoanticorpos , Rejeição de Enxerto/prevenção & controle , Mamíferos/metabolismoRESUMO
CCR5 KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5 KO mice (H-2b ) were transplanted with A/J kidneys (H-2a ); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5+ CD8+ T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5+ CD8+ T cells and CD8-mediated cytotoxicity to alloprimed IgG+ B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5+ CD8+ T cells 7 days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG+ B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5+ CD8+ T cells (but not alloprimed CXCR5- CD8+ or third-party primed CXCR5+ CD8+ T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5+ CD8+ T cells contributes to high alloantibody and AMR in CCR5 KO recipient mice, which can be rescued with ACT.
Assuntos
Transplante de Rim , Animais , Linfócitos T CD8-Positivos , Rejeição de Enxerto/patologia , Imunoglobulina G , Isoanticorpos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8+ T cells, with or without CD4+ T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type) and iNKT-deficient Jα18 knockout mice (cohorts CD4 depleted) were transplanted with allogeneic hepatocytes. Recipients were evaluated for alloprimed CD8+ T cell subset composition, allocytotoxicity, and hepatocyte rejection. We found that CD8-mediated allocytotoxicity was significantly decreased in iNKT-deficient recipients and was restored by adoptive transfer of iNKT cells. In the absence of both iNKT cells and CD4+ T cells, CD8-mediated allocytotoxicity and hepatocyte rejection was abrogated. iNKT cells enhance the proportion of a novel subset of multipotent, alloprimed CXCR3+CCR4+CD8+ cytolytic T cells that develop after hepatocyte transplant and are abundant in the liver. Alloprimed CXCR3+CCR4+CD8+ T cells express cytotoxic effector molecules (perforin/granzyme and Fas ligand) and are distinguished from alloprimed CXCR3+CCR4-CD8+ T cells by a higher proportion of cells expressing TNF-α and IFN-γ. Furthermore, alloprimed CXCR3+CCR4+CD8+ T cells mediate higher allocytotoxicity and more rapid allograft rejection. Our data demonstrate the important role of iNKT cells in promoting the development of highly cytotoxic, multipotent CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of allogeneic hepatocytes engrafted in the liver. Targeting iNKT cells may be an efficacious therapy to prevent rejection of intrahepatic cellular transplants.
Assuntos
Transplante de Fígado , Células T Matadoras Naturais , Aloenxertos , Animais , Linfócitos T CD8-Positivos , Rejeição de Enxerto , Hepatócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
CD8+ T cells have conventionally been studied in relationship to pathogen or tumor clearance. Recent reports have identified novel functions of CXCR5+CD8+ T cells that can home to lymphoid follicles, a key site of antibody production. In this review we provide an in-depth analysis of conflicting reports regarding the impact of CXCR5+CD8+ T cells on antibody production and examine the data supporting a role for antibody-enhancement (B cell "helper") and antibody-downregulation (antibody-suppressor) by CXCR5+CD8+ T cell subsets. CXCR5+CD8+ T cell molecular phenotypes are associated with CD8-mediated effector functions including distinct subsets that regulate antibody responses. Co-inhibitory molecule PD-1, among others, distinguish CXCR5+CD8+ T cell subsets. We also provide the first in-depth review of human CXCR5+CD8+ T cells in the context of clinical outcomes and discuss the potential utility of monitoring the quantity of peripheral blood or tissue infiltrating CXCR5+CD8+ T cells as a prognostic tool in multiple disease states.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Anticorpos/metabolismo , Formação de Anticorpos , Humanos , Imunomodulação , Ativação Linfocitária , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismoRESUMO
BACKGROUND: We recently reported that a novel CXCR5IFN-γCD8 T-cell subset significantly inhibits posttransplant alloantibody production in a murine transplant model. These findings prompted the current study to investigate the association of human CD8 T cells with the same phenotype with the development of de novo donor-specific antibody (DSA) after kidney transplantation. METHODS: In the current studies, we prospectively and serially analyzed peripheral blood CD8 and CD4 T-cell subsets and monitored for the development of de novo DSA in kidney transplant recipients during the first-year posttransplant. We report results on 95 first-time human kidney transplant recipients with 1-year follow-up. RESULTS: Twenty-three recipients (24.2%) developed de novo DSA within 1-year posttransplant. Recipients who developed DSA had significantly lower quantities of peripheral CXCR5IFN-γCD8 T cells (P = 0.01) and significantly lower ratios of CXCR5IFN-γCD8 T cell to combined CD4 Th1/Th2 cell subsets (IFN-γCD4 and IL-4CD4 cells; P = 0.0001) compared to recipients who remained DSA-negative over the first-year posttransplant. CONCLUSIONS: Our data raise the possibility that human CXCR5IFN-γCD8 T cells are a homolog to murine CXCR5IFN-γCD8 T cells (termed antibody-suppressor CD8 T cells) and that the quantity of CXCR5IFN-γCD8 T cells (or the ratio of CXCR5IFN-γCD8 T cells to Th1/Th2 CD4 T cells) may identify recipients at risk for development of DSA.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos HLA/imunologia , Histocompatibilidade , Interferon gama/sangue , Isoanticorpos/sangue , Transplante de Rim , Receptores CXCR5/sangue , Adulto , Idoso , Biomarcadores/sangue , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Humanos , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Prospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: We previously reported the novel activity of alloprimed CD8 T cells that suppress posttransplant alloantibody production. The purpose of the study is to investigate the expression and role of CXCR5 on antibody-suppressor CD8 T-cell function. METHODS: C57BL/6 mice were transplanted with FVB/N hepatocytes. Alloprimed CD8 T cells were retrieved on day 7 from hepatocyte transplant recipients. Unsorted or flow-sorted (CXCR5CXCR3 and CXCR3CXCR5) alloprimed CD8 T-cell subsets were analyzed for in vitro cytotoxicity and capacity to inhibit in vivo alloantibody production following adoptive transfer into C57BL/6 or high alloantibody-producing CD8 knock out (KO) hepatocyte transplant recipients. Alloantibody titer was assessed in CD8 KO mice reconstituted with naive CD8 T cells retrieved from C57BL/6, CXCR5 KO, or CXCR3 KO mice. Antibody suppression by ovalbumin (OVA)-primed monoclonal OVA-specific t-cell receptor transgenic CD8+ T cells (OT-I) CXCR5 or CXCR3 CD8 T-cell subsets was also investigated. RESULTS: Alloprimed CXCR5CXCR3CD8 T cells mediated in vitro cytotoxicity of alloprimed "self" B cells, while CXCR3CXCR5CD8 T cells did not. Only flow-sorted alloprimed CXCR5CXCR3CD8 T cells (not flow-sorted alloprimed CXCR3CXCR5CD8 T cells) suppressed alloantibody production and enhanced graft survival when transferred into transplant recipients. Unlike CD8 T cells from wild-type or CXCR3 KO mice, CD8 T cells from CXCR5 KO mice do not develop alloantibody-suppressor function. Similarly, only flow-sorted CXCR5CXCR3 (and not CXCR3CXCR5) OVA-primed OT-I CD8 T cells mediated in vivo suppression of anti-OVA antibody production. CONCLUSIONS: These data support the conclusion that expression of CXCR5 by antigen-primed CD8 T cells is critical for the function of antibody-suppressor CD8 T cells.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Linfócitos B/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/prevenção & controle , Hepatócitos/transplante , Transplante de Fígado , Receptores CXCR5/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Antígenos CD8/deficiência , Antígenos CD8/genética , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Hepatócitos/imunologia , Hepatócitos/metabolismo , Transplante de Fígado/efeitos adversos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CXCR3/deficiência , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores CXCR5/deficiência , Receptores CXCR5/genética , Transdução de Sinais , Fatores de TempoRESUMO
Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known. We hypothesized that macrophage-mediated destruction of allogeneic hepatocytes occurs by cell-cell interactions requiring FcγRs. To examine this, alloantibody-dependent hepatocyte rejection in CD8-depleted wild-type (WT) and Fcγ-chain knockout (KO; lacking all functional FcγR) transplant recipients was evaluated. Alloantibody-mediated hepatocellular allograft rejection was abrogated in recipients lacking FcγR compared with WT recipients. We also investigated anti-FcγRI mAb, anti-FcγRIII mAb, and inhibitors of intracellular signaling (to block phagocytosis, cytokines, and reactive oxygen species [ROS]) in an in vitro alloantibody-dependent, macrophage-mediated hepatocytoxicity assay. Results showed that in vitro alloantibody-dependent, macrophage-mediated hepatocytotoxicity was critically dependent on FcγRs and ROS. The adoptive transfer of WT macrophages into CD8-depleted FcγR-deficient recipients was sufficient to induce alloantibody-mediated rejection, whereas adoptive transfer of macrophages from Fcγ-chain KO mice or ROS-deficient (p47 KO) macrophages was not. These results provide the first evidence, to our knowledge, that alloantibody-dependent hepatocellular allograft rejection is mediated by host macrophages through FcγR signaling and ROS cytotoxic effector mechanisms. These results support the investigation of novel immunotherapeutic strategies targeting macrophages, FcγRs, and/or downstream molecules, including ROS, to inhibit humoral immune damage of transplanted hepatocytes and perhaps other cell and solid organ transplants.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Hepatócitos/imunologia , Macrófagos/imunologia , Receptores de IgG/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Células Cultivadas , Citotoxicidade Imunológica , DNA Helicases/genética , Humanos , Isoanticorpos/metabolismo , Transplante de Fígado , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/genética , Transdução de SinaisRESUMO
BACKGROUND: De novo alloantibodies (donor-specific antibody) contribute to antibody-mediated rejection and poor long-term graft survival. Because the development of donor-specific antibody is associated with early graft loss of cell transplants and reduced long-term survival of solid organ transplants, we hypothesized that conventional immunosuppressives, calcineurin inhibitors (CNi), and mammalian target of rapamycin inhibitors (mTORi), may not be as effective for suppression of humoral alloimmunity as for cell-mediated immunity. METHODS: Wild-type or CD8-depleted mice were transplanted with allogeneic hepatocytes. Recipients were treated with mTORi and/or CNi and serially monitored for alloantibody and graft survival. The direct effect of mTORi and CNi on alloprimed B cell function was investigated in Rag1 mice adoptively transferred with alloprimed IgG1 B cells. The efficacy of mTORi and/or CNi to suppress CD8-mediated cytotoxicity of IgG1 B cells was evaluated in in vitro and in vivo cytotoxicity assays. RESULTS: Mammalian target of rapamycin inhibitors, but not CNi, reduced alloantibody production in transplant recipients, directly suppressed alloantibody production by alloprimed IgG1 B cells and delayed graft rejection in both low and high alloantibody producers. Combination treatment with mTORi and CNi resulted in loss of the inhibitory effect observed for mTORi monotherapy in part due to CNi suppression of CD8 T cells which downregulate alloantibody production (CD8 TAb-supp cells). CONCLUSIONS: Our data support that mTORi is a potent inhibitor of humoral immunity through suppression of alloprimed B cells and preservation of CD8 TAb-supp cells. In contrast, alloantibody is readily detected in CNi-treated recipients because CNi does not suppress alloprimed B cells and interferes with downregulatory CD8 TAb-supp cells.
Assuntos
Linfócitos B/efeitos dos fármacos , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Hepatócitos/transplante , Imunidade Humoral/efeitos dos fármacos , Imunossupressores/farmacologia , Isoanticorpos/imunologia , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Inibidores de Calcineurina/farmacologia , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação para Baixo , Genótipo , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Hepatócitos/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imunidade Celular/efeitos dos fármacos , Isoanticorpos/sangue , Transplante de Fígado/efeitos adversos , Transplante de Fígado/métodos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fenótipo , Serina-Treonina Quinases TOR/metabolismo , Fatores de TempoRESUMO
BACKGROUND: The liver immune environment is tightly regulated to balance immune activation with immune tolerance. Understanding the dominant immune pathways initiated in the liver is important because the liver is a site for cell transplantation, such as for islet and hepatocyte transplantation. The purpose of this study is to examine the consequences of alloimmune stimulation when allogeneic cells are transplanted to the liver in comparison to a different immune locale, such as the kidney. METHODS: We investigated cellular and humoral immune responses when allogeneic hepatocytes are transplanted directly to the recipient liver by intraportal injection. A heterotopic kidney engraftment site was used for comparison to immune activation in the liver microenvironment. RESULTS: Transplantation of allogeneic hepatocytes delivered directly to the liver, via recipient portal circulation, stimulated long-term, high magnitude CD8 T cell-mediated allocytotoxicity. CD8 T cells initiated significant in vivo allocytotoxicity as well as rapid rejection of hepatocytes transplanted to the liver even in the absence of secondary lymph nodes or CD4 T cells. In contrast, in the absence of recipient peripheral lymphoid tissue and CD4 T cells, CD8-mediated in vivo allocytotoxicity was abrogated, and rejection was delayed when hepatocellular allografts were transplanted to the kidney subcapsular site. CONCLUSIONS: These results highlight the CD8-dominant proinflammatory immune responses unique to the liver microenvironment. Allogeneic cells transplanted directly to the liver do not enjoy immune privilege but rather require immunosuppression to prevent rejection by a robust and persistent CD8-dependent allocytotoxicity primed in the liver.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepatócitos/transplante , Imunidade Celular , Imunidade Humoral , Transplante de Fígado/métodos , Fígado/cirurgia , Animais , Linfócitos T CD4-Positivos/imunologia , Microambiente Celular , Citotoxicidade Imunológica , Genótipo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Hepatócitos/imunologia , Rim/imunologia , Fígado/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Fatores de Tempo , Transplante HomólogoRESUMO
Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with ß-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.
Assuntos
Colesterol/metabolismo , Células Endoteliais/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , RNA Mensageiro/genética , Receptores Depuradores Classe B/genética , Animais , Transporte Biológico , Células COS , Linhagem Celular , Separação Celular , Chlorocebus aethiops , Células Endoteliais/citologia , Hepatócitos/citologia , Fígado/citologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microtomia , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores Depuradores Classe B/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
INTRODUCTION: Although IL-1ß is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1ß blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1ß signaling in chondrocytes. METHODS: Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1ß-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1ß signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes. RESULTS: SOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1ß stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1ß-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-ß-activated kinase 1 (TAK1) expression. CONCLUSIONS: Our results show that SOCS1 is induced by IL1-ß in OA chondrocytes and suppresses the IL-1ß-induced synthesis of matrix-degrading enzymes by inhibiting IL-1ß signaling at multiple levels. It suggests that the IL-1ß-inducible SOCS1 acts as a negative regulator of the IL-1ß response in OA cartilage.
Assuntos
Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Osteoartrite do Joelho/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Western Blotting , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Imunoprecipitação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de CitocinaRESUMO
Allospecific T memory cell responses in transplant recipients arise from environmental exposure to previous transplantation or cross-reactive heterologous immunity. Unfortunately, these memory responses pose a significant barrier to the survival of transplanted tissue. We have previously reported that concurrent inhibition of CD154 and LFA-1 suppresses primary CD8-dependent rejection responses that are not controlled by conventional immunosuppressive strategies. We hypothesized that CD154- and LFA-1-mediated inhibition, by targeting activation as well as effector functions, may also be efficacious for the control of alloreactive CD8+ T-cell responses in sensitized hosts. We found that treatment with anti-LFA-1 mAb alone enhanced transplant survival and reduced CD8-mediated cytotoxicity in sensitized CD4 KO recipients. However, treatment with anti-CD154 mAb alone did not have an effect. Notably, when both CD4- and CD8-dependent rejection pathways are operative (wild-type sensitized recipients), LFA-1 significantly inhibited CD8-mediated in vivo allocytotoxicity but did not correspond with enhanced hepatocyte survival. We hypothesized that this was due to alloantibody-mediated rejection. When anti-LFA-1 mAb treatment was combined with macrophage depletion, which we have previously reported impairs alloantibody-mediated parenchymal cell damage, in vivo cytotoxic effector function was significantly decreased and was accompanied by significant enhancement of hepatocyte survival in sensitized wild-type recipients. Therefore, LFA-1 is a potent therapeutic target for reduction of CD8-mediated cytotoxicity in sensitized transplant recipients and can be combined with other treatments that target non-CD8-mediated recall alloimmunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Isoanticorpos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD4/genética , Antígenos CD4/metabolismo , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Hepatócitos/citologia , Hepatócitos/transplante , Imunoterapia , Isoanticorpos/farmacologia , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante HomólogoRESUMO
BACKGROUND: Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms. METHODS: Mice were transplanted with allogeneic hepatocytes. In vivo cytotoxicity assays and various gene-deficient recipient mice and target cells were used to determine the development of Fas-, TNF-α-, and perforin-dependent cytotoxic effector mechanisms after transplantation. RESULTS: CD8(+) T cells maturing in CD4-sufficient hepatocyte recipients develop multiple (Fas-, TNF-α-, and perforin-mediated) cytotoxic mechanisms. However, CD8(+) T cells, maturing in the absence of CD4(+) T cells, mediate cytotoxicity and transplant rejection that is exclusively TNF-α/TNFR-dependent. To determine the kinetics of CD4-mediated help, CD4(+) T cells were adoptively transferred into CD4-deficient mice at various times posttransplant. The maximal influence of CD4(+) T cells on the magnitude of CD8-mediated in vivo allocytotoxicityf occurs within 48 hours. CONCLUSION: The implication of these studies is that interference of CD4(+) T cell function by disease or immunotherapy will have downstream consequences on both the magnitude of allocytotoxicity as well as the cytotoxic effector mechanisms used by allospecific CD8(+) cytolytic T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Rejeição de Enxerto/imunologia , Hepatócitos/transplante , Transplante de Fígado/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Transferência Adotiva , Animais , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/metabolismo , Sobrevivência de Enxerto , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Transplante de Fígado/efeitos adversos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fatores de Tempo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production. To investigate this possibility, alloantibody isotype profiles were examined in CD8-deficient and wild-type hepatocyte recipients. We found that IgG1 (IL-4-dependent isotype) was the dominant alloantibody isotype in wild-type recipients as well as in CD8-deficient recipients, although the amount of alloantibody in the latter group was substantially higher. Utilizing real-time PCR we found that CD4(+) T cells from wild-type recipients significantly upregulated IFN-γ but not IL-4 mRNA. In contrast, in the absence of CD8(+) T cells, CD4(+) T cells switched to significantly upregulate IL-4 mRNA, while IFN-γ was downregulated. IL-4 knockout mice do not produce any posttransplant alloantibody. However, adoptive transfer of wild-type CD4(+) T cells into CD8-depleted IL-4 knockout mice restores high alloantibody levels observed in CD8-depleted wild-type recipients. This suggests that IL-4-producing CD4(+) T cells are critical for posttransplant alloantibody production. Additionally, this CD8-mediated regulation of posttransplant alloantibody production is IFN-γ-dependent. Further elucidation of the mechanisms by which CD8(+) T cells influence Ab production will significantly contribute to development of therapies to manipulate humoral responses to Ag.
Assuntos
Formação de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Hepatócitos/transplante , Imunoglobulina G/imunologia , Interleucina-4/imunologia , Isoanticorpos/imunologia , Transferência Adotiva/métodos , Animais , Formação de Anticorpos/genética , Linfócitos T CD4-Positivos/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Hepatócitos/imunologia , Imunoglobulina G/genética , Interferon gama/genética , Interferon gama/imunologia , Interleucina-4/genética , Isoanticorpos/genética , Camundongos , Camundongos Knockout , Transplante Homólogo , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
BACKGROUND: Endogenously produced interferons can regulate the growth of melanoma cells and are administered exogenously as therapeutic agents to patients with advanced cancer. We investigated the role of negative regulators of interferon signaling known as suppressors of cytokine signaling (SOCS) in mediating interferon-resistance in human melanoma cells. METHODS: Basal and interferon-alpha (IFN-alpha) or interferon-gamma (IFN-gamma)-induced expression of SOCS1 and SOCS3 proteins was evaluated by immunoblot analysis in a panel of n = 10 metastatic human melanoma cell lines, in human embryonic melanocytes (HEM), and radial or vertical growth phase melanoma cells. Over-expression of SOCS1 and SOCS3 proteins in melanoma cells was achieved using the PINCO retroviral vector, while siRNA were used to inhibit SOCS1 and SOCS3 expression. Tyr701-phosphorylated STAT1 (P-STAT1) was measured by intracellular flow cytometry and IFN-stimulated gene expression was measured by Real Time PCR. RESULTS: SOCS1 and SOCS3 proteins were expressed at basal levels in melanocytes and in all melanoma cell lines examined. Expression of the SOCS1 and SOCS3 proteins was also enhanced following stimulation of a subset of cell lines with IFN-alpha or IFN-gamma. Over-expression of SOCS proteins in melanoma cell lines led to significant inhibition of Tyr701-phosphorylated STAT1 (P-STAT1) and gene expression following stimulation with IFN-alpha (IFIT2, OAS-1, ISG-15) or IFN-gamma (IRF1). Conversely, siRNA inhibition of SOCS1 and SOCS3 expression in melanoma cells enhanced their responsiveness to interferon stimulation. CONCLUSIONS: These data demonstrate that SOCS proteins are expressed in human melanoma cell lines and their modulation can influence the responsiveness of melanoma cells to IFN-alpha and IFN-gamma.
Assuntos
Interferon-alfa/farmacologia , Interferon gama/farmacologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Humanos , Interferon alfa-2 , Melanoma/genética , Fosforilação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteínas Recombinantes , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/genética , TransfecçãoRESUMO
Interleukin-29 (IL-29) is a member of the type III IFN family that has been shown to have antiviral activity and to inhibit cell growth. Melanoma cell lines were tested for expression of the IL-29 receptor (IL-29R) and their response to IL-29. Expression of IL-28R1 and IL-10R2, components of IL-29R, was evaluated using reverse transcription-PCR. A combination of immunoblot analysis and flow cytometry was used to evaluate IL-29-induced signal transduction. U133 Plus 2.0 Arrays and real-time PCR were used to evaluate gene expression. Apoptosis was measured using Annexin V/propridium iodide staining. In situ PCR for IL-29R was done on paraffin-embedded melanoma tumors. Both IL-28R1 and IL-10R2 were expressed on the A375, 1106 MEL, Hs294T, 18105 MEL, MEL 39, SK MEL 5, and F01 cell lines. Incubation of melanoma cell lines with IL-29 (10-1,000 ng/mL) led to phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2. Microarray analysis and quantitative reverse transcription-PCR showed a marked increase in transcripts of IFN-regulated genes after treatment with IL-29. In the F01 cell line, bortezomib-induced and temozolomide-induced apoptosis was synergistically enhanced following the addition of IL-29. In situ PCR revealed that IL-10R2 and IL-28R1 were present in six of eight primary human melanoma tumors but not in benign nevi specimens. In conclusion, IL-29 receptors are expressed on the surface of human melanoma cell lines and patient samples, and treatment of these cell lines with IL-29 leads to signaling via the Jak-STAT pathway, the transcription of a unique set of genes, and apoptosis.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Interleucinas/metabolismo , Janus Quinase 1/metabolismo , Melanoma/metabolismo , Fatores de Transcrição STAT/metabolismo , Neoplasias Cutâneas/metabolismo , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Humanos , Interferons , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Pirazinas/farmacologia , Transdução de Sinais , TemozolomidaRESUMO
PURPOSE: The precise molecular targets of IFN-alpha therapy in the context of malignant melanoma are unknown but seem to involve signal transducers and activators of transcription 1 signal transduction within host immune effector cells. We hypothesized that the in vitro transcriptional response of patient peripheral blood mononuclear cells (PBMC) to IFN-alpha would be similar to the in vivo response to treatment with high-dose IFN-alpha. EXPERIMENTAL DESIGN: The gene expression profiles of PBMCs and immune cell subsets treated in vitro with IFN-alpha were evaluated, as were PBMCs obtained from melanoma patients receiving adjuvant IFN-alpha. RESULTS: Twenty-seven genes were up-regulated in PBMCs from normal donors after treatment with IFN-alpha in vitro for 18 hours (>2-fold, P < 0.001). A subset of these genes (in addition to others) was significantly expressed in IFN-alpha-treated T cells, natural killer cells, and monocytes. Analysis of gene expression within PBMCs from melanoma patients (n = 13) receiving high-dose IFN-alpha-2b (20 MU/m(2) i.v.) revealed significant up-regulation (>2-fold) of 21 genes (P < 0.001). Also, the gene expression profile of in vitro IFN-alpha-stimulated patient PBMCs was similar to that of PBMCs obtained from the same patient after IFN-alpha therapy. CONCLUSIONS: This report is the first to describe the transcriptional response of T cells, natural killer cells, and monocytes to IFN-alpha and characterize the transcriptional profiles of PBMCs from melanoma patients undergoing IFN-alpha immunotherapy. In addition, it was determined that microarray analysis of patient PBMCs after in vitro stimulation with IFN-alpha may be a useful predictor of the in vivo response of immune cells to IFN-alpha immunotherapy.
Assuntos
Interferon-alfa/farmacologia , Leucócitos Mononucleares/imunologia , Melanoma/imunologia , Doadores de Sangue , Feminino , Perfilação da Expressão Gênica , Humanos , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Células Matadoras Naturais/imunologia , Masculino , Melanoma/tratamento farmacológico , Análise em Microsséries , Monócitos/imunologia , Proteínas Recombinantes , Linfócitos T/imunologia , Ativação Transcricional , Regulação para CimaRESUMO
Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Rejeição de Enxerto/imunologia , Hepatócitos/imunologia , Transplante de Fígado/imunologia , Macrófagos/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência de Enxerto/imunologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Isoanticorpos/biossíntese , Isoanticorpos/sangue , Isoanticorpos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Transplante HomólogoRESUMO
PURPOSE: The precise molecular targets of IFN-alpha therapy of melanoma are unknown but likely involve signal transducer and activator of transcription (STAT) 1 signal transduction within host immune effector cells. We hypothesized that intermediate and high doses of IFN-alpha would be equally effective in activating patient immune cells. EXPERIMENTAL DESIGN: Eleven metastatic melanoma patients who were enrolled in a clinical trial of bevacizumab in combination with escalating doses of IFN-alpha-2b (5 megaunits/m(2) and then 10 megaunits/m(2)) were included in the study. Peripheral blood mononuclear cells (PBMC) were procured from patient blood just before therapy and again 1 h after each dose of IFN-alpha-2b and analyzed for the presence of phosphorylated STAT1, phosphorylated STAT2, and the induction of IFN-stimulated gene (ISG) transcripts. RESULTS: Phosphorylated STAT1 was significantly greater at the 5 megaunits/m(2) dose compared with the 10 megaunits/m(2) dose of IFN-alpha-2b (P = 0.02). In contrast, no significant difference in phosphorylated STAT2 was observed at a dose of 5 megaunits/m(2) compared with 10 megaunits/m(2) (P = 0.20). There were also no significant differences in the induction of ISGs within PBMCs between the two doses (P > 0.4 for all ISGs). Suppressor of cytokine signaling 1 and 3 (two inhibitors of IFN-alpha signaling) transcripts were significantly higher among patient PBMCs following the 10 megaunits/m(2) dose of IFN-alpha (P < 0.001). CONCLUSION: These results suggest that lower doses of IFN-alpha-2b are as effective as higher doses with respect to the induction of Janus-activated kinase-STAT signal transduction and the transcription of ISGs within immune effector cells.