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1.
Hum Immunol ; 69(9): 543-51, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18639598

ABSTRACT

Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.


Subject(s)
Clone Cells/immunology , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/immunology , Islets of Langerhans/immunology , T-Lymphocytes, Cytotoxic/metabolism , Cell Line, Tumor , Clone Cells/drug effects , Clone Cells/metabolism , Epitopes, T-Lymphocyte/metabolism , Fas Ligand Protein/immunology , Fas Ligand Protein/metabolism , HLA Antigens/immunology , HLA Antigens/metabolism , Humans , Interferon-gamma/pharmacology , Islets of Langerhans/metabolism , Macrolides/pharmacology , Perforin/immunology , Perforin/metabolism , T-Lymphocytes, Cytotoxic/immunology
2.
Novartis Found Symp ; 292: 68-78; discussion 78-84, 122-9, 202-3, 2008.
Article in English | MEDLINE | ID: mdl-19203093

ABSTRACT

CD8+ T cells are the principal cellular mediators of beta cell destruction in the NOD mouse. Molecular mediators include perforin and granzymes from the cytotoxic granule, Fas ligand and pro-inflammatory cytokines. Our studies in NOD mice have shown that beta cell-specific CD8+ T cells use both the perforin and Fas pathway in vitro. Reducing antigen presentation on beta cells, for example by reducing class I MHC expression by overexpression of SOCS1, protects beta cells in vivo. Perforin deficiency effectively reduces diabetes in NOD mice but in NOD8.3 mice other mechanisms compensate. We have been unable to identify a major role for direct toxicity of cytokines in NOD mice. However, in the LCMV glycoprotein model they may be more important. Deficiency of IL1 or TNF or Fas has a protective effect (greatest for TNF deficiency) but this appears to be due to effects of these cytokines on the immune response rather than on the beta cell. Combinations of interventions, for example, beta cell overexpression of SOCS1 combined with IL1 deficiency may be highly protective. It should be possible to define all the molecular mediators of beta cell destruction, and it may be possible to inhibit at least some of these.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/pathology , Insulin-Secreting Cells/immunology , Insulin-Secreting Cells/pathology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cytokines/genetics , Cytokines/immunology , Cytotoxicity, Immunologic , Disease Models, Animal , Mice , Mice, Inbred NOD , Perforin/genetics , Perforin/immunology , fas Receptor/genetics , fas Receptor/immunology
3.
Diabetes ; 55(9): 2412-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16936188

ABSTRACT

Cytotoxic T-cells are the major mediators of beta-cell destruction in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contribution of perforin and Fas ligand to beta-cell destruction using islet-specific CD8(+) T-cells from T-cell receptor transgenic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for beta-cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with beta-cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta-cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell-mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill beta-cells and the surprising blockade of NOD8.3 T-cell-mediated beta-cell death by SOCS-1 overexpression may be due in part to reduced target cell recognition.


Subject(s)
Insulin-Secreting Cells/cytology , Membrane Glycoproteins/physiology , Suppressor of Cytokine Signaling Proteins/physiology , T-Lymphocytes, Cytotoxic/physiology , fas Receptor/physiology , Adaptor Proteins, Signal Transducing/genetics , Animals , CD8-Positive T-Lymphocytes/physiology , Diabetes Mellitus, Type 1/physiopathology , Fas-Associated Death Domain Protein , Glucose-6-Phosphatase/physiology , Islets of Langerhans Transplantation/physiology , Mice , Mice, Inbred NOD , Mice, Transgenic , Perforin , Pore Forming Cytotoxic Proteins , Proteins/physiology , Suppressor of Cytokine Signaling 1 Protein
4.
Diabetes ; 55(8): 2212-9, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16873683

ABSTRACT

Perforin-deficient NOD mice are protected from diabetes, suggesting that cytotoxic granule contents of CD8(+) T-cells have a significant role in killing beta-cells. Despite this, cytotoxic granule effects on human or mouse pancreatic islets have not been reported. We tested the susceptibility of human and mouse islet cells to purified recombinant perforin and granzyme B and measured apoptotic death using a number of assays. Perforin and granzyme B impaired insulin secretion from islet cells, and this was accompanied by cytochrome c release, caspase activation, and DNA fragmentation. Granzyme B-mediated apoptotic changes only occurred in the presence of perforin. When compared with hemopoietic cells, traditionally used as targets to measure cytotoxic T-cell function in vitro, islet cells were relatively resistant to perforin and granzyme B. Inhibition of caspases prevented DNA fragmentation but not cytochrome c release, indicating that mitochondrial disruption due to granzyme B is independent of caspase activation. Consistent with this, islet cells from mice deficient in the BH3-only protein Bid were resistant to cytochrome c release and were protected from apoptosis after exposure to perforin/granzyme B. Our data suggest that Bid cleavage by granzyme B precedes mitochondrial disruption and apoptosis in pancreatic islets.


Subject(s)
Apoptosis/physiology , BH3 Interacting Domain Death Agonist Protein/physiology , Islets of Langerhans/cytology , Serine Endopeptidases/physiology , Animals , Apoptosis/drug effects , BH3 Interacting Domain Death Agonist Protein/deficiency , Caspase 3 , Caspase Inhibitors , Caspases/metabolism , Cell Line, Transformed , Cell Line, Tumor , Cytochromes c/metabolism , DNA Fragmentation , Enzyme Activation/drug effects , Granzymes , Herpesvirus 4, Human , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Islets of Langerhans/physiology , Male , Mastocytoma , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mitochondria/drug effects , Perforin , Pore Forming Cytotoxic Proteins , Recombinant Proteins/pharmacology , Serine Endopeptidases/metabolism , Serine Endopeptidases/pharmacology
5.
Transplantation ; 81(2): 146-54, 2006 Jan 27.
Article in English | MEDLINE | ID: mdl-16436955

ABSTRACT

BACKGROUND: The precise effector mechanisms and molecular mediators used by alloreactive cytotoxic T lymphocytes to kill transplanted pancreatic beta cells are poorly defined. We have used mouse (H2b-anti-d) CTLs raised in strains deficient in various key cytotoxic effector molecules to assess the importance of the various signaling pathways mobilized to kill primary mouse pancreatic islet cells, the beta cell line NIT-1, and NIT-1 cells overexpressing dominant-negative FADD and Bcl-2. METHODS: Death of target cells was assessed using 51Cr release assays. RESULTS: In short-term assays (<5 hours) beta cell death did not require a functional FasL/Fas pathway, and was not inhibited by Bcl-2. However, the absence of either perforin or granzyme B resulted in cell survival. By contrast, a crucial role for granzyme B was not seen when hematopoietic P815 cells were used as targets, indicating differential regulation of apoptosis. Interestingly, coincubation with CTL for 24 hours revealed an additional but less potent "late phase" of beta cell death that did not require perforin. This delayed death was blocked by dominant-negative FADD, but not by Bcl-2, and was likely to be due to TNFalpha secretion. CONCLUSIONS: This study suggests that strategies to protect beta cells from allogeneic CTL attack will need to inhibit the perforin/granzyme and probably also the TNFalpha pathway. As there are no known pharmacological approaches to blocking perforin, therapeutic approaches based on overexpressing both dominant negative FADD and an inhibitor of granzyme B may hold promise in prolonging beta cell survival in the allogeneic setting.


Subject(s)
Islets of Langerhans/immunology , Islets of Langerhans/pathology , Membrane Proteins/immunology , Serine Endopeptidases/immunology , T-Lymphocytes, Cytotoxic/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Cell Death/immunology , Cell Death/physiology , Cell Line , Graft Rejection , Granzymes , In Vitro Techniques , Islets of Langerhans Transplantation/immunology , Islets of Langerhans Transplantation/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Pore Forming Cytotoxic Proteins , Transplantation, Homologous
6.
J Gene Med ; 8(1): 42-52, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16075436

ABSTRACT

BACKGROUND: Currently, successful transplantation of allografts requires the systemic use of immunosuppressive drugs. These can cause serious morbidity due to toxicity and increased susceptibility to cancer and infections. Local production of immunosuppressive molecules limited to the graft site would reduce the need for conventional, generalized immunosuppressive therapies and thus educe fewer side effects. This is particularly salient in a disease like type 1 diabetes, which is not immediately life-threatening yet islet allografts can effect a cure. METHODS: We studied the efficacy of locally produced anti-CD4 antibody, mediated by adenovirus (Adv-anti-CD4) transduction of islets, to enhance allograft survival. Adenovirus-transduced islets were transplanted under the kidney capsule of diabetic recipients and graft rejection determined by monitoring blood glucose levels. RESULTS: Adv-anti-CD4 transduction of mouse islets afforded protection against allogeneic rejection after transplantation into fully mismatched recipients. In some recipients, the islet allograft survival was prolonged (persisting for at least 15 weeks), corresponding to the prolonged expression of the anti-CD4 antibody. The effect was local, as absence of CD4+ T lymphocytes was observed primarily at the graft site. CONCLUSIONS: Immunosuppressive effects can be restricted locally by our strategy. Local production of a single antibody against one subset of T lymphocytes can protect mouse islets from allograft rejection during transplantation to treat diabetes. Our findings foreshadow that this strategy may be even more effective when a combination of antibodies are used and that similar strategies may prevent xenograft rejection.


Subject(s)
Antibodies/analysis , CD4 Antigens/immunology , Graft Survival , Islets of Langerhans Transplantation/immunology , Adenoviridae/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , Diabetes Mellitus, Experimental/surgery , Female , Male , Mice , Transduction, Genetic/methods , Transplantation, Homologous/immunology
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