RESUMEN
Devil's club Oplopanax horridus (DC) is a close relative of ginseng; its inner root and stem bark extract showed antiproliferation activity on human leukemia, ovarian, breast and colon cancer cells. We study here the effects of DC 70% ethanol extract alone, or in combination with cisplatin, gemcitabine, and paclitaxel on pancreatic endocrine HP62 and pancreatic ductal carcinoma PANC-1 and BxPC-3 cells. Antiproliferation activity assay, cell cycle analysis by flow cytometry, apoptosis-related markers by antibody array, and RT-PCR assay were used for this study. DC extract inhibited proliferation of HP62 with IC50 (50% inhibition concentration) at 0.037±0.002% (v/v), PANC-1 at 0.0058 ± 0.0004% and BxPC-3 at 0.021 ± 0.003%. DC at 0.0033% combined with 1 nM of paclitaxel showed inhibition synergy on PANC-1 cells with a combination index of 0.44. Apoptosis focused antibody array profile indicated upregulation of cytochrome C, claspin, cIAP-2 and HTRA2/Omi apoptosis-related markers in DC-treated HP62 and PANC-1. Our data suggest that DC acts through targeting the intrinsic mitochondrial apoptosis pathway in the pancreatic cancer cells. The high antiproliferation potency of DC on PANC-1 is potentially useful as an adjunct therapy for treating pancreatic cancer, which is known for developing resistance to conventional chemotherapeutics.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Oplopanax/química , Poliinos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Humanos , Concentración 50 Inhibidora , Paclitaxel/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Extractos Vegetales/farmacología , Raíces de Plantas/química , Transducción de Señal , GemcitabinaRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease characterized by immunological destruction of insulin-producing pancreatic ß-cells and subsequent hyperglycemia. The non-obese diabetic (NOD) mouse strain spontaneously develops a disease similar to human T1D and is commonly used as an animal model for studying this disease. We have previously shown that the administration of B7-H4-immunoglobulin fusion protein (B7-H4.Ig), a newly identified T-cell co-inhibitory signaling molecule, blocks the onset of diabetes in NOD mice. However, the mechanism(s) by which B7-H4 protects NOD mice from T1D is not fully understood. IL-17 is a pro-inflammatory cytokine, produced by Th17 cells, that activates T cells and other immune cells to produce a variety of cytokines and chemokines. Increasing evidence has shown that therapeutic agents targeting the IL-17 molecule or directly inhibiting IL-17-producing cells regulate autoimmune diabetes in NOD mice, suggesting that IL-17 is involved in the pathogenesis of this disease. In this study, we investigate whether B7-H4.Ig treatment inhibits the generation of Th17 cells which subsequently decreases IL-17 production and prevents the onset of T1D in NOD mice. Pre-diabetic female NOD mice were injected intraperitoneally with control mouse IgG or B7-H4.Ig starting at 4 weeks of age for 12 weeks. Our data showed that the frequency of Th17 cells in B7-H4.Ig-treated mice was significantly decreased. In addition, our data showed that B7-H4.Ig-treated mice had decreased levels of pro-inflammatory cytokines and Th17-associated cytokines, and an increased level of the potent Th17 inhibitor IFN-γ. To further investigate the effect of B7-H4.Ig on differentiation of Th17 cells, we co-cultured splenocytes with Th17-polarizing cytokines in the absence or presence of B7-H4.Ig. Our results indicated that splenocytes, under the Th17 driving conditions in the presence of B7-H4.Ig, had significantly decreased the numbers of Th17 cells compared to cells co-cultured in the absence of B7-H4.Ig. Together, this study suggests that blocking the generation of Th17 cells with the administration of B7-H4.Ig effectively inhibits the development of T1D in NOD mice.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Diabetes Mellitus Tipo 1/prevención & control , Proteínas Recombinantes de Fusión/farmacología , Células Th17/efectos de los fármacos , Animales , Células Cultivadas , Técnicas de Cocultivo , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Citometría de Flujo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Inmunoglobulinas/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos NOD , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/metabolismo , Células Th17/citología , Células Th17/metabolismo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/genética , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/farmacologíaRESUMEN
B7-H4, a recently described member of the B7 family of cosignal molecules, is thought to be involved in the regulation of cellular and humoral immune responses through receptors on activated T and B cells. Human islet cells express positive B7-H4 mRNA in RT-PCR assays, but not B7-H4 protein on cell surface in flow cytometric analyses. To investigate the regulatory effects of activation of the B7-H4 pathway on the function of activated T cells of patients with type 1 diabetes (T1D), we have used our in vitro human experimental system, including human beta-cell antigen-specific T-cell clones and human beta-cell lines CM and HP62, as well as primary islet cells. B7-H4.Ig protein was purified from the culture supernatant of 293T cells transfected by a B7-H4.Ig plasmid (pMIgV, containing a human B7-H4 cDNA and a mouse IgG2a Fc cDNA). Our preliminary studies showed that immobilized fusion protein human B7-H4.Ig (coated with 5 microg/ml for 2 h at 37 degrees C), but not control Ig, clearly inhibited the proliferation of activated CD4+ and CD8+ T cells of patients induced by anti-CD3 antibody in CFSE assays. B7-H4.Ig also arrested cell cycle progression of T cells in G0/G1 phase and induced T-cell apoptosis as measured by BrdU-7-AAD flow cytometric analysis. To determine the cytoprotective effects of B7-H4, we developed transfectants of human beta-cell lines CM and HP62 and islet cells transfected with the B7-H4.Ig plasmid, using empty vector transfectants as controls. The results demonstrate that cell-associated B7-H4.Ig expressed on human beta-cells clearly inhibits the cytotoxicity of the T-cell clones to targeted human beta-cells in 51Cr release cytotoxicity assays. Activation of the B7-H4 pathway may represent a novel immunotherapeutic approach to inhibit T-cell responses for the prevention of beta-cell destruction in T1D.
Asunto(s)
Antígeno B7-1/metabolismo , Células Secretoras de Insulina/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Humanos , Inmunoglobulinas/metabolismo , Inmunoglobulinas/fisiología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Interferón gamma/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Linfocitos T/metabolismo , Inhibidor 1 de la Activación de Células T con Dominio V-SetRESUMEN
To better understand the cytokine death-signal transduction pathways in human beta cells, we investigated the inhibitory effects of Bcl-2 (protooncogene bcl-2) and X-linked inhibitor of apoptosis (XIAP) on TRAIL (TNF-related apoptosis-inducing ligand)-induced human beta-cell destruction. A panel of Bcl-2-overexpressing transfectants of the human beta-cell lines NES2Y and CM was developed by transfection with a pEFpGKpuro vector containing Bcl-2 or an empty vector as a control. TRAIL-induced cytotoxicity and apoptosis of Bcl-2-overexpressing beta cells were clearly decreased, in comparison with wild-type cells and the empty vector transfectants. XIAP-overexpressing CM, NES2Y, and primary islet cells were generated by exposing cells to recombinant adenovirus-expressing XIAP (AdXIAP) or AdLacz as a control. TRAIL-induced cytotoxicity and apoptosis of CM, NES2Y, and primary islet cells infected with AdXIAP were clearly reduced compared with controls. Interestingly, cytotoxicity induced by TRAIL in human beta cells transfected with both Bcl-2 and AdXIAP was much less than that observed in human beta cells transfected with either Bcl-2 or XIAP alone (p < 0.005 in CM and p < 0.03 in NES2Y). Overexpression of both Bcl-2 and XIAP inhibited TRAIL-induced activation of caspases as well as TRAIL-mediated damage of mitochondrial function in cells, suggesting possible regulatory mechanisms. These results indicate that Bcl-2 and XIAP synergistically inhibit TRAIL-mediated death pathways in human beta cells.
Asunto(s)
Apoptosis/fisiología , Islotes Pancreáticos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Reguladoras de la Apoptosis , Caspasa 7 , Caspasa 9 , Caspasas/metabolismo , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Mitocondrias/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Studies of type 1 diabetes indicate that autoaggressive T cells specific to beta-cell antigens, reaching certain threshold levels, may play critical roles in the development of the disease. Flow cytometric analyses found that autoreactive T-cell lines from patients induced by beta-cell antigens consisted of four major subsets (CD4(+)CD56(-), CD4(+)CD56(+), CD8(+)CD56(-), and CD8(+)CD56(+)) and that CD56(+) NKT cells might be derived from CD56(-) T cells. Moreover, the proportion of CD56(+) NKT cells in the T-cell lines was influenced by time course of repeated antigen stimulation. beta-cell antigen-specific CD56(+) NKT (CD4(+) or CD8(+)) cells were more aggressive (HLA-restricted and -unrestricted) effector cells lysing target cells such as K562, Jurkat, P815 plus anti-CD3 antibody, and autologous B cells sensitized by beta-cell peptides, when compared with their CD56(-) counterparts. beta-cell antigen- specific CD4(+)CD56(+) NKT cells showed non-HLA-restricted cytotoxicity to human beta cells, insulinoma cell line CM, and to islet cell lines TRM-6 and HP62 expressing CD56 but not to four CD56(-) pancreatic cell lines of non- islet origin. The CD4(+)CD56(+) NKT cells showed stronger cytotoxicity to CM, TRM-6 and HP62 cells than did CD4(+)CD56(-) T cells. Moreover, isotope-unlabelled CD56(+) cells and anti-CD56 antibodies were able to inhibit cytotoxicity of CD4(+)CD56(+) NKT to CD56(+) target cells. These results suggest that CD56(+) NKT cells are aggressive cytotoxic cells to beta cells and that CD56 expression might be associated with the aggressiveness of effector T cells and the susceptibility of target cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígeno CD56/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Células Asesinas Naturales/inmunología , Adolescente , Biomarcadores , Células Cultivadas , Niño , Citotoxicidad Inmunológica , Femenino , Glutamato Descarboxilasa/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Isoenzimas/inmunología , Masculino , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Auto- and alloreactive T cells are major culprits that damage ß-cells in type 1 diabetes (T1D) and islet transplantation. Current immunosuppressive drugs can alleviate immune-mediated attacks on islets. T cell co-stimulation blockade has shown great promise in autoimmunity and transplantation as it solely targets activated T cells, and therefore avoids toxicity of current immunosuppressive drugs. An attractive approach is offered by the newly-identified negative T cell co-signaling molecule B7-H4 which is expressed in normal human islets, and its expression co-localizes with insulin. A concomitant decrease in B7-H4/insulin co-localization is observed in human type 1 diabetic islets. B7-H4 may play protective roles in the pancreatic islets, preserving their function and survival. In this review we outline the protective effect of B7-H4 in the contexts of T1D, islet cell transplantation, and potentially type 2 diabetes. Current evidence offers encouraging data regarding the role of B7-H4 in reversal of autoimmune diabetes and donor-specific islet allograft tolerance. Additionally, unique expression of B7-H4 may serve as a potential biomarker for the development of T1D. Future studies should continue to focus on the islet-specific effects of B7-H4 with emphasis on mechanistic pathways in order to promote B7-H4 as a potential therapy and cure for T1D.
RESUMEN
OBJECTIVES: B7-H4 is a negative coregulatory molecule known to be involved in immune response. We study here B7-H4 expression and its possible role in diabetes and cancer development. METHODS: Formalin-fixed, paraffin-processed pancreas samples from patients with type 1 diabetes (T1D), insulinoma, pancreatic ductal adenocarcinoma (PDAC), and normal organ donors were studied by bright-field and multifluorescence immunohistochemistry to examine B7-H4 expression and its colocalization with islet endocrine hormones. Quantitative RT-PCR and Western blot assay were used to examine B7-H4 mRNA and protein expression in the islet and exocrine tissues from normal donors and pancreatic cancer cell lines. RESULTS: B7-H4 protein expression in islet ß cells is decreased in T1D and PDAC, but increased in insulinoma patients when compared to normal controls; the changes in B7-H4 expression are concomitant with insulin expression on the islet ß cells. The insulin/B7-H4 colocalization on the ß cells, expressed in colocalization coefficient Pearson r, is also changed in these islets. CONCLUSIONS: Our observation of altered B7-H4 expression, concomitant with insulin expression, in the pancreatic islets of T1D, PDAC, and insulinoma patients when compared to normal controls suggests that B7-H4 pathway might play an important role in maintenance of ß-cell function, but its exact role remains to be explored.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular , Línea Celular Tumoral , Diabetes Mellitus Tipo 1/genética , Expresión Génica , Humanos , Inmunohistoquímica , Insulina/genética , Insulina/metabolismo , Insulinoma/genética , Insulinoma/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor 1 de la Activación de Células T con Dominio V-Set/genéticaRESUMEN
BACKGROUND: Allograft rejection is one of the main obstacles for islet transplantation. B7-H4 plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. In this study, we investigated whether the endogenous expression of B7-H4 in ß cells from B7-H4 transgenic mice enhances islet allograft survival. METHODS: B7-H4 transgenic C57BL/6 (B6) mice (RIP.B7-H4) were developed by inserting the entire B7-H4 open reading frame under the rat insulin promoter (RIP). B7-H4 protein expression was examined by flow cytometric analysis and immunohistochemical staining. Islet allograft survival was investigated in streptozotocin-induced diabetic recipient BALB/c (H-2d) mice transplanted with 400 islets from RIP.B7-H4 (H-2b) mice under the kidney capsule. The recipient control group received islets from wild-type B6 donors. RESULTS: B7-H4 protein was significantly up-regulated in isolated islets from RIP.B7-H4 compared with wild-type B6 mice (56%±23% vs. 3%±1.2%). B7-H4 was coexpressed with insulin, but not glucagon, suggesting that B7-H4 is expressed in a ß-cell-specific manner. Recipient BALB/c mice transplanted with RIP.B7-H4 islets established euglycemia for 42.3±18.4 days (mean±SD; n=9) compared with controls at 23.1±7.8 days (mean±SD; n=12; P<0.004, log-rank test). CONCLUSIONS: The endogenous expression of B7-H4 in donor ß cells from transgenic mice prolongs islet allograft survival, confirming the negative role of B7-H4 in regulating alloreactive T-cell responses.
Asunto(s)
Supervivencia de Injerto , Trasplante de Islotes Pancreáticos , Inhibidor 1 de la Activación de Células T con Dominio V-Set/fisiología , Animales , Antígenos CD28/fisiología , Antígeno CTLA-4/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
Negative cosignaling molecules play an important role in regulating T-cell responses to alloantigen stimulation. We recently reported that adenoviral-mediated transduction of islet allografts with B7-H4 inhibits allograft rejection. In this study, we investigate the mechanism for B7-H4-induced prolongation of mouse islet allograft survival. Streptozotocin-induced diabetic C57BL/6 mice were rendered normoglycemic by renal subcapsular implants of B7-H4-transduced BALB/c islets. Grafts and spleens were removed after days 2, 10, and 60 (n = 8 each) for characterization of kinetics of Foxp3 and interleukin 10 (IL-10) expression. Mixed lymphocyte reaction (MLR) was done at day 60. Ten mice were subjected to nephrectomy at 60 days and then five were implanted with secondary BALB/c islets and five were given third-party CBA/J islets. An increase in Foxp3 and IL-10 mRNA expression was detected in recipients' spleens at day 60 and this was associated with increased quantities of Foxp3(+) cells. Splenocytes at day 60 showed hyporesponsiveness during MLR to alloantigen stimulation. Proliferation was partially restored after CD25(+) T-cell depletion. Secondary BALB/c islets survived for 79 ± 29 days compared with 21 ± 3.6 days for CBA/J islets (p < 0.001). Local expression of B7-H4 induces long-term unresponsiveness to donor-specific alloantigens, and is associated with T regulatory cells, suggesting the development of tolerance.
Asunto(s)
Diabetes Mellitus Experimental/cirugía , Supervivencia de Injerto , Trasplante de Islotes Pancreáticos/inmunología , Tolerancia al Trasplante , Inhibidor 1 de la Activación de Células T con Dominio V-Set/genética , Animales , Factores de Transcripción Forkhead/biosíntesis , Interleucina-10/biosíntesis , Islotes Pancreáticos/citología , Islotes Pancreáticos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Nefrectomía , Linfocitos T Reguladores/inmunología , Transducción Genética , Trasplante Homólogo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/inmunologíaRESUMEN
B7-H4 is a newly identified B7 homolog that plays an important role in maintaining T-cell homeostasis by inhibiting T-cell proliferation and lymphokine-secretion. In this study, we investigated the signal transduction pathways inhibited by B7-H4 engagement in mouse T cells. We found that treatment of CD3(+) T cells with a B7-H4.Ig fusion protein inhibits anti-CD3 elicited T-cell receptor (TCR)/CD28 signaling events, including phosphorylation of the MAP kinases, ERK, p38, and JNK. B7-H4.Ig treatment also inhibited the phosphorylation of AKT kinase and impaired its kinase activity as assessed by the phosphorylation of its endogenous substrate GSK-3. Expression of IL-2 is also reduced by B7-H4. In contrast, the phosphorylation state of the TCR proximal tyrosine kinases ZAP70 and lymphocyte-specific protein tyrosine kinase (LCK) are not affected by B7-H4 ligation. These results indicate that B7-H4 inhibits T-cell proliferation and IL-2 production through interfering with activation of ERK, JNK, and AKT, but not of ZAP70 or LCK.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteína Oncogénica v-akt/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Inhibidor 1 de la Activación de Células T con Dominio V-Set/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Anticuerpos/metabolismo , Anticuerpos/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína Oncogénica v-akt/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T/fisiología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Costimulation blockade is an effective way to prevent allograft rejection. In this study, we tested the efficacy of two negative co-signaling molecules in protecting islet allograft function. We used local expression of B7-H4 by adenoviral transduction of islets (Ad-B7-H4) and systemic administration of CTLA-4.Ig to investigate the outcomes of allograft survival. Five groups of streptozotocin-induced diabetic C57BL/6 mice received 400 islets each from BALB/c donors. The groups consisted of control (G1); CTLA-4.Ig (G2); Ad-LacZ (G3); Ad-B7-H4 (G4); and Ad-B7-H4 and CTLA-4.Ig combined (G5). G1 and G3 developed graft failure on average of two weeks. G2, G4 and G5 survived for 43.8 ± 34.8, 54.7 ± 31.2 and 77.8 ± 21.5 d, respectively. Activated T and B cells in the lymph nodes were significantly controlled by CTLA-4.Ig treatment. Significantly reduced infiltrates were also detected in the allografts of G2 compared with G1. By contrast, B7-H4 significantly inhibited Th1-associated IFN-gamma secretion in the early stage and increased Foxp3 (+) T cells in the long-term surviving allografts. Our study suggests that CTLA-4 and B7-H4 inhibit alloimmune responses through distinct mechanisms, and that combination therapy which activates two negative co-signaling pathways can further enhance islet allograft survival.
Asunto(s)
Antígeno CTLA-4/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Islotes Pancreáticos , Transducción de Señal , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Análisis de Varianza , Animales , Linfocitos B/efectos de los fármacos , Antígenos CD28/metabolismo , Antígeno CTLA-4/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/cirugía , Femenino , Factores de Transcripción Forkhead/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Inmunoglobulinas/farmacología , Insulina/metabolismo , Secreción de Insulina , Interferón gamma/metabolismo , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estadísticas no Paramétricas , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células TH1/metabolismo , Factores de Tiempo , Transducción Genética , Trasplante Homólogo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/genéticaRESUMEN
Islet cell transplantation is currently the only feasible long-term treatment option for patients with type 1 diabetes. However, the majority of transplanted islets experience damage and apoptosis during the isolation process, a blood-mediated inflammatory microenvironment in the portal vein upon islet infusion, hypoxia induced by the low oxygenated milieu, and poor-revascularization-mediated lack of nutrients, and impaired hormone modulation in the local transplanted site. Strategies using genetic modification methods through overexpression or silencing of those proteins involved in promoting new formation of blood vessels or inhibition of apoptosis may overcome these hurdles and improve islet engraftment outcomes.
RESUMEN
OBJECTIVE: Autoimmune diabetes is a T cell-mediated disease in which insulin-producing ß-cells are destroyed. Autoreactive T cells play a central role in mediating ß-cell destruction. B7-H4 is a negative cosignaling molecule that downregulates T-cell responses. In this study, we aim to determine the role of B7-H4 on regulation of ß-cell-specific autoimmune responses. RESEARCH DESIGN AND METHODS: Prediabetic (aged 3 weeks) female NOD mice (group 1, n = 21) were treated with intraperitoneal injections of B7-H4.Ig at 7.5 mg/kg, with the same amount of mouse IgG (group 2, n = 24), or with no protein injections (group 3, n = 24), every 3 days for 12 weeks. RESULTS: B7-H4.Ig reduced the incidence of autoimmune diabetes, compared with the control groups (diabetic mice 28.6% of group 1, 66.7% of group 2 [P = 0.0081], and 70.8% of group 3 [group 1 vs. 3, P = 0.0035]). Histological analysis revealed that B7-H4 treatment did not block islet infiltration but rather suppressed further infiltrates after 9 weeks of treatment (group 1 vs. 2, P = 0.0003). B7-H4 treatment also reduced T-cell proliferation in response to GAD65 stimulation ex vivo. The reduction of diabetes is not due to inhibition of activated T cells in the periphery but rather to a transient increase of Foxp3(+) CD4(+) T-cell population at one week posttreatment (12.88 ± 1.29 vs. 11.58 ± 1.46%; n = 8; P = 0.03). CONCLUSIONS: Our data demonstrate the protective role of B7-H4 in the development of autoimmune diabetes, suggesting a potential means of preventing type 1 diabetes by targeting the B7-H4 pathway.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Inhibidor 1 de la Activación de Células T con Dominio V-Set/uso terapéutico , Animales , Autoinmunidad/efectos de los fármacos , Femenino , Factores de Transcripción Forkhead/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos NOD , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Estado Prediabético/tratamiento farmacológico , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Type 1 diabetes (T1D) is a chronic autoimmune disease and characterized by absolute insulin deficiency. ß-cell replacement by islet cell transplantation has been established as a feasible treatment option for T1D. The two main obstacles after islet transplantation are alloreactive T-cell-mediated graft rejection and recurrence of autoimmune diabetes mellitus in recipients. T cells play a central role in determining the outcome of both autoimmune responses and allograft survival. B7-H4, a newly identified B7 homolog, plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. The relationship between B7-H4 and allograft survival/autoimmunity has been investigated recently in both islet transplantation and the nonobese diabetic (NOD) mouse models. B7-H4 protects allograft survival and generates donor-specific tolerance. It also prevents the development of autoimmune diabetes. More importantly, B7-H4 plays an indispensable role in alloimmunity in the absence of the classic CD28/CTLA-4 : B7 pathway, suggesting a synergistic/additive effect with other agents such as CTLA-4 on inhibition of unwanted immune responses.
RESUMEN
BACKGROUND: Allogeneic pancreatic islet transplantation has the potential to cure type 1 diabetes. One of the barriers to islet transplantation is the alloreactive T-cell response between donors and recipients. Costimulatory molecules, which play a major role in the regulation of the immune response to antigens during graft rejection, may be used to inhibit allograft destruction. B7-H4 is one such member in the costimulatory family, which has established negative regulatory function of T-cell responses. METHODS: To determine whether local expression of B7-H4 protein can protect beta cells from damage in islet allotransplantation, we have constructed a recombinant adenovirus expressing a B7-H4 complementary deoxyribonucleic acid (Ad-B7-H4). To study the in vivo effects of B7-H4 expression on islet graft survival, adenovirus-transduced islets from donor Balb/c mice were transplanted into streptozotocin-diabetic C57BL/6 mice (n=12). RESULTS: Expression of B7-H4 in islets by Ad-B7-H4 transduction at an optimized condition did not inhibit glucose-stimulated insulin secretion of the treated islets. The recipient mice transplanted with Ad-B7-H4-transduced islets established euglycemia for a longer time (mean 56.5 days), compared with control mice transplanted with Ad-LacZ-transduced islets (mean 14.5 days, [n=12, P<0.001]). Splenocytes isolated from the recipients of Ad-B7-H4-transduced islets showed hyporesponsiveness to alloantigenic stimulation, compared with control recipients. CD45 and insulin staining of the graft transplanted with Ad-B7-H4-transduced islets indicated the preservation of beta cells and decrease of infiltrating immune cells. CONCLUSIONS: Local expression of B7-H4 prolongs islet allograft survival in vivo, suggesting translational potential for beta-cell replacement with reduced immune injury.
Asunto(s)
Antígeno B7-1/genética , Supervivencia de Injerto/fisiología , Trasplante de Islotes Pancreáticos/métodos , Trasplante de Islotes Pancreáticos/fisiología , Trasplante Homólogo/fisiología , Adenoviridae/genética , Animales , Glucemia/metabolismo , Clonación Molecular , Femenino , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/fisiología , Células Secretoras de Insulina/trasplante , Islotes Pancreáticos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor 1 de la Activación de Células T con Dominio V-SetRESUMEN
Canadian surgical contributions to the field of islet transplantation have a rich heritage and a promising future. In this article, some seminal Canadian contributions to this field are reviewed, including contributions at the basic research laboratory and translational applications to bedside therapy of type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos , Animales , Canadá , Humanos , Trasplante de Islotes Pancreáticos/métodos , RatasRESUMEN
B7-H4, a recently described member of the B7 family of cosignal molecules, is thought to be involved in the regulation of cellular and humoral immune responses through receptors on activated T and B cells. Human islet cells express positive B7-H4 mRNA in RT-PCR assays, but not B7-H4 protein on cell surface in flow cytometric analyses. To investigate the regulatory effects of activation of the B7-H4 pathway on the function of activated T cells of patients with type 1 diabetes (T1D), we have used our in vitro human experimental system, including human ß-cell antigen-specific T-cell clones and human ß-cell lines CM and HP62, as well as primary islet cells. B7-H4.Ig protein was purified from the culture supernatant of 293T cells transfected by a B7-H4.Ig plasmid (pMIgV, containing a human B7-H4 cDNA and a mouse IgG2a Fc cDNA). Our preliminary studies showed that immobilized fusion protein human B7-H4.Ig (coated with 5 µg/ml for 2 h at 37°C), but not control Ig, clearly inhibited the proliferation of activated CD4+ and CD8+ T cells of patients induced by anti-CD3 antibody in CFSE assays. B7-H4.Ig also arrested cell cycle progression of T cells in G0/G1 phase and induced T-cell apoptosis as measured by BrdU-7-AAD flow cytometric analysis. To determine the cytoprotective effects of B7-H4, we developed transfectants of human ß-cell lines CM and HP62 and islet cells transfected with the B7-H4.Ig plasmid, using empty vector transfectants as controls. The results demonstrate that cell-associated B7-H4.Ig expressed on human ß-cells clearly inhibits the cytotoxicity of the T-cell clones to targeted human ß-cells in 51Cr release cytotoxicity assays. Activation of the B7-H4 pathway may represent a novel immunotherapeutic approach to inhibit T-cell responses for the prevention of ß-cell destruction in T1D.
RESUMEN
OBJECTIVES: Our previous report showed that beta-cell antigen-specific CD56+ T-cells and cytokine TRAIL mediate destruction of human pancreatic [beta] cells in vitro. To determine whether CD56 and TRAIL are present during islet cell destruction at the onset of clinical symptoms of type 1 diabetes mellitus (T1D), we studied cell marker and cytokine expression in the pancreatic islets of 2 children who died at presentation of acute-onset T1D and in T-cell lines derived from a group of children with new-onset T1D. METHODS: TRAIL, CD56, and other T-cell markers and cytokine expression were studied using immunohistochemistry on pancreatic sections from 2 children with acute-onset T1D. TRAIL and CD56 expression was analyzed by flow cytometry in the antigen-activated T-cell lines derived from 29 children with new-onset T1D. RESULTS: TRAIL+, CD56+, CD45RO+, and CD3+ cells were present in the islets of acute-onset T1D patients, while none were present in the normal islets. T-cell lines from new-onset T1D expressed TRAIL and CD56 in response to stimulation with beta-cell antigens GAD, IA-2 and insulin beta chain. CONCLUSION: The presence of TRAIL and CD56 markers is part of the T-cell response repertoire in beta-cell destruction.