RESUMEN
The induction of tolerance to transplanted organs is a major objective in transplantation immunology research. Lymphocyte function-associated antigen-1 (LFA-1) interactions have been identified as a key component of the T-cell activation process that may be interrupted to lead to allograft tolerance. In mice, αLFA-1 mAb is a potent monotherapy that leads to the induction of donor-specific transferable tolerance. By interrogating important adaptive and innate immunity pathways, we demonstrate that the induction of tolerance relies on CD8+T-cells. We further demonstrate that αLFA-1 induced tolerance is associated with CD8+CD28-T-cells with a suppressor phenotype, and that while CD8 cells are present, the effector T-cell response is abrogated. A recent publication has shown that CD8+CD28- cells are not diminished by cyclosporine or rapamycin, therefore CD8+CD28- cells represent a clinically relevant population. To our knowledge, this is the first time that a mechanism for αLFA-1 induced tolerance has been described.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Supervivencia de Injerto/inmunología , Tolerancia Inmunológica/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Tolerancia al Trasplante/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD28/inmunología , Ciclosporina/farmacología , Femenino , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/inmunología , Supervivencia de Injerto/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Sirolimus/farmacología , Tolerancia al Trasplante/efectos de los fármacos , Trasplante Homólogo/métodosRESUMEN
The autoimmune condition is a primary obstacle to inducing tolerance in type 1 diabetes patients receiving allogeneic pancreas transplants. It is unknown how autoreactive T cells that recognize self-MHC molecules contribute to MHC-disparate allograft rejection. In this report, we show the presence and accumulation of dual-reactive, that is autoreactive and alloreactive, T cells in C3H islet allografts that were transplanted into autoimmune diabetic NOD mice. Using high-throughput sequencing, we discovered that T cells prevalent in allografts share identical TCRs with autoreactive T cells present in pancreatic islets. T cells expressing TCRs that are enriched in allograft lesions recognized C3H MHC molecules, and five of six cell lines expressing these TCRs were also reactive to NOD islet cells. These results reveal the presence of autoreactive T cells that mediate cross-reactive alloreactivity, and indicate a requirement for regulating such dual-reactive T cells in tissue replacement therapies given to autoimmune individuals.
Asunto(s)
Aloinjertos/inmunología , Autoantígenos/inmunología , Rechazo de Injerto/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Animales , Diabetes Mellitus Tipo 1/cirugía , Ratones , Ratones Endogámicos NOD , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Diabetes is prevalent among solid organ transplant recipients and is universal among islet transplant recipients. Whereas diabetes is often considered to result in an immune-compromised state, the impact of chronic hyperglycemia on host alloimmunity is not clear. Potential immune-modifying effects of obesity, autoimmunity, or diabetogenic agents like streptozotocin may confound understanding alloimmunity in experimental models of diabetes. Therefore, we sought to determine the role of chronic hyperglycemia due to insulinopenia on alloimmunity using the nonautoimmune, spontaneously diabetic H-2b-expressing C57BL/6 Ins2Akita mice (Akita). Akita mice harbor a mutated Ins2 allele that dominantly suppresses insulin secretion, resulting in lifelong diabetes. We used BALB/c donors (H-2d) to assess alloimmunization and islet transplantation outcomes in Akita recipients. Surprisingly, chronic hyperglycemia had little effect on primary T-cell reactivity after alloimmunization. Moreover, Akita mice readily rejected islet allografts, and chronic hyperglycemia had no impact on the magnitude or quality of intragraft T-cell responses. In contrast, allospecific IgM and IgG were significantly decreased in Akita mice after alloimmunization. Thus, whereas diabetes influences host immune defense, hyperglycemia itself does not cause generalized alloimmune impairment. Our data suggest that immune compromise in diabetes due to hyperglycemia may not apply to cellular rejection of transplants.
Asunto(s)
Diabetes Mellitus/inmunología , Rechazo de Injerto/inmunología , Hiperglucemia/inmunología , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Linfocitos T/inmunología , Animales , Diabetes Mellitus/cirugía , Insulina/genética , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Trasplante HomólogoRESUMEN
The complement system is proposed to play an important role in the pathogenesis of rheumatoid arthritis (RA). The complement system mannan-binding lectin-associated serine proteases (MASP)-1/3 cleave pro-factor D (proDf; inactive) into Df (active), but it is unknown where this cleavage occurs and whether inhibition of MASP-1/3 is a relevant therapeutic strategy for RA. In the present study, we show that the cleavage of proDf into Df by MASP-1/3 can occur in the circulation and that inhibition of MASP-1/3 by gene silencing is sufficient to ameliorate collagen Ab-induced arthritis in mice. Specifically, to examine the cleavage of proDf into Df, MASP-1/3-producing Df-/- liver tissue (donor) was transplanted under the kidney capsule of MASP-1/3-/- (recipient) mice. Five weeks after the liver transplantation, cleaved Df was present in the circulation of MASP-1/3-/- mice. To determine the individual effects of MASP-1/3 and Df gene silencing on collagen Ab-induced arthritis, mice were injected with scrambled, MASP-1/3-targeted, or Df-targeted small interfering RNAs (siRNAs). The mRNA levels for MASP-1 and -3 decreased in the liver to 62 and 58%, respectively, in mice injected with MASP-1/3 siRNAs, and Df mRNA decreased to 53% in the adipose tissue of mice injected with Df siRNAs; additionally, circulating MASP-1/3 and Df protein levels were decreased. In mice injected with both siRNAs the clinical disease activity, histopathologic injury scores, C3 deposition, and synovial macrophage/neutrophil infiltration were significantly decreased. Thus, MASP-1/3 represent a new therapeutic target for the treatment of RA, likely through both direct effects on the lectin pathway and indirectly through the alternative pathway.
Asunto(s)
Artritis Experimental/terapia , Artritis Reumatoide/terapia , Factor D del Complemento/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Interferencia de ARN , Animales , Artritis Experimental/genética , Artritis Reumatoide/genética , Factor D del Complemento/genética , Humanos , Masculino , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteolisis , ARN Interferente Pequeño/genéticaRESUMEN
A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD20/inmunología , Linfocitos B/efectos de los fármacos , Insulina/administración & dosificación , Proinsulina/administración & dosificación , Proinsulina/genética , Vacunas de ADN/administración & dosificación , Administración Oral , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Quimioterapia Combinada/métodos , Femenino , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/inmunología , Insulina/genética , Insulina/inmunología , Interleucina-4/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Páncreas/efectos de los fármacos , Páncreas/inmunología , Proinsulina/inmunología , Bazo/efectos de los fármacos , Bazo/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunologíaRESUMEN
OBJECTIVE: Because of reduced antioxidant defenses, beta-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant. RESEARCH DESIGN AND METHODS: The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation. RESULTS: Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant-treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection. CONCLUSIONS: Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.
Asunto(s)
Diabetes Mellitus Experimental/cirugía , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Islotes Pancreáticos/métodos , Metaloporfirinas/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Humanos , Trasplante de Islotes Pancreáticos/inmunología , Masculino , Ratones , Trasplante Heterólogo/inmunología , Trasplante Homólogo/inmunologíaRESUMEN
NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients' pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin-positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9-23 (insulin B:9-23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9-23 sequence in their pancreata. Additionally, CD4(+) T cells from B16:A-dKO mice immunized with native insulin B:9-23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9-23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9-23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9-23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Insulina/inmunología , Insulina/metabolismo , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Animales , Autoanticuerpos/inmunología , Trasplante de Médula Ósea , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Trasplante de Células , Reactividad Cruzada/inmunología , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Inmunización , Insulina/genética , Ratones , Ratones Endogámicos NOD , Fragmentos de Péptidos/genética , Bazo/inmunología , Bazo/metabolismo , Bazo/trasplante , Tasa de SupervivenciaAsunto(s)
Trasplante de Islotes Pancreáticos/métodos , Animales , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/terapia , Supervivencia de Injerto , Humanos , Isoantígenos/química , Activación de Linfocitos , Complejo Mayor de Histocompatibilidad/inmunología , Modelos Biológicos , Linfocitos T/inmunologíaRESUMEN
CD154 and LFA-1 (CD11a) represent conceptually distinct pathways of receptor/ligand interactions (costimulation and adhesion/homing, respectively) that have been effectively targeted to induce long-term allograft acceptance and tolerance. In the current study, we determined the relative efficacy and nature of tolerance induced by mAbs specific for these pathways. In vitro analysis indicated that simultaneous targeting of CD154 and LFA-1 resulted in profound inhibition of alloreactivity, suggesting that combined anti-CD154/anti-LFA-1 therapy could be highly effective in vivo. Thus, we evaluated combining mAb therapies targeting CD154 and LFA-1 for inducing transplantation tolerance to pancreatic islet allografts. Monotherapy with either anti-CD154 or anti-LFA-1 was partially effective for inducing long-term allograft survival, whereas the combination resulted in uniform allograft acceptance in high-responder C57BL/6 recipients. This combined therapy was not lymphocyte depleting and did not require the long-term deletion of donor-reactive T lymphocytes to maintain allograft survival. Importantly, combined anti-CD154/anti-LFA therapy uniquely resulted in "dominant" transplantation tolerance. Therefore, simultaneous perturbation of CD154 and LFA-1 molecules can result in profound tolerance induction not accomplished through individual monotherapy approaches. Furthermore, results show that such regulatory tolerance can coexist with the presence of robust anti-donor reactivity, suggesting that active tolerance does not require a corresponding deletion of donor-reactive T cells. Interestingly, although the induction of this regulatory state was highly CD4 dependent, the adoptive transfer of tolerance was less CD4 dependent in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Transducción de Señal/inmunología , Tolerancia al Trasplante , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/fisiología , Supresión Clonal/genética , Quimioterapia Combinada , Epítopos Inmunodominantes/inmunología , Inyecciones Intraperitoneales , Activación de Linfocitos/genética , Prueba de Cultivo Mixto de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/genética , Tolerancia al Trasplante/genéticaRESUMEN
BACKGROUND: Although many transplantation studies have implicated a graft-destructive role for T helper (Th)1 cytokines and a graft-protective role for Th2 cytokines, more recent studies have challenged this paradigm by showing that long-term allograft survival can actually require the presence of Th1 cytokines, such as interleukin 2 and interferon (IFN)-gamma. The purpose of this study was to examine the requirement for IFN-gamma in the induction of islet allograft acceptance after monoclonal antibody therapy targeting conceptually distinct molecular pathways: the costimulatory molecule CD154, the CD4 coreceptor, or the beta2 integrin lymphocyte function-associated antigen (LFA)-1 (CD11a). METHODS: Diabetic C57Bl/6 (B6; H2b) mice were grafted with fully MHC mismatched BALB/c (H2d) islets, or reciprocally, diabetic BALB/c mice underwent transplantation with B6 islets and were treated with anti-CD154, anti-CD4, or anti-LFA-1. RESULTS: When IFN-gamma gene knockout mice were used as graft recipients, the requirement for IFN-gamma in allograft survival was found to be highly conditional, depending on both the host strain and the induction therapy used. In both strain combinations studied, anti-CD154 was effective in the presence or absence of IFN-gamma, whereas anti-CD4 lost therapeutic potential in the absence of this cytokine. Alternatively, the requirement for IFN-gamma for allograft prolongation by anti-LFA-1 therapy was noted only in B6 transplant recipients. CONCLUSIONS: IFN-gamma is not always requisite in islet allograft survival but rather varies according to the molecular target of induction therapy and the genetic background of the transplant recipient.