RESUMEN

We have previously shown that 12 days of high-dose calcineurin inhibition induced tolerance in MHC inbred miniature swine receiving MHC-mismatched lung, kidney, or co-transplanted heart/kidney allografts. However, if lung grafts were procured from donation after brain death (DBD), and transplanted alone, they were rejected within 19-45 days. Here, we investigated whether donor brain death with or without allograft ischemia would also prevent tolerance induction in kidney or heart/kidney recipients. Four kidney recipients treated with 12 days of calcineurin inhibition received organs from donors rendered brain dead for 4 hours. Six heart/kidney recipients also treated with calcineurin inhibition received organs from donors rendered brain dead for 4 hours, 8 hours, or 4 hours with 4 additional hours of cold storage. In contrast to lung allograft recipients, all isolated kidney or heart/kidney recipients that received organs from DBD donors achieved long-term survival (>100 days) without histologic evidence of rejection. Proinflammatory cytokine gene expression was upregulated in lungs and hearts, but not kidney allografts, after brain death. These data suggest that the deleterious effects of brain death and ischemia on tolerance induction are organ-specific, which has implications for the application of tolerance to clinical transplantation.

Asunto(s)

Muerte Encefálica/fisiopatología , Rechazo de Injerto/inmunología , Trasplante de Corazón , Isquemia/fisiopatología , Trasplante de Riñón , Trasplante de Pulmón , Tolerancia al Trasplante/inmunología , Animales , Citocinas/genética , Citocinas/metabolismo , Supervivencia de Injerto , Especificidad de Órganos , Porcinos , Porcinos Enanos , Donantes de Tejidos

RESUMEN

The emergence of skin-containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin-resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II-mismatched chimeras were tolerant of VCAs. MHC class I-mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient-type CD8+ lymphocytes. Systemic donor-specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long-term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.

Asunto(s)

Aloinjertos Compuestos/trasplante , Rechazo de Injerto/prevención & control , Complejo Mayor de Histocompatibilidad/inmunología , Trasplante de Piel/efectos adversos , Quimera por Trasplante/inmunología , Tolerancia al Trasplante/inmunología , Alotrasplante Compuesto Vascularizado/efectos adversos , Animales , Aloinjertos Compuestos/inmunología , Aloinjertos Compuestos/patología , Rechazo de Injerto/etiología , Supervivencia de Injerto/inmunología , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Porcinos , Porcinos Enanos

RESUMEN

The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, anti-interleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor-specific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.

Asunto(s)

Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Inmunosupresores/uso terapéutico , Trasplante de Islotes Pancreáticos/inmunología , Isoantígenos/inmunología , Transfusión de Linfocitos/métodos , Linfocitos T Reguladores/inmunología , Animales , Quimioterapia Combinada , Rechazo de Injerto/inmunología , Proyectos Piloto , Primates

RESUMEN

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full-MHC barrier in miniature swine. However, the renal element(s) responsible for kidney-induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic-derived "passenger" cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co-transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC-mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC-matched to each other but MHC-mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC-mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC-mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC-matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

Asunto(s)

Trasplante de Corazón , Corazón/fisiología , Histocompatibilidad/fisiología , Trasplante de Riñón , Riñón/fisiología , Complejo Mayor de Histocompatibilidad/fisiología , Tolerancia al Trasplante/fisiología , Aloinjertos , Animales , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Histocompatibilidad/inmunología , Terapia de Inmunosupresión , Inmunosupresores/uso terapéutico , Complejo Mayor de Histocompatibilidad/inmunología , Modelos Animales , Porcinos , Porcinos Enanos , Tacrolimus/uso terapéutico , Obtención de Tejidos y Órganos , Tolerancia al Trasplante/inmunología

RESUMEN

The relative contribution of central and peripheral mechanisms to the generation and maintenance of allograft tolerance is of considerable interest. Here, we present new evidence that regulatory T cells (Foxp3(+) ) maintain skin and heart allograft tolerance in mixed hematopoietic chimeric mice. Transient depletion of both donor- and recipient-derived Foxp3(+) cells was necessary and sufficient to induce decisive rejection of long-accepted skin and heart allografts. In contrast, stable hematopoietic chimerism remained, and there was no detectable induction of donor-specific reactivity to hematopoietic cells. Foxp3(+) cell depletion did not result in the rejection of skin grafts of only MHC-disparate donors (B6.C-H2(d) /bByJ), indicating that MHC antigens were not the target in the graft. We conclude that two different mechanisms of tolerance are present in mixed chimeras. Hematopoietic chimerism, resistant to Foxp3(+) depletion, is probably due to deletional tolerance to MHC antigens, as supported by previous studies. In contrast, regulatory tolerance mechanisms involving Foxp3(+) cells are required to control reactivity against non-MHC antigens not present on hematopoietic lineages.

Asunto(s)

Quimera , Factores de Transcripción Forkhead/inmunología , Trasplante de Corazón , Tolerancia Inmunológica , Depleción Linfocítica , Trasplante de Piel , Linfocitos T/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Rechazo de Injerto/inmunología , Ratones

RESUMEN

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long-term immunosuppression, realigning the risk-benefit ratio for these life-enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3-immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45-day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85-150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n=4), and those transplanted at the time of HCT (n=2) accepted all components, including skin, without evidence of rejection to the experimental end point 115-504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long-term immunosuppression-free survival.

Asunto(s)

Aloinjertos Compuestos/inmunología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Células Madre Hematopoyéticas , Complejo Mayor de Histocompatibilidad/inmunología , Alotrasplante Compuesto Vascularizado , Animales , Aloinjertos Compuestos/patología , Histocompatibilidad , Técnicas para Inmunoenzimas , Inmunosupresores/uso terapéutico , Prueba de Cultivo Mixto de Linfocitos , Porcinos , Porcinos Enanos , Linfocitos T Reguladores/inmunología , Quimera por Trasplante/inmunología , Tolerancia al Trasplante/inmunología

RESUMEN

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

Asunto(s)

Rechazo de Injerto/inmunología , Trasplante de Corazón , Trasplante de Riñón , Complejo Mayor de Histocompatibilidad/inmunología , Donantes de Tejidos , Tolerancia al Trasplante , Aloinjertos , Animales , Citometría de Flujo , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Inmunosupresores , Trasplante de Piel , Porcinos , Porcinos Enanos

RESUMEN

T cells require continual presence of extrinsic signals from their in vivo microenvironment to maintain viability. T cells removed from these signals and placed in tissue culture atrophied and died in a caspase-independent manner. Atrophy was characterized by smaller cell sizes, delayed mitogenic responses, and decreased glycolytic rate. Bcl-2 expression remained constant in vitro despite ongoing cell death, indicating that endogenous Bcl-2 expression is insufficient to explain the life span and size control of lymphocytes in vivo and that cell-extrinsic signals provided may be required to maintain both cell viability and size in vivo. One such signal, IL-7, was found to maintain both the size and survival of neglected T cells in vitro. IL-7 was not unique, because the common gamma-chain cytokines IL-2, IL-4, and IL-15, as well as the gp130 cytokine IL-6, also promoted both T cell survival and size maintenance. IL-7 did not induce resting T cells to proliferate. Instead, IL-7 stimulated neglected T cells to maintain their metabolic rate at levels comparable to freshly isolated cells. The survival and trophic effects of IL-7 could be separated because IL-7 was able to promote up-regulation of Bcl-2 and maintain cell viability independent of phosphatidylinositol 3-kinase and mammalian target of rapamycin activity but was unable to prevent cellular atrophy when phosphatidylinositol 3-kinase and mammalian target of rapamycin were inhibited. These data demonstrate that T cells require the continuous presence of extrinsic signals not only to survive but also to maintain their size, metabolic activity, and the ability to respond rapidly to mitogenic signals.

Asunto(s)

Interleucina-7/farmacología , Linfocitos T/inmunología , Animales , Caspasas/fisiología , Muerte Celular , División Celular , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Glucosa/metabolismo , Subunidad gamma Común de Receptores de Interleucina , Cinética , Activación de Linfocitos , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Interleucina-7/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR

RESUMEN

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.

Asunto(s)

Antígenos CD/fisiología , Antígeno B7-1/fisiología , Citotoxicidad Inmunológica/inmunología , Interleucina-2/fisiología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD/genética , Antígeno B7-1/biosíntesis , Antígeno B7-1/genética , Antígeno B7-2 , Antígenos CD28/fisiología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/genética , Activación de Linfocitos/genética , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Noqueados , Bazo/citología , Bazo/inmunología , Células Madre/inmunología , Linfocitos T Citotóxicos/citología

RESUMEN

Antibody and cytotoxic T-lymphocyte (CTL) responses have critical roles in eliminating many viral infections. In addition to stimulation of the T-cell receptor, T cells require costimulatory signals to respond optimally. We evaluated the role of B7 costimulatory molecules (B7-1 and B7-2) in the immune response to viral infection using vesicular stomatitis virus (VSV) and mice lacking either B7-1 or B7-2 or both molecules. Mice lacking both B7-1 and B7-2 had essentially no anti-VSV immunoglobulin G1 (IgG1) response, decreased IgG2a responses, and normal IgM responses, while mice lacking either B7-1 or B7-2 had unaltered anti-VSV antibody responses compared to wild-type mice. Depletion of CD4(+) cells further reduced the IgG2a response in mice lacking both B7 molecules, suggesting that CD4(-) cells may supply help for IgG2a in the absence of B7 costimulation. The absence of both B7 molecules profoundly reduced generation of both primary and secondary VSV-specific class I major histocompatibility complex (MHC)-restricted CTL, whereas VSV-specific CTL responses in mice lacking either B7-1 or B7-2 were similar to those of wild-type animals. Class I MHC-restricted CTL in wild-type mice were not dependent on CD4(+) cells, suggesting that the failure of CTL in the absence of B7s is due to a lack of B7 costimulation directly to the CD8(+) CTL. These data demonstrate that B7-1 and B7-2 have critical, overlapping functions in the antibody and CTL responses to this viral infection.

Asunto(s)

Antígenos CD/fisiología , Antígeno B7-1/fisiología , Glicoproteínas de Membrana/fisiología , Linfocitos T Citotóxicos/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos CD/genética , Antígeno B7-1/genética , Antígeno B7-2 , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados

RESUMEN

In standard mouse strains, a high proportion (more than 90%) of epidermal tumors produced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with a variety of chemical agents contain an activating mutation in codon 61 (A182-->T) of the c-Ha-ras gene. We analyzed the ras mutational spectra in 69 tumors induced by DMBA in a unique transgenic model, the K6/ODC mouse. In this model, low-dose DMBA treatment is sufficient per se for tumor induction, so tumor promotion with chemical agents is not required. In contrast to previous studies in standard mouse strains, our study showed that less than 50% of epidermal tumors from K6/ODC mice contained an activating codon 61 c-Ha-ras mutation (A182-->T). This result was obtained in mice initiated either as newborns (when the transgene is not expressed) or as adults (when the transgene is fully expressed). Analysis of other codon hot-spots and other ras genes revealed the presence of three codon 12 and 20 codon 61 (A182-->T) mutations in the c-Ki-ras gene in the 36 tumors that did not have c-Ha-ras mutations. We concluded that promotion in this model, by means of constitutive ornithine decarboxylase expression, causes the clonal expansion of a population of initiated cells not promoted by chemical agents.

Asunto(s)

Genes ras , Mutación , Papiloma/genética , Neoplasias Cutáneas/genética , 9,10-Dimetil-1,2-benzantraceno , Animales , Animales Recién Nacidos , Secuencia de Bases , Carcinógenos , Transformación Celular Neoplásica/genética , Cartilla de ADN , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ornitina Descarboxilasa/genética , Papiloma/inducido químicamente , Neoplasias Cutáneas/inducido químicamente

Asunto(s)

Síndromes de Compresión Nerviosa/etiología , Nervios Torácicos , Adulto , Femenino , Humanos , Bloqueo Nervioso , Síndromes de Compresión Nerviosa/terapia , Embarazo , Complicaciones del Embarazo/etiología