RESUMEN
BACKGROUND: Alpha-galactosylceramide (alpha-GalCer) is an invariant natural killer T (iNKT) cell ligand that prevents type 1 diabetes in NOD mice. However, alpha-GalCer can activate or suppress immune responses, raising concern about its potential use in human diabetes. MATERIALS AND METHODS: To evaluate this therapeutic issue further, BBDR and LEW.1WR1 rats were treated with Kilham rat virus (KRV) plus polyinosinic-polycytidylic acid, with or without alpha-GalCer, and followed for onset of diabetes. RESULTS: alpha-GalCer did not prevent diabetes in inducible rat models. To investigate this discrepancy, we analyzed iNKT cell function. Splenocytes stimulated with alpha-GalCer produced similar levels of IFNgamma in all rat strains, but less than mouse splenocytes. Rat splenocytes stimulated with alpha-GalCer preferentially produced IL-12, whereas mouse splenocytes preferentially produced IL-4. CONCLUSION: alpha-GalCer elicits species-specific cytokine responses in iNKT cells. In humans with type 1 diabetes, differences in iNKT cell responses to stimulation with alpha-GalCer due to age, genetic variability and other factors may influence its therapeutic potential.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Galactosilceramidas/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/virología , Modelos Animales de Enfermedad , Femenino , Galactosilceramidas/fisiología , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratas , Factores Sexuales , Bazo/citología , Bazo/metabolismoRESUMEN
Recent studies have implicated the cell surface receptor Programmed Death-1 (PD-1) in numerous models of T cell anergy, though the specific mechanisms by which the PD-1 signal maintains tolerance is not clear. We demonstrate that the depletion of PD-1 with siRNA results in a complete reversal of clonal anergy in the A.E7 T cell model, suggesting that the mechanism by which PD-1 maintains the anergic phenotype is a T-cell-intrinsic phenomenon, and not one dependent on other cell populations in vivo. We have also shown that the neutralization of IL-2 during restimulation abrogates the effect of PD-1 depletion, suggesting that tolerance mediated by PD-1 is wholly IL-2 dependent, and likewise intrinsic to the tolerized cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Linfocitos T CD4-Positivos/inmunología , Anergia Clonal/inmunología , Animales , Antígenos/administración & dosificación , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Secuencia de Bases , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Proliferación Celular , Anergia Clonal/genética , Columbidae , Citocromos c/inmunología , Cartilla de ADN/genética , Interleucina-2/antagonistas & inhibidores , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos BALB C , Fenotipo , Receptor de Muerte Celular Programada 1 , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/inmunología , Transfección , Tolerancia al Trasplante/genética , Tolerancia al Trasplante/inmunologíaRESUMEN
TLR activation of innate immunity prevents the induction of transplantation tolerance and shortens skin allograft survival in mice treated with costimulation blockade. The mechanism by which TLR signaling mediates this effect has not been clear. We now report that administration of the TLR agonists LPS (TLR4) or polyinosinic:polycytidylic acid (TLR3) to mice treated with costimulation blockade prevents alloreactive CD8(+) T cell deletion, primes alloreactive CTLs, and shortens allograft survival. The TLR4- and MyD88-dependent pathways are required for LPS to shorten allograft survival, whereas polyinosinic:polycytidylic acid mediates its effects through a TLR3-independent pathway. These effects are all mediated by signaling through the type 1 IFN (IFN-alphabeta) receptor. Administration of IFN-beta recapitulates the detrimental effects of TLR agonists on transplantation tolerance. We conclude that the type 1 IFN generated as part of an innate immune response to TLR activation can in turn activate adaptive immune responses that abrogate transplantation tolerance. Blocking of type 1 IFN-dependent pathways in patients may improve allograft survival in the presence of exogenous TLR ligands.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Innata , Interferón Tipo I/inmunología , Receptor de Interferón alfa y beta/inmunología , Transducción de Señal/inmunología , Trasplante de Piel/inmunología , Tolerancia al Trasplante , Animales , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Inmunidad Innata/efectos de los fármacos , Inductores de Interferón/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/inmunología , Poli I-C/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunología , Tolerancia al Trasplante/efectos de los fármacos , Trasplante HomólogoRESUMEN
BACKGROUND: Blockade of T cell costimulation by treatment with donor-specific transfusion (DST) and anti-CD154 monoclonal antibody (mAb) induces prolonged allograft survival in mice. This effect is due in part to deletion of host CD8 and CD4 T cells that recognize alloantigen by direct presentation. The fate of host CD4 T cells that recognize alloantigen by indirect presentation, however, is unclear. METHODS: We studied Tg361 TCR transgenic CD4 T cells that recognize alloantigen by indirect presentation. Carboxyfluorescein diacetate, succinimidyl ester-labeled Tg361 cells were adoptively transferred into syngeneic nontransgenic recipients and their fate in the peripheral blood, spleen, and lymph nodes following treatment with DST and anti-CD154 was analyzed. RESULTS: Treatment of mice with DST plus anti-CD154 mAb does not delete Tg361 CD4 T cells, but instead renders them hyporesponsive to rechallenge with alloantigen. Mice circulating hyporesponsive CD4 T cells also fail to reject skin allografts. The hyporesponsive state of the T cells is not reversed by the addition of interleukin-2, anti-CD28 mAb, or an agonistic anti-CD134 mAb in the presence of antigen. These T cells are capable of activation, however, as evidenced by in vitro proliferation in response to anti-CD3 mAb. CONCLUSIONS: These results demonstrate that costimulation blockade can induce hyporesponsiveness of host CD4 T cells recognizing alloantigens by indirect presentation, thus prolonging graft survival by a mechanism that does not involve deletion of alloreactive T cells.
Asunto(s)
Presentación de Antígeno , Isoantígenos/química , Linfocitos T/citología , Animales , Linfocitos T CD4-Positivos/citología , Ligando de CD40/biosíntesis , Linfocitos T CD8-positivos/citología , Cricetinae , Fluoresceínas/farmacología , Supervivencia de Injerto , Tolerancia Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , RatasRESUMEN
Ag-specific immune tolerance results from the induction of cellular mechanisms that limit T cell responses to selective Ags. One of these mechanisms is characterized by attenuated proliferation and decreased IL-2 production in fully stimulated CD4(+) Th cells and is denoted T cell anergy. We report the identification of the early growth response gene (Egr-2; Krox-20), a zinc-finger transcription factor, as a key protein required for induction of anergy in cultured T cells. Gene array screening revealed high Egr-2 expression distinctly persists in anergized but not proliferating murine A.E7 T cells. In contrast, Egr-1, a related family member induced upon costimulation, displays little or no expression in the anergic state. IL-2-mediated abrogation of anergy causes rapid depletion of Egr-2 protein. Full stimulation of anergic A.E7 T cells fails to enhance IL-2 and Egr-1 expression, whereas Egr-2 expression is greatly increased. Silencing Egr-2 gene expression by small interfering RNA treatment of cultured A.E7 T cells before incubation with anti-CD3 alone prevents full induction of anergy. However, small interfering RNA-mediated depletion of Egr-2 5 days after anergy induction does not appear to abrogate hyporesponsiveness to stimulation. These data indicate that sustained Egr-2 expression is necessary to induce a full anergic state through the actions of genes regulated by this transcription factor.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Anergia Clonal/inmunología , Proteínas de Unión al ADN/fisiología , Activación de Linfocitos/inmunología , Factores de Transcripción/fisiología , Animales , Antígenos CD28/farmacología , Complejo CD3/farmacología , Linfocitos T CD4-Positivos/enzimología , Línea Celular , Proliferación Celular , Células Clonales , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteína 2 de la Respuesta de Crecimiento Precoz , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/inmunología , Silenciador del Gen , Interleucina-2/biosíntesis , Interleucina-2/farmacología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Fosforilación , ARN Interferente Pequeño/farmacología , Receptores de Antígenos de Linfocitos T/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Regulación hacia Arriba/inmunología , Dedos de Zinc/inmunologíaRESUMEN
BACKGROUND: Treatment with anti-CD154 monoclonal antibody (mAb) plus a donor-specific transfusion (DST) of spleen cells prolongs skin allograft survival in mice through a mechanism involving deletion of host alloreactive CD8(+) T cells. It is unknown if other lymphohematopoietic cell populations can be used as a DST. METHODS: Murine recipients of allogeneic skin grafts on day 0 were either untreated or given a DST on day -7 plus 4 doses of anti-CD154 mAb on days -7, -4, 0, and +4. Deletion of CD8(+) alloreactive cells was measured using "synchimeric" CBA recipients, which circulate trace populations of TCR transgenic alloreactive CD8(+) T cells. RESULTS: Transfusion of splenocytes, thymocytes, lymph node cells, or buffy coat cells led to prolonged skin allograft survival in recipients treated with anti-CD154 mAb. In contrast, bone marrow DST failed to delete host alloreactive CD8(+) T cells and was associated with brief skin allograft survival. Transfusions consisting of bone marrow-derived dendritic cells or a mixture of splenocytes and bone marrow cells were also ineffective. CONCLUSIONS: Donor-specific transfusions of splenocytes, thymocytes, lymph node cells, or buffy coat cells can prolong skin allograft survival in recipients treated with costimulation blockade. Bone marrow cells fail to serve this function, in part by failing to delete host alloreactive CD8(+) T cells, and they may actively interfere with the function of a spleen cell DST. The data suggest that transplantation tolerance induction protocols that incorporate bone marrow cells to serve as a DST may not be effective.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Transfusión Sanguínea , Células de la Médula Ósea/fisiología , Ligando de CD40/fisiología , Supervivencia de Injerto , Trasplante de Piel , Animales , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/trasplante , Depleción Linfocítica , Metrizamida/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Bazo/citología , Trasplante HomólogoRESUMEN
BACKGROUND: Donor-specific transfusion (DST) and a brief course of anti-CD154 monoclonal antibody (mAb) induces permanent islet and prolonged skin allograft survival in mice. Induction of skin allograft survival requires the presence of CD4 cells and deletion of alloreactive CD8 cells. The specific roles of CD4 and CD4CD25 cells and the mechanism(s) by which they act are not fully understood. METHODS: We used skin and islet allografts, a CD8 T cell receptor (TCR) transgenic model system, and in vivo depleting antibodies to analyze the role of CD4 cell subsets in regulating allograft survival in mice treated with DST and anti-CD154 mAb. RESULTS: Deletion of CD4 or CD25 cells during costimulation blockade induced rapid rejection of skin but only minimally shortened islet allograft survival. Deletion of CD4 or CD25 cells had no effect upon survival of healed-in islet allografts, and CD25 cell deletion had no effect upon healed-in skin allograft survival. In the TCR transgenic model, DST plus anti-CD154 mAb treatment deleted alloreactive CD8 T cells, and anti-CD4 mAb treatment prevented that deletion. In contrast, injection of anti-CD25 mAb did not prevent alloreactive CD8 T cell deletion. CONCLUSIONS: These data document that (1) both CD4CD25 and CD4CD25 cells are required for induction of skin allograft survival, (2) CD4CD25 T cells are not required for alloreactive CD8 T cell deletion, and (3) CD4CD25 regulatory cells are not critical for islet allograft tolerance. It appears that skin and islet transplantation tolerance are mediated by different CD4 cell subsets and different mechanisms.
Asunto(s)
Trasplante de Islotes Pancreáticos/inmunología , Trasplante de Piel/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Transfusión Sanguínea , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Experimental/cirugía , Femenino , Terapia de Inmunosupresión/métodos , Activación de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Modelos Animales , Trasplante Homólogo/inmunologíaRESUMEN
Treatment of mice with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb to block CD40-mediated signaling uniformly induces donor-specific transplantation tolerance. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. The nature of the cellular mechanisms involved and the basis for the difference in survival of islet vs skin allografts are not known. In this study, we used CD40 knockout mice to investigate the role of CD40-mediated signaling in each component of the tolerance induction protocol: the DST, the graft, and the host. When CD40-mediated signaling was eliminated in only the DST or the graft, islet allografts were rapidly rejected. However, when CD40 signaling was eliminated in the host, approximately 40% of the islet allografts survived. When CD40 signaling was eliminated in the DST, the graft, and the host, islet grafts survived long term (>84 days), whereas skin allografts were rapidly rejected ( approximately 13 days). We conclude that transplantation tolerance induction in mice treated with DST and anti-CD154 mAb requires blockade of CD40-mediated signaling in the DST, the graft, and the host. Blockade of CD40-mediated signaling is necessary and sufficient for inducing islet allograft tolerance and is necessary but not sufficient for long-term skin allograft survival. We speculate that a requirement for regulatory CD4(+) T cells in skin allograft recipients could account for this differential response to tolerance induction.
Asunto(s)
Anticuerpos Bloqueadores/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD40/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Transducción de Señal/inmunología , Trasplante de Piel/inmunología , Tolerancia al Trasplante , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/trasplante , Antígeno B7-1/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/genética , Antígenos CD40/fisiología , Ligando de CD40/biosíntesis , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Terapia Combinada , Refuerzo Inmunológico de Injertos/métodos , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Inyecciones Intraperitoneales , Trasplante de Islotes Pancreáticos/métodos , Linfopenia/genética , Linfopenia/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Quimera por Radiación/inmunología , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/genética , Transducción de Señal/genética , Trasplante de Piel/métodos , Especificidad de la Especie , Donantes de Tejidos , Acondicionamiento Pretrasplante , Tolerancia al Trasplante/genéticaRESUMEN
Defects in the gene that encodes SHP-1 protein tyrosine phosphatase result in multiple hematopoietic abnormalities and generalized autoimmunity in viable motheaten (me(v)) mice. These mice also exhibit early thymic involution and abnormalities in T cell development. Here, we describe the use of fetal thymic organ culture (FTOC) and bone marrow adoptive transfer to study the effects of SHP-1 deficiency on thymocyte development. Chimeric FTOC established with normal bone marrow placed onto deoxyguanosine-treated fetal thymic lobes or onto scid fetal thymic lobes generated T cells. Bone marrow from SHP-1-deficient me(v)/ me(v) mice generated decreased numbers of T cells in chimeric FTOC established using deoxyguanosine-treated thymi but generated normal numbers in chimeric FTOC established using scid thymi. However, scid fetal thymi seeded with me(v)/ me(v) bone marrow also exhibited morphological abnormalities and contained elevated numbers of macrophages. Addition of IL-7 to me(v)/ me(v) bone marrow-seeded scid FTOC led to increased cell numbers, particularly of macrophages. Intrathymic injection of IL-7 partially restored the ability of progenitor cells in me(v)/ me(v) bone marrow to populate the thymus of adoptive recipients. We conclude that abnormal T cell development in me(v)/ me(v) mice may in part be due to defects in the ability of bone marrow-derived accessory cells to provide bioavailable IL-7 to developing thymocytes.