RESUMEN
OBJECTIVES: Mixed hematopoietic chimerism is associated with islet allograft tolerance and may reverse autoimmunity. We developed low intensity regimens for the induction of mixed chimerism and examined the effects on autoimmunity in prediabetic nonobese diabetic (NOD) mice. RESEARCH DESIGN AND METHODS: NOD mice received various combinations of total body irradiation, anti-CD154, anti-CD8alpha, anti-CD4, and anti-Thy1.2 monoclonal antibodies, with or without transplantation of C57BL/6 bone marrow cells and were followed up for development of diabetes, chimerism, and donor skin graft survival. Autoimmunity was assessed by histologic examination of salivary glands and pancreata. RESULTS: Although conditioning alone prevented or delayed the onset of diabetes, stable mixed chimerism was required for the reversal of isletitis. Mixed chimerism and skin graft tolerance were achieved in NOD mice receiving anti-CD154 with bone marrow transplantation as the means of tolerizing peripheral CD4 T cells to alloantigens. However, isletitis was not reversed in allotolerant mixed chimeras prepared with this regimen. CONCLUSIONS: Partial depletion of peripheral autoreactive NOD CD4 T cells is needed to achieve full reversal of isletitis by mixed chimerism induction from a protective donor strain, but it is not required for induction of specific tolerance to donor alloantigens. Thus, the requirements for tolerizing alloreactive and autoreactive NOD CD4 cells are distinct.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Ratones Endogámicos NOD/inmunología , Trasplante de Piel/inmunología , Quimera por Trasplante/inmunología , Trasplante Homólogo/inmunología , Traslado Adoptivo , Animales , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/inmunología , Ligando de CD40/efectos de la radiación , Antígenos CD8/inmunología , Antígenos CD8/efectos de la radiación , Femenino , Citometría de Flujo , Islotes Pancreáticos/inmunología , Isoantígenos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Estado Prediabético/inmunología , Antígenos Thy-1/inmunología , Antígenos Thy-1/efectos de la radiación , Irradiación Corporal TotalRESUMEN
Previously, we reported that 100 Gy X-ray irradiation followed by 24 hr incubation up-regulates CD80 expression in murine B lymphoma cells, A20-2J. In the present study, we analysed the underlying mechanisms of such up-regulation using A20-HL cells derived from A20-2J cells. Irradiation of A20-HL cells with 100 Gy enhanced CD80 expression. Incubation of untreated A20-HL cells with those 100 Gy irradiated induced up-regulation of CD80 expression. Irradiation of A20-HL cells also up-regulated the expression of tumour necrosis factor-alpha (TNF-alpha) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF-alpha and CD40L in cell lysates. The addition of anti-TNF-alpha or anti-CD40L monoclonal antibody (mAb) to the incubation of irradiated A20-HL cells partially inhibited up-regulation of CD80 expression, and the addition of both antibodies together almost completely inhibited the up-regulation, suggesting that irradiation up-regulated the CD80 expression through the induction of TNF-alpha and CD40L expression. Irradiation also increased the accumulation of CD80, TNF-alpha and CD40L mRNA. n-tosyl-l-phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)-kappaB inhibitor, markedly decreased irradiation-induced accumulation of CD80 mRNA and CD80 expression. FK506, a calcineurin inhibitor, and nifedipine, a calcium channel inhibitor, inhibited not only the expression of TNF-alpha and CD40L, but also the up-regulation of CD80 on irradiated A20-HL cells. These results strongly suggested that irradiation induced TNF-alpha and CD40L expression, which then up-regulated CD80 mRNA and CD80 expression through activation of NF-kappaB transcription factor in A20-HL cells.
Asunto(s)
Antígenos de Neoplasias/efectos de la radiación , Antígeno B7-1/efectos de la radiación , Linfoma de Células B/inmunología , Regulación hacia Arriba/efectos de la radiación , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Ligando de CD40/inmunología , Ligando de CD40/efectos de la radiación , Células Dendríticas/inmunología , Ratones , FN-kappa B/inmunología , ARN Mensajero/genética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/efectos de la radiaciónRESUMEN
Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.