RESUMEN
Treatment of autoimmune diseases remains a challenge for immunological research. An ideal therapy should inhibit the immune reaction against the diseased organ and leave the rest of the immune response intact. Our previous studies showed that donor-derived dendritic cells (DCs) treated in vitro with mitomycin C (MMC) suppress rat heart allograft rejection if injected into recipients before transplantation. Here we analyze their efficacy in controlling autoimmunity. MMC-DCs loaded with myelin-basic-protein (MBP) inhibited specific T cells derived from multiple sclerosis patients in vitro. If coincubated with MMC-DCs, T cells were arrested in the G(0)/G(1) cell cycle phase. Microarray gene scan showed that MMC influences the expression of 116 genes in DCs, one main cluster comprising apoptotic and the second cluster immunosuppressive genes. Apparently, the combination of apoptosis with expression of tolerogenic molecules renders MMC-DCs suppressive. MBP-loaded MMC-DCs also inhibited mouse T cells in vitro and, in contrast to MBP-loaded naïve DCs, did not induce experimental autoimmune encephalitis. Most importantly, mice vaccinated with inhibitory DCs became resistant to the disease. Whereas this is not the first report on generation of suppressive DCs, it delineates a method using a clinically approved drug at nontoxic concentrations, which yields irreversibly changed DCs, effective across species in vitro and in vivo.
Asunto(s)
Enfermedades Autoinmunes/terapia , Células Dendríticas/efectos de los fármacos , Mitomicina/farmacología , Linfocitos T/efectos de los fármacos , Vacunas/inmunología , Animales , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Fase G1 , Ratones , Ratones Transgénicos , Fase de Descanso del Ciclo Celular , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The most important antigen-presenting cells are dendritic cells (DCs), which play a central role in the initiation of immunity and tolerance. Their immunoregulatory properties offer the potential of donor-specific control of graft rejection after organ transplantation. It has not been clarified which DC subpopulations mediate tolerance, and the use of natural DCs for therapeutic applications is therefore problematic. Suppressive DCs can be generated in vitro by treating the cells with biologic, pharmacologic, or genetic agents. Here we discuss approaches for generating inhibitory DCs and present DC-based animal models for control of allograft rejection. A prerequisite of suppressive DCs for therapeutic application in clinical transplantation is a reproducible method for their generation as well as the induction of irreversible suppressive function. Based on lessons learned from the use of DCs as tools in clinical vaccine trials in cancer, we discuss the unknown aspects and risks of DC therapy in transplantation.
Asunto(s)
Células Dendríticas/inmunología , Rechazo de Injerto/inmunología , Trasplante de Órganos , Tolerancia al Trasplante/inmunología , Animales , Presentación de Antígeno/inmunología , Diferenciación Celular/inmunología , Inhibición de Migración Celular/inmunología , Ciclosporina/farmacología , Ingeniería Genética , Rechazo de Injerto/prevención & control , Humanos , Inmunoterapia Activa , Ratones , Modelos Animales , RatasRESUMEN
We showed previously that dendritic cells (DCs) treated with mitomycin C (MMC) tolerize allogeneic T cells in vitro and this might be mediated by downregulation of CD80, CD86, and ICAM-1. Here we analyze the suppression of the T-cell response induced by MMC-DCs in vivo. Rats injected with allogeneic DCs developed a strong lymph node reaction, whereas MMC-DCs induced no reaction. The same effect was obtained when CD80, CD86, and ICAM-1 expressed by DCs were blocked with antibodies. One injection of donor MMC-DCs strongly prolonged heart allograft survival in a donor-specific manner. Suppression of rejection was also achieved when donor DCs were pretreated with a combination of anti-CD80, anti-CD86, and anti-ICAM-1 antibodies, showing that downregulation of these molecules confers the DCs inhibitory properties. We conclude that allogeneic MMC-DCs specifically inhibit the T-cell response in vivo and that downregulation of CD80, CD86, and ICAM-1 is a potential mechanism of this effect.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Rechazo de Injerto/prevención & control , Trasplante de Corazón , Terapia de Inmunosupresión/métodos , Mitomicina/farmacología , Linfocitos T/inmunología , Animales , Anticuerpos/farmacología , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Regulación hacia Abajo , Molécula 1 de Adhesión Intercelular/metabolismo , Activación de Linfocitos , Ratas , Ratas Endogámicas , Linfocitos T/efectos de los fármacos , Donantes de TejidosRESUMEN
Mitomycin C (MMC) is an alkylating agent which suppresses allogeneic T-cell responses. We analyzed the effect of graft perfusion with MMC on transplant survival. Hearts from Brown-Norway (BN) rats were perfused ex vivo with MMC-containing solution, stored and implanted into Lewis (LEW) rats. In order to analyze the in vivo effect of MMC, recipients received MMC posttransplantation or were pretreated with MMC-incubated donor-derived peripheral blood mononuclear cells (PBMCs). The results show that MMC-perfusion significantly prolongs graft survival. Treatment of recipients with MMC has no effect, whereas MMC-treated donor PBMCs injected into the recipient prolong graft survival. Our findings indicate that the targeted perfusion of donor hearts with MMC-containing solution protects the graft from rejection.
Asunto(s)
Supervivencia de Injerto/efectos de los fármacos , Trasplante de Corazón , Corazón/efectos de los fármacos , Mitomicina/farmacología , Animales , Apoptosis , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Perfusión , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Soluciones/farmacología , Donantes de TejidosRESUMEN
Previous animal studies showed that donor-derived blood cells treated with mitomycin C (MMC) prolong allograft survival when injected into recipients. This model was effective with whole blood, peripheral blood mononuclear cells (PBMC) (monocytes being the active cell subpopulation) or dendritic cells. In view of a potential clinical application, we study now the immunosuppressive properties of human myeloid cells in vitro. Mature dendritic cells (generated from naïve monocytes) or monocytes treated with mitomycin C do not or only weakly inhibit allogeneic T cells in vitro, whereas cells in an early differentiation state between monocytes and DC exert suppressive activity when treated with MMC. In contrast, DC generated from MMC-treated monocytes show the morphology and phenotype of early immature DC (iDC) and suppress T-cell responses. It is known that untreated monocytes injected into a recipient encounter a cytokine milieu which differentiates them to stimulatory DC. In our in vitro experiment MMC-treated monocytes cultured in a DC-maturing milieu transform themselves into suppressive early iDC. This reproduces a process which takes place when administering MMC-monocytes to a recipient. In conclusion, human MMC-DC or MMC-monocytes are not or only weakly suppressive in vitro. When MMC-monocytes are differentiated to DC the resulting cells become suppressive.
Asunto(s)
Inmunosupresores/farmacología , Mitomicina/farmacología , Células Mieloides/efectos de los fármacos , Apoptosis/efectos de los fármacos , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Humanos , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/efectos de la radiación , Linfocitos T/inmunologíaRESUMEN
INTRODUCTION: The demonstration of anaplastic lymphoma kinase (ALK) positivity in non-small-cell lung cancer (NSCLC) has been hindered by the technical complexity and interpretative challenges of fluorescence in situ hybridization methods for detection of ALK gene rearrangement and by the inadequate sensitivity of existing immunohistochemistry (IHC) methods for ALK protein detection. In this study, we sought to increase the sensitivity of ALK IHC detection and to develop a brightfield assay for concurrent detection of ALK protein expression and ALK gene rearrangement. METHODS: We developed a horseradish peroxidase-based IHC detection system using the novel, nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) and tyramide. We also developed a dual gene protein ALK assay combining a brightfield break-apart in situ hybridization ALK assay with another sensitive IHC method using the novel, nonendogenous hapten 5-nitro-3-pyrazole. We examined the sensitivity and accuracy of these methods using surgically resected NSCLC cases examined with ALK fluorescence in situ hybridization. RESULTS: The new HQ-tyramide IHC detection system offered readily interpretable staining with substantially greater sensitivity than conventional ALK IHC, and produced heterogeneous and homogeneous patterns of ALK protein staining among ALK-positive NSCLC surgical cases. The new 5-nitro-3-pyrazole-based IHC detection system was similar in ALK detection sensitivity to the HQ-tyramide IHC system and was compatible with the brightfield in situ hybridization assay. CONCLUSION: The new HQ-tyramide IHC reagent system allows more sensitive assessment of ALK protein status in NSCLC cases. The new ALK gene-protein assay allows the concurrent visualization of ALK gene and ALK protein status in single cells, allowing more accurate ALK status determination even in heterogeneous specimens.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/química , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/química , Proteínas Tirosina Quinasas Receptoras/análisis , Quinasa de Linfoma Anaplásico , Línea Celular Tumoral , Haptenos/inmunología , HumanosRESUMEN
Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases.