RESUMEN
Vascular endothelial growth factor (VEGF) is a proangiogenic mediator that promotes tumor growth. The role of VEGF in T lymphocytes is unknown. We found that T lymphocytes activated by either anti-CD3 monoclonal antibody (mAb) plus anti-CD28 mAb or by antigens on antigen-presenting cells transcribed mRNA for VEGF receptor 1 (VEGFR1) and VEGFR2. However, only VEGFR1 was expressed on the T cell surface. The addition of VEGF to either resting or activated T cells did not affect their proliferation, but VEGF increased IL-10 production and slightly decreased IFN-gamma production. A chemotaxis assay revealed that activated T lymphocytes migrate in response to VEGF. Our data suggest that VEGF has a direct immunomodulatory effect on T cells. Engagement of a high concentration of VEGF with VEGFR1 on T cells may cause T cells to migrate to tumor sites, and this interaction may play a role in IL-10-mediated immune evasion by tumor cells.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Factores Inmunológicos/farmacología , Interleucina-10/biosíntesis , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Secuencia de Bases , Línea Celular , Proliferación Celular/efectos de los fármacos , Cartilla de ADN/genética , Humanos , Inmunoterapia , Técnicas In Vitro , Interferón gamma/biosíntesis , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Neoplasias/inmunología , Neoplasias/terapia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/citología , Linfocitos T/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either na?ve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Asunto(s)
Interleucina-10/biosíntesis , Activación de Linfocitos , Células Madre Mesenquimatosas/metabolismo , Linfocitos T/inmunología , Animales , Proliferación Celular , Células Cultivadas , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Linfocinas/farmacología , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Interleucina-10/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Triptófano/análogos & derivados , Triptófano/farmacologíaRESUMEN
Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3+ T cells. Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.
Asunto(s)
Rechazo de Injerto/genética , Hiperglucemia/cirugía , Trasplante de Islotes Pancreáticos , Macaca mulatta/cirugía , ARN , Sus scrofa , Animales , Biología Computacional , Regulación de la Expresión Génica , Rechazo de Injerto/sangre , Macaca mulatta/genética , Macaca mulatta/inmunología , ARN/sangre , ARN/genética , Análisis de Secuencia de ARN , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Trasplante HeterólogoRESUMEN
CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.
RESUMEN
Mesenchymal stem cells (MSCs) are multipotent, non-hematopoietic stem cells that exhibit the capacity to inhibit the proliferation of a variety of immune cells. However, the underlying mechanisms of the immunosuppressive effects of MSCs are still obscure. Therefore, we attempted to identify the mechanisms underlying immunosuppression toward the activated T lymphocytes by MSCs in a murine model. In particular, we aimed to find possible factors derived from MSCs that drive this phenomenon. We found that T lymphocytes incubated with conditioned media of MSCs (MSC CM) entered into apoptosis and were subjected to cell cycle arrest during the course of activation, and these phenomena were accompanied by the reduction of IL-2 production. Specifically, matrix metalloproteinases (MMPs) derived from MSCs caused cleavage of IL-2 receptor α (CD25) from the surface of activated T cells, and as a consequence, IL-2 signaling in response to engagement of the IL-2 receptor (IL-2R) was downregulated. The inhibition of MMP activity in the MSC CM by GM6001 abrogated CD25 cleavage and restored IL-2 production from the activated splenocytes. However, the blockade of MMP activity could not fully restore the proliferative response and apoptosis of T cells altered by MSC CM. In conclusion, MSC-derived MMPs have a significant role in the suppression of IL-2 production through induction of CD25 cleavage and have a partial role in the suppression of T cell proliferation.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Metaloproteinasas de la Matriz/metabolismo , Células Madre Mesenquimatosas/metabolismo , Linfocitos T/metabolismo , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Medios de Cultivo Condicionados , Ciclina D2/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Dipéptidos/farmacología , Terapia de Inmunosupresión , Inhibidores de la Metaloproteinasa de la Matriz , Células Madre Mesenquimatosas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción STAT5/metabolismo , Linfocitos T/inmunologíaRESUMEN
As alkylating agents, cyclophosphamides (CTX) are used to treat various cancers and, ironically, to boost immune responses. In the present study, we attempted to elucidate the mechanism responsible for the immunomodulatory effect of high-dose CTX in an established tumor model. A single injection of high-dose CTX increased the survival rate of immunocompetent, but not immunodeficient, mice. Notably, 10 days after CTX injection, the number of CD44(high) memory T cells significantly increased, without a selective decrease in the actual number and percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the proportion of Tregs among CD4+ T cells decreased due to expansion of memory and other CD4+ T cell subtypes. This outcome was accompanied by an increase in IL-15 mRNA and up-regulation of IL-15 receptors in the CD44+CD8+ T cell compartment. We postulate that the CTX-induced change in T cell balance may increase anti-tumor immunity.