Prolonged survival of mouse skin allografts after transplantation of fetal liver cells transduced with hIL-10 gene.
ABSTRACT Interleukin-10 (IL-10) is a cytokine with a moleculary weight of 18 kDa, that was first identified as being produced by Th2 cells. It appears to have anti-inflammatory action by diminishing the production of pro-inflammatory cytokines produced by Th1 cells. IL-10 also regulates the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells, mast cells and granulocytes. Recent data suggest, however, that IL-10 also has immunostimulatory properties with important consequences on the prognosis of disease. In this study, we demonstrate the importance of injection of hematopoietic fetal liver cells transduced with the human IL-10 (hIL-10) gene into an allogenic recipient subsequently transplanted with allogenic skin grafts. The immaturity of stem cells and precursor cells from fetal liver and their transient survival in the host, due to the production of hIL-10, may afford 'prope' tolerance. It also explains the lack of graft-vs.-host reaction (GvHR) and the delay in rejection of the specific donor skin grafts after virtual disappearance of donor hematopoietic cells. Objectives: Transduction of CBA hematopoietic fetal cells with the human IL-10 gene was used with the aim of inducing tolerance to donor antigen in recipient BALB/c mice. The observed effects were prolonged IL-10 production, donor cell chimerism in the host and delayed rejection of skin grafts from the specific donor strain.
To prevent or delay rejection of highly incompatible skin allografts, we used IL-10 gene transfer to establish chimerism with donor hematopoietic cells. Fetal liver cells from CBA mice were transduced with the human IL-10 gene and injected into BALB/c mice.
Human IL-10, which is active in mice but does not cross-react with murine IL-10 in ELISA, was produced in vivo for 3 weeks. Donor cells were identified in the recipients during the same time period, on the basis of presence of the H-2 k gene and human IL-10 intracellular protein. Skin allografts from CBA or C57BL/6 mice survived for a mean of 9.5 days in recipient mice injected with non-transduced cells. In contrast, survival of CBA allograft was extended to 18.9+/-1.8 days in recipients injected with hIL-10-transduced fetal liver cells from CBA mice. Human IL-10 alone, without donor hematopoietic cell engraftment, did not prolong graft survival (9.6+/-1.2 days).
IL-10 transduction of donor hematopoietic stem cells resulted in production of IL-10, cell engraftment and chimerism. Although full tolerance was not obtained at this level of donor cell development in the host, a specific and highly significant (P<0.001) prolongation of the survival of donor skin allografts was observed.
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ABSTRACT: Interest in mixed chimerism has evolved from its role in the induction of alloantigen tolerance. However, its precise impact on the host organism remains to be elucidated. In the present work, we analyzed cytokine secretion from chimeric mice cells to assess the influence of different mixed chimerism induction protocols on immune system function in recipient mice. To our knowledge, there have been no reports on using this parameter for the optimization of the mixed chimerism induction method. B6.SJL-PtprcaPep3b or C57BL/6J mice were used as recipients and Balb/c as donors. We utilized four protocols consisted of: 3Gy total body irradiation (Day -1), the injection of 20-30×10(6) bone marrow cells (Day 0), and a combination of CD40L (Day 0, and 4), CD8 (Day -2), and NK1.1 (Day -3) blocking antibodies and cyclophosphamide (175mg/kg - Day 2). The concentrations of cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF) were evaluated in the supernatants of unstimulated or phytohemagglutinin-stimulated chimeric spleen, bone marrow and peripheral blood cells in the 8th week of experiment. The induction of tolerance to Balb/c mouse antigens was initially tested in chimeric mice by assessing the presence of Vβ5 and Vβ11 TCR-expressing lymphocytes. The cytokine production was considerably increased, especially in chimeric mice treated by cyclophosphamide. Also the mixed chimerism itself seems to affect IFN-γ, IL-2, IL-4, IL-6, IL-17A, and TNF secretion. Using the optimized induction protocol, we established that chimeric mice cells secreted lower IFN-γ, IL-2, IL-4 and higher IL-6, IL-17A, and TNF levels as compared to control animals. We found that both donor and recipient cells markedly participated in the cytokines production. In conclusion, our optimization study based on cytokine assessment contributes to establishing an effective protocol of mixed chimerism induction with no cyclophosphamide use and better understanding of the influence of this phenomenon on the recipient organism.Transplant Immunology 09/2013; · 1.83 Impact Factor
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ABSTRACT: We previously reported that transduction of the human interleukin (IL)-10 gene into the total fetal liver stem cells (hIL-10-TFLs) of mice protects against their rejection in an allogeneic host. In this study, we explored the effects of these cells in two different models of organ transplantation. Balb/c mice were sublethally irradiated before receiving skin or vascularized heterotopic heart grafts from C57Bl/6 mice. TFLs from C57Bl/6 mice transduced with hIL-10 or untransduced TFLs were injected on the day of transplantation into recipient mice once or also every 20 days thereafter. Skin allograft survival was prolonged for up to 17.8±0.6 days, vs. 9.0±0.4 days, in mice that received hIL-10-TFLs or untransduced TFLs, respectively. Allogeneic heart transplants survived for 86.25±13.8, 46.3±4.6, 28.1±6.1, or 11.5±0.6 days in mice that received repeated injections of hIL-10-TFLs, a single injection of hIL-10-TFLs, repeated injections of untransduced TFLs, or controls, respectively. Histological analyses of the grafts showed fewer inflammatory foci and CD8+ infiltrating cells in mice injected with hIL-10-TFLs compared with untreated mice. Expressions of H-2b and hIL-10 were found in several organs, including the thymus, liver, and the transplant, in hIL-10-TFL-injected mice. Finally, in hIL-10-TFL-injected mice, FoxP3 T cells were present inside the transplanted heart as late as 140 days after transplantation. In this study, we showed that repeated injections of hIL-10-TFLs are efficient in mitigating transplant rejection. This "prope" tolerance was associated with survival of donor hematopoietic cells in the host.Transplantation 04/2012; 93(8):761-8. · 3.78 Impact Factor
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ABSTRACT: In transgenic mice over-expressing CD200 (CD200(tg)) graft survival is associated with increased intra-graft expression of mRNAs for genes associated with altered T cell subset differentiation (Foxp3; TGFβ; IL-10). Grafts are rejected in recipients lacking the inhibitory receptor for CD200, CD200R1. We compared grafts of C57BL/6 skin taken from control, CD200KO, CD200(tg), CD200R1KO or CD200(tg).CD200R1KO C57BL/6 donor mice transplanted to control or CD200(tg) BALB/c recipients. Animals received either low-dose rapamycin (0.5mg/Kg), which only enhanced survival in CD200(tg) mice, or high dose rapamycin (1.5mg/Kg) which increased graft survival in all recipients. Recipient draining lymph nodes (DLNs) were analyzed at 14d post grafting in mixed leukocyte cultures (MLCs) with irradiated BL/6 or C3H/HeJ stimulator cells, assaying antigen-specific CTL at d5. MLC responses were correlated with changes in mRNA gene expression in skin tissue harvested from the same recipients, focusing on genes altered in "graft-accepting" CD200(tg) recipients. Tissue histology was used to assess graft infiltrating Foxp3(+)Tregs, mast cells (MCs) and their degranulation. CD200(tg) grafts were accepted in control but not CD200KO/CD200R1KO recipients, along with decreased degranulation in graft MCs, diminished DLN MLC responses, and augmented intragraft Foxp3, TGFβ, IL-10 and mast cell gene expression. Skin grafts from either CD200KO or CD200R1KO donors to control mice were rejected, with no change in DLN MLC responses, no altered graft gene expression from that seen using control skin grafts, and pronounced graft MC degranulation. Our data highlight a role for both graft and host CD200/CD200R expression in increased allograft survival.Immunology letters 11/2012; · 2.91 Impact Factor