Inhibition Of Allogeneic T-cell Proliferation Responses Of Hematopoietic Stem Cell Transplant By Dendritic Cells Transduced Indoleamine2,3-dioxygenase In Vitro

allo-geneic Hematopoietic Stem Cell Transplantation (allo-HSCT) has revolutionized the treatment of hematopoietic malignancies, inherited hematopoietic disorders, and aplastic anemia. However, graft-versus-host disease (GVHD) is still a major complication of allo-gene Hematopoietic Stem Cell Transplantation(allo-HSCT) and producing multiple-organ damage,immune deficiency and infection,such that 10-15% of patiens who receive MHC-matched sibling or unrelated donor transplants die from complication of transplant procedure.Among these patients, GVHD is mediated by donor T cell that can recognize minor histoconpatibility antigens (miHAs), which are peptides derived from intracellular proteins which distinguish host from donor.Following the presentation of miHAs by antigen presenting cells(APC),donor T cell are triggering by miHAs bound to MHC class I and class II antigen , respectively, to induce GVHD. Although innovative strategies for the prediction, prevention, and treatment of this caveat in allogeneic HSCT have been proposed, prophylaxis or treatment of GVHD still remains unsatisfactory. Removal of donor-derived T lymphocytes prior to transplantation efficiently ameliorates GVHD. However, ex vivo depletion of T cells has been associated with an increased risk of viral opportunistic infections, impaired engraftment, and the loss of the graft-versus-leukemia (GVL) effect, resulting in an increased incidence of recurrence of leukemia. Several clinical and experimental studiessupport the idea that GVHD and GVL are mediated not only by the same (host-specific) T cell but also by distinct (leukemia-specific) alloreactive donor T-cell populations. Moreover, allo-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) to antigens frequently expressed at elevated levels on leukemic cells can be cloned from an allorestricted T-cell repertoire and used to mediate a GVL reaction without causing GVHD.Dendritic cells (DC), are the most potent professiongnal antigen presenting cell (APC) , are very important in immune systems for their functions to activate naive T cell , initiate a primary immune response and induce rejections.Recent studies have indicated that DCs may act as immune-stimulating cells as well as tolerance-inducing cells. Genetic modification of DC with genes encoding immunoregulatory molecules is an alternative for artificial generation of tolerogenic DC. Indeed, recent reports suggest that transfection of DC with IL-10 and TGF can increase their tolerogenic potential. Several attributes make DC ideal vehicles for the delivery of such molecules.Indoleamine 2,3-dioxygenase (IDO), a monomeric hemoprotein,is the initial and rate-limiting enzyme of the kynurenine pathway of degradation of L-tryptophan.This substance is futhur catabolized to terminal metabolites quiolinic acid. IDO oxidizes the pyrole moiety of tryptophan. .Tryptophan is necessary to the survival if some cell lines and pathogens,such as T ell.Rcently,IDO has been implicated in the regulation of feto-maternal tolerance in a mouse model.Following the blocking if IDO by 1-methy l-tryptophan,allogeneic but not syngeneic fetuses were found to be rejected. This was provoked by a single(paternally inherited) MHC- I difference and depended on the activity of maternal T-cells. Application of1-methyl-tryptophan resulted in extensive inflammation, haemorrhagic necrosis, a T cell infiltrate and deposition of C3 at the murine materno-fetal interface.IDO induced inM-CSF-derived macrophages or in monoxyte-derived dendritic cells by IFN- y ,and IDO present in human placental villous has been shown to inhibitT-cel lproliferation viat ryptophan depletion.The critical first step in the initiation of GVHD is antigen presentation to donor T cells by recipient (not donor) APCs. So we proposed that host bonemarrow-derived DC transduced to express IDO can deplete alloreactive cells from donorhaematopoietic stem cell grafts in vitro, and transplantating this manipulated grafts may prevent GVHD and keep GVL and antiviral immunity effects.In this study we transferred the bone marrow-derived DCs with AdmlDO, AdmLacz and day 7 normal DC used as control. DCs surface phenotype and functional changes were analyzed by FACS after IDOgene transduction. ion of Allogeneic T-cell proliferation responses inhibited by dendritic cells transduced indoleamine2,3-dioxygenase (IDO) in vitro and apoptosis in activated alloreactive T lymphocytes was induced in an allogeneic mixed lymphocyte reactions (MLR).IDOmRNA could be detected by RT-PCR in DCs transferred with AdmlDO, which indicated that adenovirus could effectively transfer DCs.Through DCs,Lacz-DCs and IDO-DCs analyzed by FACS,we observed that DCs transduced withlDO gene consistent with untransduced DCs surface phenotype.Naive T lymphocytes from C57BL/6 mice were cocultured with BALB/c mouse-derived IDO-DC.Mixed lymphocyte reactions (MLR) DCs^ Lacz-DCs> IDO-DCs with C57BL/6 derived T cell show that IDO-DC might inhibit activated T cell proliferation.Addition of I-MT (the inhibitor ofIDO) ,the proliferation of activated T cell was resumed.Induction of apoptosis of alloreactive T cells was specific, because the residual T lymphocytes MLR and CTL to the third part (C3H) still remained. Thus, depletion of alloreactivity by IDO-DC retained functional T-cell specificities to defined nonalloantigens. We found that IDO-DC could induce T cells apoptosis analyzed by AnnexinV/PI.In conclusion, we have established an allogeneic model system that allows elimination of alloreactive T cells by IDO-DC. Transfer a modified allogeneic donor T-lymphocyte repertoire into a BMT model demonstrated that depletion of alloreactive T cells was sufficient for preventing a lethal GVHD in recipients. Thus, utilizing IDO-DC for elimination of alloreactivity in allogeneic transplants might represent a suitable strategy for preventing a lethal GVHD and remaining GVL effect.