The Mechanism Of Inducing Immune Tolerance Of G-CSF And The Relationship Of HLA-G And GVHD

Background and ObjectivesAllogeneic hematopoietic stem cell transplantation (allo-HSCT) are now applied widely for the treatment of hematological or non-hematological malignancies, aplastic anemia and hereditary diseases. Granulocyte colony-stimulating factor (G-CSF) primed bone marrow grafts (G-BM) plus G-CSF mobilized peripheral blood grafts (G-PB) were used successfully in haploidentical transplantation and the incidence of graft-versus-host disease (GVHD) was not higher compared with that in patients with HLA-matched donors. In humans, granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell grafts (G-PB) do not cause a higher incidence of acute GVHD than marrow grafts, despite at least a 10-fold higher T cell dose. These clinical observations imply that G-CSF application modulates cellular in vivo immune functions. E.D. Carosella was the pioneer who demonstrated the protective role of the HLA-G molecule on trophoblasts, which form a shield protecting the fetus from the immune reaction of its mother and subsequent reject. Human leucocyte antigen G (HLA-G) is a nonclassical HLA classⅠmolecule, This non-classical HLA classⅠmolecule is first expressed on the fertilized ovocyte, thus enabling a uterine implantation and then on the surface of the placenta trophoblast where the classical classⅠandⅡantigens are absent. He brought the first demonstration ex vivo of the protector role of HLA-G molecule present on the surface of fetal cytotrophoblast cells versus the lysis carried out by maternal decidual uterine NK cells, in both semi-allogenic combinations (maternal uterine NK cells and their own fetal cytotropohoblast counterparts) and allogenic combinations (different maternal uterine NK cells and cytotrophoblasts from different fetuses). The blockage of this protein triggers off an important cytotoxicity towards the fetal cells. Furthermore, he showed that HLA-G molecules act as an inhibitor of the T-lymphocytes, NK cells and antigen presenting cells (APC). Through his discovery Carosella also shows for the first time the three major clinical consequences:Ⅰ) HLA-G molecules are crucial, as an altered expression of these molecules would lead to abortion and failed pregnancies, i.e. recurrent spontaneous abortions and preeclamptic disease. The embryo expression of soluble HLA-G molecules is a mandatory prerequisite to implantation.Ⅱ) In allogenic transplantation (heart, kidney and liver-kidney graft) the expression of HLA-G protein significantly reduces acute rejection and showed an absence of chronic rejections.Ⅲ) Finally, this expression on the malignant cells has a negative functional impact in the anti-tumour response. So the expression of HLA-G molecule constitutes an escape mechanism from immunosurveillance, just as the fetal cells protect themselves from the aggression of maternal immune cells. In contrast to the general belief that G-CSF acts exclusively on T cells via monocytes and dendritic cells, soluble human leukocyte antigen (sHLA) molecules seem to be promising candidates involved in immunosuppressive properties of rhG-CSF treatment,The mechanisms of HLA-G in immunotolerance include:inhibition of natural killer (NK) and CD8+ T cells cytotoxicity; inhibition of T CD4+ alloproliferative response; inhibition of antigen-presenting cell function; induction regulatory T cells (Treg cells) formed and regulation of cytokine secretion. Because of HLA-G closely related to immunotolerance we envisaged: HLA-G could be affected by rhG-CSF treatment.The soluble HLA-G5 isoform encoded by intron-4 retaining spliced transcript has been previously detected in vivo in sera and grafts from transplanted patients who had significantly better graft acceptance. These findings led us to investigate the role of HLA-G5 in tolerance induction in vitro and its biological relevance in allograft acceptance in vivo. HLA-G expression found in heart-transplant recipients, both in serum and in grafted endomyocardial cells, was associated with a decreased number of acute rejection episodes and an absence of chronic rejection, compared with patients who did not express HLA-G. Furthermore, following liver-kidney cotransplantation, ectopic expression of HLA-G in grafted liver and in serum was associated with an absence of acute and chronic rejection of both liver and kidney transplants.We detect the HLA-G in HSCT patients and try to find the relationship between HLA-G and GVHD.MethodFlow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) on donor bone marrow (BM) cells. The levels of soluble HLA-G (s HLA-G) in the bone marrow fluid and blood were determined using enzyme-linked immunosorbent assay (ELISA), Statistical analysis:All data were analyzed using SPSS 13.0 for windows. Comparison between the two groups use two independent samples t test. Comparison mHLA-G,sHLA-G at differert time use multiple independent samples t test. A P-value< 0.05 was considered to be statistically significant. ResultThe mHLA-G of CD4+ lymphocytes, CD8+ lymphocytes and sHLA-G in the post-G-CSF groups were significantly higher than the pre-G-CSF groups both in bone marrow and peripheral blood cells.Both IL-10 and IFN-γin plasma of post-G-CSF were much higher than pre-G-CSF in bone marrow and peripheral blood.The levels of HLA-G of bone marrow cells and peripheral blood cells after incubating with G-CSF for 24 h were significantly higher than the group without incubating with G-CSF. And the blocking antibody of IL-10 or the blocking antibody of IFN-γcould not reduce the expression levels of mHLA-G and sHLA-G in vitro.ConclusionThe tolerogenic role of HLA-G is highly supported in pregnancy immunization, tumor immune escape and organ transplant. HLA-G and very likely sHLA-G are capable of inhibiting alloresponses by binding to an up-to-now unknown HLA-G receptor, resulting in the differentiation of CD4+T cells into suppressive cells.Results from experimental models, in vitro, studies, and clinical data indicate that granulocyte colony-stimulating factor (GCSF) stimulation alters T-cell function and induces Th2 immune responses. Th2 immune responses. The immune modulatory effect of G-CSF on T cells results in an unexpected low incidence of acute graft-versus-host disease in peripheral stem cell transplantation. However, the underlying mechanism for the reduced reactivity and/or alloreactivity of T cells upon G-CSF treatment is still unknown.we found that G-CSF can up regulation the expression of HLA-G,not though IL-10 or IFN-γ, It show us that G-BM transplantation or G-PBSC transplantation associated with a low incidence of GVHD have something to do with the increasing expression levels of HLA-G in CD4+T lymphocytes and CD8+T lymphocytes by G-CSF acting on cells directly and independently. High levels of HLA-G in the plasma of patients who under gone transplantation have been correlated with better acceptance of the solid transplant. HLA-G5 purified from plasma of patients who underwent transplantation inhibited T-cell alloproliferation, showing the direct involvement of HLA-G in better graft acceptance status. Soluble HLA-G induces suppressive T cells of a CD41ow or CD81ow phenotype. These cells are over-represented in HLA-G positive patients and correlate with better graft acceptance. HLA-G expression is associated with a better allograft acceptance both in solid transplantation and hematopoietic stem cell transplantation. HLA-G expression has been proposed to contribute in addition to immunosuppressive therapy to human allograft acceptance. Indeed, HLA-G expression found in heart-transplant recipients, both in serum and in grafted end myocardial cells, was associated with a decreased number of acute rejection episodes and an absence of chronic rejection, compared with patients who did not express. We found that high secretion of HLA-G5 reduced the incidence of acute GVHD and chronic GVHD, The level of HLA-G5 can predict whether GVHD will happen. Although HLA-G6 and HLA-G7 were higher in the group of no GVHD, the differences were no statistical significance.