The Role of Human Leukocyte Antigen G in Inducing Immune Tolerance After Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Du Xuan , Wu Xiuli , Li Rui , Fan Zhiping , Zhang Yu , Liu Qifa 
Published: October 2019

Abstract
Abstract 4478

Background and Objective
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is now applied widely for the treatment of hematological or non-hematological malignancies, aplastic anemia and hereditary diseases. Recently, a protocol for haploidentical allo-HSCT that combines granulocyte-colony stimulating factor (G-CSF) primed bone marrow (G-BM) and peripheral blood stem cells (G-PBSC) without in vitro T-cell depletion received great success. But the mechanism of G-CSF inducing immunotolerance in haploidentical-HSCT has not yet been clarified. Human leucocyte antigen G (HLA-G) is a nonclassical HLA class I molecule, the tolerogenic role of HLA-G is highly supported in pregnancy immunization, tumor immune escape and organ transplant. Because HLA-G closely related to immunotolerance, we investigate the role of HLA-G in inducing immune tolerance after allo-HSCT and the effects of G-CSF on the expression and secretion level of HLA-G.

Methods
Flow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) on donor peripheral blood cells (PBC) or bone marrow (BM) cells. The levels of soluble HLA-G (sHLA-G) in the plasma and bone marrow fluid were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the expression and secretion level of HLA-G in bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) after G-CSF stimulated were detected by flow cytometry and ELISA, respectively; Separated T lymphocytes which expressed high level of HLA-G were cultured with allogeneic T lymphocytes and relative response index (RPI) was measured with MTT assay; Furthermore, separated T lymphocytes were cultured with allogeneic BMMCs and the levels of IFN-γ and IL-10 in culture supernatant were determined by ELISA.

Results
The mean level of mHLA-G after G-CSF mobilization in the PBC or BM cells was significantly higher than that before G-CSF mobilization (P<0.05). The level of mHLA-G or sHLA-G in BM cells was higher than that in PBC after G-CSF mobilization (P<0.05). The level of mHLA-G or sHLA-G in BMMCs or PBSCs which were stimulated by G-CSF was higher than that of the controls (P<0.05), and the level of HLA-G in BMMCs was higher than that in PBSCs. HLA-G predominant expressed in CD3+ T cells; The results of allogeneic mixed lymphocyte culture revealed that immunological function of the separated T lymphocytes which expressed high level of HLA-G was inhibited (RPI: 54.3%). The separated T lymphocytes co-cultured with allogeneic BMMCs, the levels of IFN-γ and IL-10 in culture supernatant were significantly higher than the controls (P<0.05).

Conclusions
HLA-G is rich in G-BM that might be interpret G-BM could induce better immunotolerance than G-PBSC. The G-CSF could regulate HLA-G expression and secretion directly. The mechanism of G-CSF inducing immunotolerance might be related to the inhibition of allogeneic T cell reactivity and the increase of IFN-γ and IL-10 secretion through HLA-G.

Disclosures:
No relevant conflicts of interest to declare.