Study Of Ly49A Expression In T Lymphocytes And Its Regulation In Mouse Acuse Acute Graft-Versus-Host Disease Model

Hematopoietic stem cell transplantation (HSCT) provides a curative therapy for patients with hematological malignancies and some refractory non-hematological diseases but even when the donor and recipient are major histocompatibility complex (MHC) matched siblings, graft-versus-host disease (GVHD) occurs frequently from minor antigenic differences. GVHD, acute GVHD (aGVHD) in particular, is addition to treatment-related mortality, therefore approaches that have the capacity to reduce the ability of donor T cell to respond to alloantigens , while not increasing transplant related toxicity have great potential to improve the outcome of allogeneic HSCT. Killer cell inhibitory receptors (KIR) is a group of glycoproteins expressed on NK and some activated T cells. KIR can interact with MHC type I molecule, which is self/non-self peptide complex expressed on self cell surface, and produces inhibitory signal which can prevent the activation of killer cell. The mechanism is an important factor to maintain self-immune tolerance in normal. Mouse KIR was Ly49 family, belong to C-type lectin superfamily, and recognize mice11MHC (H-2 system). Ly49A was one of the typical inhibitory receptors in this family and connected with H-2d, which leads to the inhibition of killer cell function. Utilizing the special-immune tolerance of KIR to the recipient cells which expressed corresponding ligands, we conceived that up-regulation of specific KIR expression in donor T lymphocyte or NK cell could induce the tolerance of CTL to recipient , thus alleviating aGVHD.Up to now there has been no comprehensive research on the effect of KIR+ T cells on aGVHD occurrence and the regulation of KIR expression by cytokines. Based on mouse aGVHD models, the distribution of Ly49A+T lymphocyte and dynamic profile change of its expression in normal, syngeneic, semimatched and allogeneic transplanted mice, the relationship between aGVHD onset and Ly49A expression in T cells, and the effects of cytokines on the regulation of its expression were investigated to explore the potential to control aGVHD by the induction of specific KIR expression.Objective: The aim of the research was to investigate the relationship between Ly49A expression and aGVHD incidence, up-regulation of Ly49A expression in C57BL/6 T lymphocytes by some cytokines, and the change in expression of Ly49A in T cells exposed to CsA, IL-4 and IL-15, respectively, based on mouse hematopoietic system models.12Methods: 1 Induction of aGVHD Specific pathogen-free (SPF) inbred mouse BALB/cH2d C57BL/6H2b (B6)> CB6FlH2dxb (BALB/c XC57BL/6, CB6F1) at age of 8 to 12 weeks were used to induce aGVHD model through splenocyte and bone marrow cell mixed transplantation. Animal groups:(Dsyngenic transplantation group, B6-B6 or BALB/c-BALB/c;semi-syngeneic transplantation group, B6-CB6F1; (3) allogeneic transplantation group, B6 -BALB/c; (D TBI without transplantation group; (5) normal control group. Recipients were prepared for transplant with 950-1050 rads v lethal irradiation using 60Co source at day 0. Donor cells were injected intravenously into recipients in four hours after irradiation (BM cells 3 X 106+splenocytes 3X 106)/0.4ml. All animals were monitored during the experiment and euthanized when needed, obtained spleen and bone marrow MNC and pathological samples were leaved over. aGVHD was determined by the onset of diarrhea, weight loss, alopecia, dermatitis, hunched posture, cachexia, eye inflammation and death, which was connected with histopathology analyses and WBO1.5X 109/L at the same time. The signs of aGVHD were observed in d+10 as super-aGVHD, and in d+10~d+30 as aGVHD. Recipients were weighted tertian, and WBC and platelet of periphery blood counted from d-1 to d+60. 2 X 105/ml non-adherent bone marrow MNCs were planted in 1% methyl cellulose in 35-mm dishes containing13optimal concentration of recombinant murine cytokines: mGM-CSF for CFU-GM, EPO+IL-3 for BFU-E and CFU-E, mGM-CSF + EPO + IL-3 + SCF for CPU-Mix. Dishes were incubated in a fully humidifie...