Primary Study Of The Role Of Alloreactive Natural Killer Cell On The Transfusion-associated Immunodepression

[Objective] Allogeneic blood transfusion will lead to changes in immune status, which in the literatures of clinical blood transfusion study is termed transfusion-associated immunomodulation(TRIM).The natural killer cells(NK cells)is an important element in transfusion-related immunomodulation. Killer cell immunoglobulin-like receptors (KIRs) expressed on the surface of NK cells. It can identify specific target cells in combination with the surface of HLA-Ⅰmolecules, and conduct inhibit or active signal. It is like a set of effective switch system regulating the function of NK cells, playing an essential role in anti-infection, anti-tumor, etc. KIRs are divided into two groups of inhibiting type (DL) and activating type (DS). There is a particular structural domain outside its cell in every group. Each group has its own specific extracellular domain, related to a specific ligand binding, mediating inhibitory or activate response. KIR can identify different specific HLA molecules, for example, KIR2DLl/2DS1 identify HLA-Cw2, Cw4, Cw5, Cw6, Cw15, its common characteristic is that the HLA 80th amino acid residues is lysine; KIR2DL2/2DL3/2DS2/2DS3 identify HLA–Cw1, Cw3, Cw7, Cw8, Cw12, Cw14, its common characteristic is that the HLA 80th amino acid residues is aspartic acid; KIR3DL1 identify HLA-Bw4, 3DL2 identify HLA-A3 /-A11; 2DL4 identify HLA-G ; the rest of KIR ligands have not been found. When KIR could not identify individuals from different HLA-I molecules, it will generate the heterologous reactivity, on the contrary, when KIR can identify individuals from different HLA-I molecules ,it will generate immune tolerance. At present, majority of studies proceed with NK cell's quantity and active aspect change. Allogeneic blood transfusion causes NK cell quantity and activeness reducing passively, the initiative role of NK cells does not involve. The appearance of the alloreactive natural killer cell concept offered the new thinking on the prevention and reversion for the immunological suppression of blood transfusion. Up to now, there is no report about the role of the alloreactivity of NK cells in TRIM. Referring to the research experience that NK cells employed in the hematopoietic stem cell transplants, we put forward such an imagination: as same as the cross matching, matching KIR and HLA- I molecular before blood transfusion, select the donor whose KIR is unmatched with recipient's HLA-I molecule, to induce the alloreactivity of NK cells, to prevent or reverse the immune inhibition. It will be used in conventional and individual blood transfusion therapy for tumors, hematologic disease and transplant patients. In this experiment, we simulated clinical external blood transfusion, aiming to investigate the role of alloreactive natural killer cell on the immunological suppression of blood transfusion when blood donor's KIR and receptor's HLA-I molecular in varying degree matching situation.[Methods] NK cells from PBMC were isolated by using miniMACS(Magnetic cell-selection) and NK Cell Isolation KitⅡ(Miltenyi Biotec ,Germany), then cultured in the specific conditions in vitro. Five kinds of inhibitory KIRs were genotyped by PCR-SSP method. NK cells known KIR types were mixed with the blood known HLA- I type in certain proportion(equal to inputs the 800ml whole blood to person who is 60 kilogram body weight), sample were taken on different time point and the immunologic change were detected. A total of 20 groups were analyzed on the degree of mismatching of KIR and HLA. The degree of mismatching on the immune function of blood was compared.[Result] NK cells with high purity were obtained using the method of immuno-magnetic separation. The purity rose 13.8±2.24% to 94.5±2.61% after separation. The mean survival rate is > 95%. NK cells proliferated up to 13 times on the 21d culture. The faster proliferation is during 7d to 15d culture. They reached the highest killing activity of 94% on 14d with an efficiency-target ratio of 10:1. Based on the analysis of the immunologic indexes of different level of KIR and HLA, mixed and incubated from 0h to 24h, we found that the concentration of IL-2,IL-4,PGE2,TNF-α,TNF-β,IFN-γand GM-CSF were increased (P <0.05); the concentration of IgA,IgM and IgG were decreased(P <0.05) at different level. There is no correlation between KIR-HLA matching level with the concentration changing of IL-2,IL-4,PGE2,IgA,IgM,IgG (P> 0.05 ). However, the concentration of TNF-α,TNF-β,IFN-γand GM-CSF were increased with the raising of mismatching degree between KIR and HLA (P <0.05).[Conclusion] The higher degree of mismatching of KIR and HLA is, the more can stimulate the immunological function of alloreactivity of NK cells. It will be better to reverse the transfusion-associated immunodepression.