Mechanisms Of Umbilical Cord Blood Hematopoietic Stem Cells Escaping From NK Cell-mediated Cytotoxicity

Objective:Previous studies have shown that allogeneic umbilical cord blood hematopoietic stem cells(UCB-HSCs) were demonstrated to be able to engraft into a host with intact innate immunity.While the underlying mechanisms of UCB-HSCs' immune escaping ability have not been clarified.We hypothesized that allogeneic UCB-HSCs are able to escape from the cytolysis of natural killer cells(NK cells) which play a pivotal role in eliminating transplanted allogeneic HSCs,and this immune escaping ability may be associated with the expression of killer cell activatory receptors and killer cell inhibitory receptors on the cell surface. Methods:(1) HSCs were isolated from PB,BM and UCB by sequentially incubating the cells with biotin-conjugated anti-CD34 antibody and the streptavidin-conjugated microbeads,followed by magnetic selection.(2) NK cells isolated from PB and NK-92 cells were used as effector cells,and HSCs were used as target cells.The cytolytic activity of PB-NK cells and NK-92 cells against the HSCs was assessed in 4-hours lactate dehydrogenase release assay;The numbers of HSCs with the ability to form colony in vitro assay of NK cells were measured using CFC Assay.(3) Immunofluorescence analyses of cell surface phenotypes were performed by FACS Aria.Detection of NCR ligands was performed by sequentially incubating cells with NKp/Fc fusion proteins and anti-IgG1 antidoby.Dectection of HLA-G was performed by directly incubating cells with anti-HLA-G antibody.(4) In NCRs blocking experiments,targets were preincubated with NKp30/Fc chimera,NKp44/Fc chimera, and NKp46/Fc chimera respectively or in combination,as indicated.When in HLA-G blocking experiments,targets were preincubated with anti-HLA-G monoclonal antibodies.Percentage of lysis was calculated with the same formula as that used for 4-hours lactate dehydrogenase release assay.Results:(1) UCB-HSCs are able to escape from the cytolysis of NK cells;(2) UCB-HSCs express low levels of NKp30L,Nkp44L and NKp46L on the surface;(3) UCB-HSCs express high level of HLA-G on the surface.Conclusion:(1) In summary,our data provide the evidence that three kinds of HSCs all have the characteristic of immune escaping from NK cells.But they differ from each other,with PB-HSCs,BM-HSCs and UCB-HSCs in ascending order.(2)Low levels of NKp30L,Nkp44L and NKp46L on the surface of HSCs may be the public mechanisms of their immune escaping from NK cells.(3) High level of HLA-G may be the reason why UCB-HSCs has more ability of immune escaping from NK cells than PB-HSCs and BM-HSCs.