Distribution And Function Of Platelet And T Cell Activation Antigen 1 (CD226)on NK Cells

Platelet and T cell activation antigen 1 (PTA1) was initially reported by Bums in 1985. PTA1 was mainly expressed on activated T cells, platelets and megekaryocyte lineage, and was involved in the activation and differentiation of T cells and platelet activation and aggregation. In 1997, PTA1 cDNA was cloned from cDNA library of TPA activated Jurkat cells, which belongs to immunoglobulin superfamily (IgSF) with only two Ig-V like domains. In the same year, the PTA1 homologous cDNA of gibbon and monkey was cloned in our lab. Homology analysis showed that PTA1 molecule is highly conserved among human, gibbon and monkey, sharing the same sequence about 93%~95% of the total. In the year of 2000, PTAlwas designated as CD226 at the 7th HLDA.NK cell is a kind of lympohcyte with natural cytotoxicity which palys an important role in innate immunity. It has been widely accepted that NK cell is involved in the effect of anti-infection, anti-tumor and immunol ogical regulation in immune system. In general, the target cells of NK cell aretransformed cells (tumor) or virus-transfected cells which are lost or downregulated the expression of MHC I molecules. NK cell receptors are divided into three groups according to their function, activation receptor, inhibition receptor and receptor without influence on the cytotoxicity of NK cells mainly determined by RCA (redirected cytotoxicity assay, RCA). Recently, we found that PTA1 is expressed on NK cells and some kinds of NK cell lines. So, it is important to investigate the distribution and function of PTA 1 on NK cells, NK cell lines as well as NK cell clones.First, we separated NK cells from mixed lymphocyte culture (MLC) generated effector killer cells by CD56mAb positive immunobeads. Using limited dilution assay, we established a NK cell clone with the phenotype of CD56+CD3~. The distribution of PTA1 molecule on NK cell, NK cell lines and NK cell clone was detected by indirected immunofluorescence staining and FCM analysis. The results showed that PTA1 expression rate on activated NK cell in MLC was above 60%. The expression rate on NKL was 97.9%, while there were no expression on NK92, YT and NK3.3 cell lines. On the NK cell clone, the expression rate was 61 .2%.Then we used activated NK cell and NK cell clone as effect cell in RCA respectively. We found that PTA1 mAb could upregulate the cytotoxicity of both these two kind of cells. The cytokines including IFN- y ,GM-CSF and TNF- a in the supernatants from RCA culture at different time point during the killing phase were measured by ELISA. The results showed that PTA1 mAb could increase the levels of IFN- Y and GM-CSF obviously, and the levels of these two cytokines came up during the first 4 hours, and almost reached to the peak between 8 to 12 hours. As a positive control, CD16 mAb could upregulate the release of all these three kinds of cytokines in RCA. In comparation, during the first 4 hours of RCA culture, the level of GM-CSF was much higher in PTA1 mAb group than that in CD 16 mAb group, while during the following hours the level of GM-CSF in CD 16 mAb group surpassed that in PTA1 mAb group which suggested that the mechanisms of NK cells activated by PTA1 and CD 16 may be different. The rudiment investigation showed that PTA1 (CD226) was anovel membrane activation receptor on NK cells.