NKG2D overexpression by mRNA transfection with CART ONA in human primary NK cells

We are investigating the function of natural killer (NK) cell receptor, NKG2D, in the functional response of NK cells to HIV-infected autologous CD4+ T cells. We will overexpress NKG2D in human primary NK cells by transfecting them with charge-altering releasable transporters (CARTs) to deliver the mRNA that encodes NKG2D. DAP10 is the natural signaling adaptor of NKG2D in human NK cells, and co-transfection of DAP10 can enhance the expression of exogenous NKG2D on the surface of NK cells. In this experiment, we are testing which ratio of co-transfecting NKG2D and DAP10 mRNA allows the highest expression level of NKG2D on the surface of the transfected NK cells. To distinguish NK cells that have translated the exogenous NKG2D mRNA from the ones that naturally express NKG2D, we will co-transfect NK cells with GFP mRNA and GFP expression will indicate the transfected NK cells.
Conclusion:
We tested co-transfection of NK cells with NKG2D+DAP10+GFP mRNA at 2:1:2, 3:1:3, 4:1:4, and 1:1 (1:1 ratio of NKG2D:GFP, no DAP10) mass ratios. Among all the four ratios tested, we identified that 3:1:3 allowed slightly higher expression level of NKG2D in GFP+ cells, compared to 2:1:2 and 4:1:4, indicated by the mean fluorescent intensity of NKG2D staining. When no DAP10 mRNA was used for co-transfecting NK cells, there is no considerable increase of NKG2D expression in GFP+ cells compared to GFP- NK cells, suggesting that exogenous DAP10 is required for the surface expression of the exogenous NKG2D.
Notes:
In this experiment, we tested the co-transfection of NKG2D+DAP10+GFP mRNA at 4 different mass ratios. 4:1:4 means that 44.4 ng of NKG2D mRNA, 11.1 ng of DAP10 mRNA and 44.4 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. 3:1:3 means that 42.9 ng of NKG2D mRNA, 14.3 ng of DAP10 mRNA and 42.9 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. 2:1:2 means that 40 ng of NKG2D mRNA, 20 ng of DAP10 mRNA and 40 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. 1:1 means that 50 ng of NKG2D mRNA and 50 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. The expression of NKG2D and GFP was analyzed 16 hours after transfection.