Evaluating the function of NK cells that overexpress NKp30 or NKG2D or CD244 in the co-culture with autologous HIV-infected CD4+ T cells

In our previous experiments, we demonstrated that NKp30, or NKG2D, or CD244 can be overexpressed in human primary NK cells through charge-altering releasable transporters (CART)/mRNA transfection and the transfected NK cells can be indicated with the co-expression of GFP. In this experiment, we are comparing the functional response of NK cells (indicated with degranulation marker CD107a, and cytokines, IFNgamma and TNFalpha) that overexpress NKp30, or NKG2D, or CD244 and the ones with natural levels of expression of these receptors, when co-cultured with autologous HIV-infected and mock-infected CD4+ T cells. This experiment will indicate the function of NKp30, NKG2D and CD244 in NK cell recognition of HIV.
Conclusion:
In transfected NK cells (GFP+) where NKp30 or NKG2D were overexpressed, the percentage of functional NK cells (positive for any of CD107a, IFNgamma, and TNFalpha) was higher than in untransfected NK cells (GFP-) in the same well of co-culturing with HIV-infected or mock-infected CD4+ T cells. CD244 overexpression resulted in increased percentage of functional NK cells compared to NK cells that had natural level of CD244 expression. When NK cells were co-cultured with resting CD4+ T cells (the ones that were not activated to become susceptible to HIV infection), NKp30, or NKG2D, or CD244 overexpression didn't cause any noticeable change to the percentage of functional NK cells. These data suggest that NKp30 and NKG2D facilitate NK cell recognition of CD4+ T cells when their ligands are upregulated on CD4+ T cells during HIV infection. Increase of ligand expression caused by in vitro activation may also cause elevation of NK cell function. CD244 overexpression resulted in decreased NK cell function when interacting with their ligands on CD4+ T cells.
Notes:
Overexpression of NKp30, or NKG2D, or CD244 in NK cells was achieved through charge-altering releasable transporters (CARTs) transfection to deliver the encoding mRNAs. NK cells were co-transfected with GFP mRNA to distinguish the NK cells that have translated the exogenous mRNA from the ones that naturally express these receptors. When transfected with NKG2D mRNA, NK cells were co-transfected with DAP10 mRNA, which is essential for the surface expression of the exogenous NKG2D. Three control groups were included, one with untransfected NK cells, another with NK cells transfected with GFP mRNA alone, and the other with NK cells co-transfected with DAP10+GFP mRNA. The mass ratio of co-transfected mRNA in each group is 1:1, 3:1:3, 1:1, and 1:6 for NKp30+GFP, NKG2D+DAP10+GFP, CD244+GFP, and DAP10+GFP, respectively. NK cell transfection was performed by adding CART/mRNA complex that contains 100 ng of total mRNA into each 500,000 NK cells in a non-tissue culture treated U-bottom 96-well plate. NK cells were cultured overnight after the transfection and co-cultured with autologous HIV infected CD4+ T cells or mock-infected CD4+ T cells at effector-to-target ratio 1:4 for 4-4.5 hours. Analysis of NK cells that were cultured alone indicates the background level of NK cell function. NK cell function elevation due to interaction with CD4+ T cells was evaluated by background subtraction calculated as the percentageo of functional NK cells in the presence of CD4+ T cells minus the percentage of functional NK cells in the absence of CD4+ T cells. The experiment was performed in 2 batches. The first batch contains the fcs files whose names start with 240126, with cells from 3 donors, donor RP47 (donor 3), donor RP48 (donor 4), donor RP50 (donor 6). The second batch contains the fcs files whose names start with 240203, with cells from 3 donors, donor RP44 (donor 2), donor RP49 (donor 5), and donor RP28 (donor 1).