Evaluating the role of individual NK cell ligands in HIV-specific recognition by NK cells

In our previous experiments, we performed phenotypic analysis on NK cells that had been co-cultured with HIV-infected or mock-infected CD4+ T cells to identify the NK cell receptors that are predictive of NK cells that are functionally responding to HIV-infected CD4+ T cells. We also analyzed CD4+ T cells for the activating NK cell ligands that are upregulated during HIV infection. We identified 8 candidate receptor-ligand pairs that can potentially contribute to the recognition of HIV by NK cells. In this experiment, we applied CRISPR editing on primary CD4+ T cells to knockout individual genes that encode the candidate ligands, infected the edited CD4+ T cells with HIV, co-cultured autologous NK cells with these CD4+ T cells at effector-to-target ratio 1:1 and evaluated the functional response of NK cells. Reduction in NK cell function compared to the unedited control group would indicate a facilitating role of the knocked-out gene in NK cell recognition during HIV infection.
Conclusion:
In this experiment, we identified 6 individual ligands, including B7-H6, LFA-3, MICA, MICB, ULBP1, and ULBP2 to facilitate the HIV-specific response in autologous NK cells.
Notes:
The functional response of NK cells was evaluated through the staining of functional markers, including CD107a, IFNgamma, and TNFalpha, and functional NK cells are defined as the ones that are stained positive for any of the three markers. The percentage of functional NK cells were analyzed in the groups where NK cells were co-cultured with HIV-infected or mock-infected CD4+ T cells. The level of HIV-specific response was evaluated by subtracting the percentage of functional NK cells when co-cultured with mock-infected CD4+ T cells from the percentage of functional NK cells when co-cultured with HIV-infected CD4+ T cells. HIV-specific response to edited CD4+ T cells was compared to unedited CD4+ T cells, which indicated the role of each NK cell ligand in HIV recognition. In this experiment, we used HIV strain Q23-17 to infect CD4+ T cells (Poss & Overbaugh, 1999). For donor 1 and 2, expression of each ligand was analyzed in the unedited CD4+ T cells and CD4+ T cells that were knocked out of the respective gene.