FIG. 2.

NK cells lyse autologous HIV-infected T cells when DNAM-1 and NKG2D are triggered by HIV-infected T cells. (A, B) anti-CD3 and -CD28 antibody-treated primary CD4⁺ T cells were infected with VSV-G-pseudotyped DHIV3 for 3 days. The infected cells were then separated from the uninfected cells using anti-CD4 beads. The unattached cells that remained behind were used as target cells. As a control target cell, we utilized anti-CD3 and -CD28 antibody-treated primary CD4⁺ T cells (white bar). Target cells were labeled with ⁵¹Cr for 2 h and then mixed with NK cells at effector cell to target cell (E:T) ratios of 2.5:1, 5:1, or 10:1. In some groups, NK cells were incubated with blocking antibodies to DNAM-1 (hashed line bar), NKG2D (gray bar), DNAM-1, and NKG2D (horizontal line bar) or control antibody of similar isotype as blocking antibody (black bar). After 4 h, fluids were removed and the remaining ⁵¹Cr in the fluid was measured. Each group was done in triplicate. The mean % specific lysis ±SD was determined as described in Material and Methods section. Each Figure (A, B) represents data from experiments involving two separate donors. (C) Purified NK cells cultured in a medium or 200 U/ml of IL-2 were stained with CD56 and DNAM-1. (D) Similar to A and B except NK cells were treated overnight with 200 U/ml of IL-2 before their use in the ⁵¹Cr release assay and involved a different donor. (E) Ability of NK cells to degranulate when exposed to autologous HIV-infected T cells (NK cell:HIV-infected cell ratio of 2:1) when exposed to anti-DNAM-1, anti-NKG2D, or a combination of both blocking antibody when compared with NK cells exposed to Ig of irrelevant specificity. Each symbol represents a different donor. Bars represent mean percent decrease in frequency of CD107a⁺ NK cells in the presence of blocking antibody to DNAM-1 and/or NKG2D compared to frequency of CD107a⁺ NK cells treated with antibody of irrelevant specificity. The statistical difference between treatment groups was determined using the Mann–Whitney U-test. (F) Degranulation of NK cells from a donor involved in the study presented in (E) following exposure to target cells in the presence of control of blocking antibodies. NK cell degranulation following exposure to control target cells (i.e., uninfected CD4+ T cells and K562 cells) is displayed. (G) Degranulation of NK cells when triggered with antibodies to DNAM-1, NTB-A, NKG2D, or a combination of the various antibodies. Antibody-labeled P815 cells were treated with NK cells for 4 h and the extent to which the NK cells degranulated was measured by determining the frequency of NK cells that expressed CD107a on their surface. Each figure represents data from experiments involving three separate donors. NK, natural killer.