uNK cells that inhibited HIV-1 infection produced CXCL12 and not IFN-γ. (A) Cell-free uNK CM from clones that demonstrated anti-HIV- activity (+) or lacking anti-HIV-1 activity (−) were analyzed for IFN-γ. There was a negative correlation between anti-HIV-1 activity and amounts of IFN-γ. ***, P < 0.001 (n > 6 uNK clones). (B) CM from uNK and bNK clones were analyzed for the presence of CXCL12. There was a positive correlation between CXCL12 production and anti-HIV-1 activity. ***, P < 0.001 (n = 8). Data are shown as means ± SEM. (C) Comparison between amounts of CXCL12 and IFN-γ in uNK CM. These data show that uNK CM with large amounts of CXCL12 had small amounts of IFN-γ and vice versa. Statistical analysis was performed using the Mann-Whitney U test.

Neutralizing CXCL12 abrogates uNK cell anti-HIV activity. (A) uNK clones were stimulated with IL-12 plus IL-15 for 3 days, and uNK CM (black bars) or culture medium (open bars) was preincubated with or without CXCL12 neutralizing antibodies or the isotype control (IgG). This mixture was then added to TZM-bl cells for 1 h before infecting the cells with HIV-1_IIIB. β-Gal expression was determined after 48 h. (B) uNK CM from four different individuals were treated in four different experiments as in panel A, and the data were normalized to media (M). Values represent means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 4). The y axis shows RLU values, converted into percentages with “media” as 100%. Statistical analysis was performed using the Mann-Whitney U test.

Anti-HIV-1 activity of CM from uNK cells is increased by cytokine activation. (A) CM from uNK and bNK clones collected after 3 days of stimulation with IL-12 and IL-15 (S) or in the absence of cytokine stimulation (U) were preincubated for 1 h with TZM-bl cells prior to the addition of HIV-1_IIIB. The percentage of inhibition of HIV-1 infection was determined by comparing RLU values with those of the medium control. Each data point represents an individual clone, and the average of each group is shown. Stimulated uNK clones showed significantly higher inhibition than unstimulated uNK clones (**, P < 0.01; n = 31), and CM from bNK clones did not inhibit HIV-1_IIIB (n = 10). These data represent uNK clones from 15 individuals and bNK clones from 6 individuals. (B) Comparison of anti-HIV activities between CM from paired IL-12- plus IL-15-stimulated and unstimulated uNK clones. These data show that stimulation of uNK cells resulted in an increase in HIV-1 inhibitory activity. ***, P < 0.001.

CM from uNK cells inhibit infection of PBMC and primary FRT cells by HIV-1. (A) PHA-activated PBMCs were incubated with media or with CM from IL-12- plus IL-15-stimulated uNK cells (uNK CM), followed by infection with HIV-1 (IIIB +). Following infection, supernatant was collected from the PBMC cultures on days 3, 5, and 7 and added to TZM-bl cell cultures to quantify the amount of infectious HIV-1. After 48 h of incubation with TZM-bl cells, β-Gal activity was determined. Controls included supernatants from uninfected PBMCs (−). These data are representative of three independent experiments and are means ± SEM (n = 4). ***, P < 0.001. (B) PHA-activated endometrial cells from human uterine tissues were treated as in panel A, and cell-free supernatants collected on days 3 and 5 were tested for the presence of infectious HIV using TZM-bl reporter cells. Results are shown as means ± SEM (***, P < 0.001) and represent three independent experiments, and samples from different donors were used in each experiment.

Human uNK cells produce soluble factors with anti-HIV activity. (A) uNK cells were cultured with or without IL-12 plus IL-15 for 3 days. The resulting cell-free uNK CM was incubated with TZM-bl cells for 1 h and then infected with HIV-1_IIIB at an MOI of 1. β-Gal activity was measured after 48 h. Results are means ± SEM of a representative experiment in which each condition was tested in quadruplicate. Media (open bars) and uNK CM (black bars), in the absence (−) or presence of HIV-1_IIIB (IIIB), are shown. uNK CM significantly reduced HIV-1 infection in TZM-bl cells. ***, P < 0.001. (B) uNK CM from four different individuals in three different experiments were treated in quadruplicate as in panel A and infected with either HIV-1_IIIB or HIV-1_BaL (BaL). The data are normalized to RLU from the medium-plus-virus (HIV-1_IIIB or HIV-1_BaL) conditions and show that the uNK anti-HIV activity is specific for HIV-1_IIIB. ***, P < 0.001 (Mann-Whitney test). Values represent means ± SEM (n = 7 different uNK clones). ns, not significant. The y axis shows RLU values, converted into percentages with media as 100%. (C) CM from primary NK cells were treated in quadruplicate as in panel B. Data are representative of two experiments with two different individuals. (D) TZM-bl cells were preincubated for 1 h with tissue culture media (open bars), bNK CM (hatched bars), or uNK CM (black bars). bNK and uNK CM were prepared from the blood and endometrial tissue of the same individual. One hour later, TZM-bl cells were infected with the indicated X4-tropic HIV-1 strains. After 48 h in culture, β-gal activity was measured. Data are expressed as the percentage of β-gal expression relative to the media control. Values represent means ± SEM, with n = 4 different clones. ***, P < 0.001 (one-way analysis of variance).