NK cells in blood can directly kill HIV-1-infected targets though cell-to-cell contact or through the secretion of CC chemokines that block CCR5 HIV-1 coreceptors (5
). However, HIV-1 is predominantly transmitted heterosexually (34
), and the primary entry site of virus is through the mucosa of the FRT. Moreover, NK cells present in the FRT are unique and distinct from those found in the peripheral blood (28
). We therefore examined whether uNK cells can mediate anti-HIV activity. In this study, we activated uNK cells with IL-12 and IL-15 as these are proinflammatory mediators which are present within the human endometrium, produced by macrophages during an initial phase of an infection, and have been shown to increase the cytolytic activity of NK cells (11
). Our study demonstrates that uNK cells secrete soluble mediators that exhibit anti-HIV activity for X4-tropic strains of HIV-1 and that this anti-HIV activity correlates with the amount of secreted CXCL12 but not IFN-γ.
Although the anti-HIV effects of bNK cells have been reported by others (26
), the ability of uNK cells from the FRT to secrete soluble mediators that inhibit HIV-1 infection has not been previously described. To the best of our knowledge, this study is the first showing that CM from uNK cells blocks HIV-1 infection of target cells. We found that bNK cell CM did not inhibit HIV infection under similar experimental conditions. In contrast, previous studies have shown that supernatants from NK cell cultures (both primary NK cells and NK cell lines YTS and NK 92) inhibited HIV activation in chronically HIV-infected immune cells (PBMCs) from HIV-infected subjects (46
). In the present study, we used NK cell clones and uninfected TZM-bl cells, PBMCs, and primary endometrial cells to measure the inhibitory effects of HIV infection and found that CM from bNK cells lacked the ability to inhibit HIV-1 infection of target cells. These observed differences between uNK and bNK cells are likely due to the fact that bNK and uNK cells are phenotypically and functionally distinct NK cell subsets (15
). While the anti-HIV-1 activity shown in some uNK clones was modest, a limited number of NK cells were used in these experiments. Moreover, NK cells are present in large numbers in the human FRT (31
); thus, we believe that the anti-HIV activity in vivo will be biologically relevant.
NK cells are present in the human FRT, where they play a critical role during implantation as well as in conferring protection against potential pathogens (14
). Our studies used uNK cells isolated from reproductive tissues from patients undergoing hysterectomy for benign conditions. To obtain sufficient numbers of fresh uNK cells for these studies, the uNK cells were cloned and expanded in the presence of IL-2. Although it is possible for the uNK cell clones to express effector functions that differ from primary uNK cells, we have found that uNK cell clones are quite similar to activated primary uNK cells in many effector functions (15
). In this study, we demonstrated that a significant proportion of the uNK clones secrete soluble mediators that inhibit HIV infection, an activity that is enhanced upon cytokine activation and that is blocked by neutralizing antibody to CXCL12. The ability of anti-CXCL12 antibody to block the anti-HIV activity of uNK CM demonstrated that the anti-HIV activity was likely due to CXCL12. Furthermore, this inhibitory activity was effective against both laboratory-adapted as well as primary X4 HIV-1 strains, but was not effective at preventing an infection with an R5-tropic strain of HIV-1. Moreover, CM from cytokine-activated bNK cells did not have anti-HIV activity against any of the viruses tested. Other studies have shown that noncytolytic bNK cells have anti-HIV activity that is limited to R5, but not X4, HIV-1 strains (17
CXCL12 is the natural ligand for the chemokine receptor CXCR4, and this chemokine can effectively block the entry of X4-, but not R5-tropic viruses. The blocking activity of this chemokine is consistent with our data in which we show that the anti-HIV activity was limited to uNK cells that produced larger amounts of CXCL12 and that the anti-HIV effect was restricted to X4 viruses. Marechal et al. reported that CXCL12 has dual role in HIV-1 in that it can block the entry of the X4 strain of HIV-1 by binding to CXCR4 and also can increase HIV replication through augmenting HIV LTR transcription (29
). Thus, CXCL12 may have competing roles during infection so that a large amount of CXCL12 may block X4-tropic strains, but it may enhance replication of R5-tropic strains of HIV. In addition, there may be other factors present in uNK CM that contribute to inhibition of HIV-1 infection. The data show that CXCL12 is required, but we cannot exclude that other factors may be involved. R5-tropic viruses infect cells based on CCR5 and other cell surface molecules. The mechanisms that regulate how different R5-tropic viruses infect different cell types remain unclear. The data presented showed that uNK cells did not inhibit HIV-1BaL
infection, but it cannot be concluded that uNK cells are unable to affect infection by other R5-tropic viruses. It is possible that uNK cells may prevent (or enhance) infection by other R5-tropic viruses by mechanisms as yet undiscovered.
HIV-1 that is sexually transmitted is typically R5 tropic, although the mechanism responsible for the selective transmission of R5 and the inhibition of X4 is not well characterized. Whether this is due in part to the susceptibility of X4-tropic strains to the host's mucosal immune cell defenses within mucosal tissue sites or to selective virulence of R5-tropic strains is not yet resolved. In contrast to reports by others (37
), our study showed that the presence of large amounts of IFN-γ did not correlate with anti-HIV activity, suggesting that the ability of uNK cells to inhibit HIV is independent of IFN-γ (26
). It is likely that there is heterogeneity within the population of uNK cells, so that some uNK cell clones are able to mediate CXCL12 production, while others can produce IFN-γ. Thus, depending on their differentiation state, some uNK cells may produce CXCL12 and inhibit HIV-1, while others lack the ability to secrete this chemokine. Unique subsets of NK cells with different effector potential have been reported. Recently, a mucosal NK cell subset that expresses NKp44 was found in the tonsil and gut that produce IL-22 but not IFN-γ upon stimulation (8
). Our findings in the present study that uNK cells did not produce IL-22 upon stimulation or express cell surface proteins or transcription factors associated with this unique mucosal NK cell subset suggest that uNK cells are unique with a different effector potential from those found at other mucosal sites. It has been shown that human bNK cells can be differentiated in vitro into cell subsets with different patterns of cytokine secretion: an NK1 subset that releases IFN-γ and an ΝΚ2 subset that secretes type 2 cytokines, including IL-5 and IL-13 (12
In summary, this report demonstrates that uNK cells can be induced to secrete soluble factors that can inhibit HIV-1. This anti-HIV activity is specific for X4-tropic strains, is increased by cytokine activation, and is mediated by CXCL12. These findings imply that uNK cells may play a role in the protection against HIV-1 within the FRT and that modulating uNK cells with inflammatory cytokines may augment this antiviral activity.