Reduced NK cell activity and a decrease in NK cell numbers have been implicated in HIV disease progression [2
] and their function is known to be augmented by exogenous cytokines, particularly IL-15 [31
]. We have recently demonstrated that IL-21 is a potent inducer of perforin in CD8+ T cells and that this activity is independent of CD8+ T cell proliferation [33
]. We tested the hypothesis that the cytokine IL-21 would have similar effects on NK cells as on T cells in HIV-infected patients. Our data shows that IL-21 increases expression of perforin in NK cells, enhances degranulation and induces NK cell cytotoxicity without augmenting NK cell proliferation.
Among NK cell subsets, the CD56 negative subset is expanded in viremic HIV-infected subjects, but following control of viremia with ART, it decreases to a low frequency that is the norm in healthy HIV-uninfected persons [2
]. In aviremic HIV-infected people on ART, the CD56dim
NK subset is described to be defective [2
]. As all subjects in this study were on potent antiretroviral therapy and were virologically controlled (plasma HIV RNA <50 copies/mL) and immunologically stable (CD4 counts >200), we focused our attention on CD56dim
NK subset. This NK cell subset was significantly decreased in peripheral blood of patients as compared to healthy control subjects. The CD56bright
NK cell subset was well preserved, and even increased in some patients. One potential explanation for the maintenance of CD56bright
and decrease of CD56dim
NK cell subset could be that these two distinct subsets of mature NK cells are differentially affected by HIV-1, possibly due to alterations in the cytokine milieu. The development of CD56bright
NK cells is dependent on IL-15 [12
], which is mainly produced by monocytes. On the contrary, the development of CD56dim
NK cells is dependent upon IL-21 [12
], which is produced by CD4+ T lymphocytes. It could be hypothesized that CD4 T cell deficiency negatively influences IL-21 production whereas IL-15 activity is sustained in HIV-infected patients. The positive effect of IL-15 on the CD56bright
NK cell population was evident in our study. There was an increase in CD56bright
cells and a concomitant decrease in CD56dim
NK cells following culture of PBMC with IL-15. This effect of IL-15 may represent a maturation induced transition of CD56dim
phenotype to CD56bright
NK phenotype as described for IL-2 [16
] or might result from proliferation of CD56bright
cells, as IL-15 induces NK cell proliferation. The IL-21 effect on NK cells was distinctly different from that of IL-15. Cell cultures with IL-21 led to minimal changes in frequency of CD56dim
NK cells without significant cellular proliferation. As the CD56dim
subset is the primary NK cell subset with cytolytic properties, we examined perforin expression in both NK subsets. Perforin content was higher in the CD56dim
subset as compared to the CD56bright
subset, supporting its increased cytolytic potential. Although perforin-expressing CD56dim
NK cells from HIV-infected individuals and uninfected subjects were comparable, short-term culture with IL-21, but not IL-15, augmented perforin expression in this subset. Both, IL-21 and IL-15 were found to upregulate perforin expression in the CD56bright
subset of NK cells. Ex vivo culture with IL-15 and IL-21 resulted in modulation of effector functions of NK cells as tested by coculturing them with target MHC-devoid K562 cells. This was manifested by an increase in surface CD107a expression, intracellular content of perforin and IFN-γ, and cytotoxicity in NK cells. CD107a is located within membrane-bound lytic lysosomal vesicles containing proteins such as granzymes and perforin, and up-regulation of CD107a has been shown to occur in synchrony with secretion of perforin [36
]. We have shown that NK cells from HIV-infected individuals express more CD107a on their surface following stimulation with K562 than uninfected individuals. This finding is in agreement with Alter et al
] who found that CD107a on NK cells is elevated in viremic HIV-infected individuals and represents a practical marker of NK activity in HIV infection. Furthermore, we found that both IL-15 and IL-21 induced CD107a expression in NK cells mediating cytotoxicity in healthy and HIV-infected individuals. These results suggest that both cytokines not only increase perforin content, but they also induce efficient release of cytotoxic granules and subsequent target cell killing. However, IL-15 induced cytotoxicity was more potent and was accompanied by selective induction of NKG2D, a well-known activating receptor on NK and CD8+ T cells (data not shown).
As was true with CD8+ T cells, IL-21 had a minimal effect in inducing proliferation whereas IL-15 had a potent effect on NK cell proliferation. This observation is consistent with the known proliferative effect that IL-15 has on NK cells [8
]. Notably, proliferation of NK cells of HIV-infected individuals was higher in comparison to uninfected controls. Moreover, cell division patterns for HIV-infected samples were different from uninfected samples. This finding is reminiscent of common γ–chain cytokine effects on CD8+ T cells [32
] of HIV-infected subjects which are more responsive to cytokine-induced proliferation than cells of healthy donors.
Perforin gene activation has been linked to STAT3 and STAT5 activation in NK cells [29
] and to STAT5 activation in CTL [30
] via upstream enhancers of the perforin gene. Binding of IL-21 to the IL-21 receptor (IL-21R) results in the activation of STAT proteins, which translocate to the nucleus and initiate transcription of IL-21-responsive genes. IL-21-induced expression of IFN-γ in NK cells and T cells has been shown to be dependent upon the activation of STATs [11
]. In agreement with Roda et al
] we found that IL-21 induced activation of STAT3 and STAT5, but not STAT4 as reported by Strengell et al.
]. Interestingly, as was true for T cells, IL-15 predominantly activated STAT5 in CD3negCD56+ NK cells from healthy and HIV-infected individuals, while IL-21 activated STAT5 and STAT3 [33
]. Furthermore, IL-21-stimulated NK cells from HIV-infected individuals demonstrated higher phosphorylation of STAT3 than uninfected subjects. STAT5 phosphorylation was also higher in HIV-infected individuals, correlating with greater perforin induction by IL-21 in patient cells compared to control cells.
IL-21 is currently proposed as an adjuvant in cancer immunotherapy [39
]. Our results suggest that the IL-21 needs to be investigated further for its ability to modulate immune responses in HIV-infected individuals.