IL-10 induces aberrant deletion of dendritic cells by natural killer cells in the context of HIV infection (original) (raw)
Accumulation of partially mature DC populations in LNs from HIV-infected individuals. Chronic viral infections are associated with declining adaptive immune function (20, 21, 31), including the accumulation of dysfunctional T cells that express inhibitory receptors on their surface, including programmed death receptor 1 (PD-1; ref. 31). However, it is uncertain whether the observed dysfunction is caused by direct effects of chronic viral replication on T cells or indirect mechanisms that act to induce poorly functional T cell responses. It is possible that early changes in the repertoire of DCs may result in the generation of progressively dysfunctional T cell responses, specifically at inductive sites. To test whether chronic HIV infection was associated with changes in DC populations in the peripheral circulation and LNs, we characterized the maturation phenotype of DCs in the peripheral blood (PB) and LNs of 10 individuals within the first year of untreated HIV infection as well as 5 HIV-negative controls. DCs were remarkably different in the PB compared with those in the LNs (Figure 1). Interestingly, DCs in the LNs exhibited significantly higher levels of HLA-DR and HLA class I (HLA-I) expression — indicating that these cells had received maturation signals — compared with those in PB (P = 0.001 and P = 0.007, respectively; Figure 1A). However, despite elevated levels of HLA molecules, these cells expressed significantly fewer of the costimulatory molecules CD40 and CD83 in the LNs of HIV-infected versus HIV-negative individuals (P = 0.001 and P = 0.01, respectively; Figure 1B). These data suggest that untreated early HIV infection is associated with aberrant accumulation of DCs that pass NK cell quality control because of their normal levels of MHC-I and -II, but exhibit a more immature/tolerogenic phenotype as a result of reduced levels of costimulatory molecules. These data potentially reflect earlier selection of aberrant DCs in vivo as a result of differential deletion of particular DC populations in the LN, or because of novel recruitment of these aberrant DCs after exposure to viral replication.
Accumulation of aberrant DC populations in the LNs of HIV-infected individuals. Shown are mean ± SEM of (A) HLA-DR and HLA-I (i.e., HLA-A, -B, or -C) and (B) costimulatory molecules CD40, CD83, CD80, and CD86 on the surface of CD3–CD14–CD56–CD11c+ DCs in PBMCs and LNs from HIV-infected and HIV-negative donors. *P < 0.05.
IL-10 modulates HLA-I expression on DCs. Central to the immunologic dysfunction observed in chronic viral infections, such as HIV, HCV, and LCMV, are persistent elevated levels of the immunoregulatory cytokine IL-10 (20, 32). Previous work has demonstrated that in vitro culture of both immature and mature DCs with IL-10 results in profound phenotypic changes (30). To characterize the effect of IL-10 on DC expression of MHC-I, we cocultured immature and mature monocyte-derived DCs in the presence or absence of IL-10 for 2 days and then measured MHC-I surface expression. As expected, MHC-I expression was significantly lower on immature than mature DCs in the absence of IL-10 (P = 0.0002; Figure 2, A and B). In the presence of IL-10, mature DCs lost MHC-I expression (P = 0.008); however, MHC-I level increased on the surface of IL-10–treated immature DCs compared with untreated immature DCs (P = 0.02; Figure 2, A and B). Thus, IL-10 has opposite effects on MHC-I expression levels on immature and mature DCs, potentially rendering them differentially susceptible to NK cell–mediated lysis.
IL-10 reverses DC subset susceptibility to NK cell–mediated lysis via altered MHC-I expression levels. (A) Changes in MHC-I (i.e., MHC-A, -B, or -C) expression on the surface of immature and mature monocyte-derived DCs in the presence of medium alone or IL-10. (B) Box and whisker plot denotes median and range of MHC-I expression on immature and mature DCs in the presence or absence of IL-10 (n = 6 per group). (C) Mean ± SEM percent lysis of immature or mature DCs at 1:10, 1:20, and 1:50 effector/target ratios with NK cells after coculture with medium alone or with IL-10 (using 6 independent donors).
IL-10 modifies DC susceptibility to NK cell–mediated deletion. Given the profound differences in MHC-I expression on immature and mature DCs after treatment with IL-10, we tested the hypothesis that these changes render IL-10–treated DCs differentially susceptible to NK cell–mediated lysis. Thus, IL-10–treated and untreated immature and mature chromium-labeled DCs were cocultured with autologous NK cells for 6 hours. As reported previously, immature DCs were susceptible to NK cell–mediated lysis, whereas mature DCs were resistant to lysis (Figure 2C). After IL-10 treatment, immature DCs became resistant to NK cell–mediated elimination at all effector/target ratios compared with the untreated immature DCs (P < 0.05, all comparisons; Figure 2C). In contrast, IL-10 treatment rendered mature DCs susceptible to elimination by autologous NK cells compared with untreated mature DCs (P < 0.05, all comparisons; Figure 2C). Thus, it is likely that IL-10–induced changes in MHC-I expression renders immature DCs resistant while rendering mature DCs susceptible to elimination by autologous NK cells.
IL-10 skews DCs toward a tolerogenic phenotype. In addition to changes in MHC-I expression, we determined whether IL-10 treatment of immature and mature DCs affects the expression of other maturation markers. Similar to its effects on MHC-I, IL-10 slightly increased MHC-II expression on immature DCs, while downregulating it on mature DCs (Figure 3, A and B), as previously reported (30). Although MHC-II expression on immature DCs did not reach the levels expected based on ex vivo DCs observed in the LNs of HIV-infected individuals (Figure 1), in vitro treatment of DCs resulted in the upregulation of this molecule on these cells, which typically express little MHC-I. Thus, it is possible that in vivo immature DCs may be generated with a greater dynamic range of MHC-II expression, allowing NK cells to specifically select for those with higher levels of this molecule on their surface, or that additional signals in the LN may stimulate enhanced expression of MHC-II, but not of other molecules. Moreover, increasing concentrations of IL-10 appeared to increase the expression of MHC-II on the surface of immature DCs (Supplemental Figure 1; available online with this article; doi:10.1172/JCI40913DS1). This indicates that at higher concentrations of IL-10 — which may occur in particular microenvironments — immature DCs may upregulate this molecule to a greater extent. Furthermore, IL-10 treatment of immature DCs resulted in reduced expression of CD86, which suggests that despite the increase in HLA-DR, these DCs do not upregulate the required costimulatory molecules expressed on fully mature immunogenic DCs. Conversely, IL-10 stimulation of mature DCs resulted in reduced CD11c, CD83, CD86, CD40, and HLA-DR expression. Furthermore, IL-10 was able to alter both immature and mature DC phenotypes at even 100-fold lower concentrations of the immunoregulatory cytokine (Supplemental Figure 1), demonstrating the substantial impact of IL-10, even at low doses, on the phenotype of DCs.
IL-10 treatment of immature and mature DCs results in a more tolerogenic DC phenotype. (A) Expression pattern of a range of phenotypic markers on the surface of immature and mature monocyte-derived DCs in the presence of medium alone or IL-10. (B) Mean ± SEM of the phenotypic markers for all 5 experiments. *P < 0.05.
Recently, 2 negative regulators of T cell function, programmed death ligand 1 (PD-L1) and PD-L2, have been implicated in regulating the quality of T cell responses induced by DCs. We therefore monitored IL-10–treated immature and mature DCs for changes in these 2 molecules. Immature DCs significantly upregulated PD-L1 (P = 0.004) and exhibited an overall shift in the expression of PD-L2 after treatment with IL-10 (Figure 3, A and B). Interestingly, PD-L1 and PD-L2 were both downregulated on mature DCs after treatment with IL-10. Thus, IL-10 treatment results in a partially mature tolerogenic phenotype on immature DCs.
NKG2D-dependent elimination of IL-10–treated DCs. NK cell–mediated elimination of target cells is controlled by a delicate balance of inhibitory and activating signals (19). The loss of inhibitory signals tips the balance away from inhibition, but is not sufficient to induce killing by an NK cell; a second activating signal is critically required to induce the functional killing of the target cell. The activating C-type lectin NKG2D has been implicated in NK cell–mediated control of several viral infections and tumors (33). Given the multifunctional role of this activating NK cell receptor, we examined whether it could also be involved in activation of NK cells by IL-10–treated DCs. We stained IL-10–treated or untreated immature and mature DC populations with an NKG2D-fusion construct. The NKG2D-fusion construct did not bind to either of the immature DC populations (Figure 4A), which suggests that IL-10 was not inducing NKG2D ligands on the surface of immature DCs. However, the NKG2D-fusion construct bound to the IL-10–treated mature DC population, but not the untreated mature DCs, which demonstrated that NKG2D ligands are upregulated on the surface of mature DCs only after IL-10 treatment.
NK cells eliminate IL-10–treated mature DCs in an NKG2D-dependent manner, which is blocked by IL-10R blockade. (A) Binding capacity of an NKG2D-fusion construct and expression of MIC-A/B on immature and mature DCs in the presence of medium alone or IL-10. (B) Coexpression levels of MIC-A/B and MHC-I on the surface of immature or mature DCs in the presence and absence of IL-10. (C) Mean ± SEM capacity of NK cells to lyse IL-10–treated mature DCs in the absence or presence of an NKG2D-blocking antibody (n = 5). (D) Mean ± SEM percent lysis of mature DCs by autologous NK cells after maturation in the presence of medium alone, IL-10 alone, and IL-10 plus IL-10R–blocking antibody — either during maturation in vitro (culture) or during the NK cell/DC killing assay (killing). *P < 0.05; **P < 0.01.
NKG2D binds to a number of different stress-inducible ligands, including MHC-1 homolog A (MIC-A), MIC-B, and UL-16–binding proteins (ULBP-1–ULBP-3; ref. 33). To identify whether MIC-A or -B were upregulated on the surface of IL-10–treated mature DCs, we stained IL-10–treated and untreated immature and mature DCs with antibodies against MIC-A/B. Although the antibody did not bind to either of the immature DC populations, it did bind to the IL-10–treated mature DCs, but not to IL-10–treated immature DCs (Figure 3B). These data suggest that the MIC-A and MIC-B are upregulated on mature, but not immature, DCs after treatment with IL-10.
As the upregulation of MIC-A/B was modest on the whole IL-10–treated mature DC population, we sought to ascertain whether there was any relation between DCs that upregulate MIC-A/B expression and those that lose MHC-I expression, as these would represent the most vulnerable population of DCs for NK cell–mediated elimination. We costained the different DC populations for MIC-A/B and MHC-I simultaneously in the presence or absence of IL-10 in 4 separate individuals (Figure 4B). The level of MHC-I increased remarkably on the surface of IL-10–treated immature DCs, in the absence of any changes in MIC-A/B levels (Figure 4B). In contrast, MHC-I expression declined dramatically in nearly half the mature DCs in the presence of IL-10, of which nearly half potently upregulated MIC-A/B (Figure 4B). These data suggest that the NKG2D ligands MIC-A and MIC-B are upregulated specifically on IL-10–treated mature DCs that have lost MHC-I expression.
NK cell–induced lysis of IL-10–treated mature DCs is mediated by NKG2D. The above data demonstrated that IL-10 treatment of DCs results in the upregulation of NKG2D ligands and downregulation of MHC-I on the surface of mature DCs. To define the potential role of these NKG2D ligands in NK cell–mediated lysis of autologous IL-10–treated mature DCs, we attempted to measure the involvement of NKG2D in NK cell–mediated DC killing by blocking this receptor with a blocking antibody. Whereas IL-10–treated mature DCs were lysed efficiently, NKG2D blockade prior to coculture with autologous IL-10–treated mature DCs resulted in a nearly 80% reduction in NK cell–mediated killing of autologous DCs (P = 0.0001; Figure 4C). Thus, NKG2D-dependent activation of NK cells constitutes a principal mechanism by which NK cells recognize and eliminate IL-10 treated mature DCs.
IL-10 receptor blockade during coculture restores mature DC resistance to NK cell–mediated elimination. To definitively determine whether IL-10 is responsible for modulating DC susceptibility to NK cell–mediated elimination, as well as to define at which step IL-10 blockade reverses mature DC susceptibility to NK cell–mediated elimination, mature DCs were treated with an IL-10 receptor–blocking (IL-10R–blocking) antibody, either during maturation in vitro or during the NK cell/DC killing assay. Although IL-10R blockade had no impact on mature DC elimination during the chromium release assay, treatment of mature DCs with the IL-10R–blocking antibody during the maturation process dramatically reduced DC susceptibility to elimination by autologous NK cells (Figure 4D). These data strongly suggest that IL-10 renders mature DCs susceptible to NK cell–mediated lysis indirectly via alterations in DC phenotype rather than through potentiation of NK cell–mediated lysis of DCs during coculture.
HIV alters DC maturation and phenotype analogously to IL-10. To assess whether the observed differences in IL-10–mediated DC phenotypic modifications are also observed after DC coculture with HIV, we characterized the surface expression of the above markers on both immature and mature monocyte-derived DCs cultured in the presence or absence of an R5 virus. Coculture with HIV induced partial maturation of immature DCs, resulting in elevated levels of CD83, HLA-DR, and HLA-I expression, but not CD80 or CD86 expression, on immature DCs (Figure 5A). Remarkably, this phenotype of HIV-treated immature DCs was similar to the observed DC populations in the LNs of HIV-infected individuals (Figure 1). In contrast, HIV-treated mature DCs exhibited reduced expression of CD83, CD86, HLA-DR, and HLA-I (Figure 5A). Additionally, both immature and mature DCs upregulated PD-L1 after coculture with HIV. Furthermore, coculture of DCs with HIV resulted in potent secretion of IL-10 by both subsets of DCs (Figure 5B), as previously described (34), and supernatant from these cocultures induced similar changes in DC phenotype (data not shown). These data suggest that HIV-induced modulation of DC phenotype may be attributable in part to autocrine IL-10–induced effects.
Coculture of DCs with HIV results in IL-10 secretion and an altered DC phenotype. (A) Expression patterns of phenotypic markers on the surface of immature and mature monocyte-derived DCs in the presence of medium alone or HIV JRCSF. (B) Range of IL-10 secretion measured by ELISA in the supernatant of immature and mature DCs cocultured in the presence of or absence of HIV JRCSF (n = 5). (C) Mean ± SEM percent lysis of immature and mature DCs at 1:10, 1:20, and 1:50 effector/target ratios with NK cells after coculture with HIV (using 5 independent donors).
To determine whether monocyte-derived DC exposure to HIV renders immature and mature DCs differentially susceptible to NK cell–mediated elimination, we performed a similar NK cell/DC killing assay in the presence of HIV. Immature and mature DCs were cocultured for 2 days in the presence of HIV, then placed in coculture with autologous NK cells. As we showed for IL-10 treatment of DCs, HIV pretreatment resulted in an inverted susceptibility of DCs for elimination by NK cells: immature DCs were resistant to elimination, whereas mature DCs were robustly eliminated by autologous NK cells (Figure 5C). These data demonstrate that HIV itself, which induces IL-10 secretion from DCs, can reverse DC susceptibility to NK cell–mediated elimination.