Defective plasmacytoid dendritic cell-NK cell cross-talk in HIV infection

Abstract

HIV viremia is associated with a wide range of immune dysfunctions that contribute to the immunocompromised state. HIV viremia has been shown to have a broad effect on several immune cell types and/or their interactions that are vital for mounting an effective immune response. In this study, we investigated the integrity of plasmacytoid dendritic cell (pDC)-NK cell interactions among HIV viremic, aviremic, and seronegative individuals. We describe a critical defect in the ability of pDCs from HIV-infected individuals to secrete IFN-alpha and TNF and subsequently activate NK cells. We also describe an inherent defect on NK cells from HIV-infected individuals to respond to pDC-secreted cytokines. Furthermore, we were able to demonstrate a direct effect of HIV trimeric gp120 on NK cells in vitro similar to that described ex vivo. Finally, we were able to establish that the HIV gp120-mediated suppressive effect on NK cells was a result of its binding to the integrin alpha(4)beta(7) expressed on NK cells. These findings suggest a novel mechanism by which HIV is capable of suppressing an innate immune function in infected individuals.

FIG. 1.

NK cells from HIV-infected individuals failed to respond to CpG activation. (A) Levels of CD69 expression were measured in HIV-seronegative, HIV-viremic, and HIV-aviremic individuals. pDC-mediated stimulation of NK cells, as determined by CD69, was lower in both HIV-viremic and aviremic individuals compared to HIV seronegatives (p < 0.001). Levels in viremic and aviremic individuals were similar (p = 0.2). (B) PBMCs from all three groups of individuals were treated with blocking receptor for IFN-α and a blocking antibody for TNF. The results showed a significant decrease in CD69 expression with the addition of the antibodies, indicating that IFN-α and TNF are primarily responsible for pDC-mediated activation of NK cells. (C) To further understand the cause for this decrease in activation, PBMCs from each of the three groups of individuals were treated with or without IFN-α, TNF, and IL-12. Whereas NK cells from all three groups responded to the cytokines, NK cells from viremic and aviremic individuals responded less when compared to those from the HIV-seronegative group (p < 0.001 for each of the three cytokines). The level of CD69 on NK cells in PBMCs without any stimulation was subtracted from the levels obtained after stimulation with each cytokine.

FIG. 1.

NK cells from HIV-infected individuals failed to respond to CpG activation. (A) Levels of CD69 expression were measured in HIV-seronegative, HIV-viremic, and HIV-aviremic individuals. pDC-mediated stimulation of NK cells, as determined by CD69, was lower in both HIV-viremic and aviremic individuals compared to HIV seronegatives (p < 0.001). Levels in viremic and aviremic individuals were similar (p = 0.2). (B) PBMCs from all three groups of individuals were treated with blocking receptor for IFN-α and a blocking antibody for TNF. The results showed a significant decrease in CD69 expression with the addition of the antibodies, indicating that IFN-α and TNF are primarily responsible for pDC-mediated activation of NK cells. (C) To further understand the cause for this decrease in activation, PBMCs from each of the three groups of individuals were treated with or without IFN-α, TNF, and IL-12. Whereas NK cells from all three groups responded to the cytokines, NK cells from viremic and aviremic individuals responded less when compared to those from the HIV-seronegative group (p < 0.001 for each of the three cytokines). The level of CD69 on NK cells in PBMCs without any stimulation was subtracted from the levels obtained after stimulation with each cytokine.

FIG. 2.

CpG-induced stimulation of PBMCs from HIV-infected individuals results in lower levels of IFN-α and TNF secretion. IFN-α (A) and TNF (B) levels were measured from supernatants collected 24 h after treatment with CpG. The changes in IFN-α and TNF levels were significantly less in viremic and aviremic individuals compared to the changes in seronegative individuals (p < 0.001). Baseline levels of IFN-α present in the supernatants were not statistically different among HIV-negative, HIV-viremic, and aviremic subjects (mean 45.3 pg/ml, 38.4 pg/ml, and 33.95 pg/ml, respectively, p > 0.05). The change in the TNF levels was greater in aviremic than in viremic individuals (p = 0.05). The change in IFN-α was not (p = 0.15).

FIG. 3.

Expression of interferon-inducible genes (IFIGs) in PBMCs from viremic, aviremic, and seronegative individuals was measured as described in Materials and Methods. PBMCs from HIV viremic and aviremic individuals expressed significantly higher levels of IFIGs when compared to those in PBMCs from HIV-negative individuals (p < 0.01 and 0.02, respectively). The Y axis is log₂ expression of IFIG genes, which represents fold changes.

FIG. 4.

HIV gp120-mediated interference of pDC-NK cell cross-talk involves direct binding to the integrin α₄β₇ receptor on NK cells. We performed experiments of pDC-NK cell interactions in the presence or absence of trimeric HIV gp120 as described below. (A) PBMCs from healthy normal volunteers were treated with trimeric HIV gp120 (1 μg/ml) for 1 h at 37°C. The cells were washed and treated with CpG (1 μg/ml) overnight. CD69 expression on NK cells was determined by flow cytometry as described in Materials and Methods. Exposure to HIV gp120 markedly reduces the ability of NK cells to be activated, while gp120 by itself does not activate NK cells. (B) Purified pDCs and NK cells from healthy normal volunteers were isolated as described in Materials and Methods. Both pDCs and NK cells were exposed to trimeric HIV gp120 (1 μg/ml) for 60 min. Afterward the cells were washed and cocultured as follows. HIV untreated pDCs and NK cells with or without CpG (1 μg/ml), HIV gp120 treated or untreated pDCs with HIV gp120, and untreated or treated NK cells with or without CpG (1 μg/ml). The suppression of CD69 expression is calculated as a percentage of CD69 expression seen on NK cells with experimental conditions appropriate to that seen on NK cells cocultured with pDCs treated with CpG. Results indicate both cell types are affected by exposure to HIV gp120. (C) We performed experiments similar to those described in (B); when NK cells were pretreated with α₄ integrin antibody that blocks HIV gp120 binding to NK cells, there was a complete reversal of the suppressive effects of HIV gp120.

FIG. 4.

HIV gp120-mediated interference of pDC-NK cell cross-talk involves direct binding to the integrin α₄β₇ receptor on NK cells. We performed experiments of pDC-NK cell interactions in the presence or absence of trimeric HIV gp120 as described below. (A) PBMCs from healthy normal volunteers were treated with trimeric HIV gp120 (1 μg/ml) for 1 h at 37°C. The cells were washed and treated with CpG (1 μg/ml) overnight. CD69 expression on NK cells was determined by flow cytometry as described in Materials and Methods. Exposure to HIV gp120 markedly reduces the ability of NK cells to be activated, while gp120 by itself does not activate NK cells. (B) Purified pDCs and NK cells from healthy normal volunteers were isolated as described in Materials and Methods. Both pDCs and NK cells were exposed to trimeric HIV gp120 (1 μg/ml) for 60 min. Afterward the cells were washed and cocultured as follows. HIV untreated pDCs and NK cells with or without CpG (1 μg/ml), HIV gp120 treated or untreated pDCs with HIV gp120, and untreated or treated NK cells with or without CpG (1 μg/ml). The suppression of CD69 expression is calculated as a percentage of CD69 expression seen on NK cells with experimental conditions appropriate to that seen on NK cells cocultured with pDCs treated with CpG. Results indicate both cell types are affected by exposure to HIV gp120. (C) We performed experiments similar to those described in (B); when NK cells were pretreated with α₄ integrin antibody that blocks HIV gp120 binding to NK cells, there was a complete reversal of the suppressive effects of HIV gp120.