CD4+ NK cells can be productively infected with HIV, leading to downregulation of CD4 expression and changes in function

Abstract

NK cells mediate the innate immune response, and HIV-infected individuals demonstrate altered NK cell phenotype and function. We find that CD4+ NK cells are susceptible to HIV infection; this could account for the NK cell dysfunction seen in HIV-infected individuals. CD4+ NK cells express CXCR4 and can be infected with X4-tropic viruses and some primary R5-utilizing viral isolates. Treatment with the CXCR4 ligands AMD3100 and SDF-1alpha partially blocks infection with X4-tropic virus, treatment with anti-CCL Igs upregulates CCR5 surface expression and enables infection with HIV-Bal. HIV infection of NK cells results in CD4 downregulation and the production of infectious virus. HIV-infected CD4+ NK cells mediate NK cell cytotoxicity, however, HIV infection is associated with decreased chemotaxis towards IL-16. Thus, HIV infection of CD4+ NK cells could account for the NK cell dysfunction observed in HIV-infected individuals. Furthermore infected NK cells could serve as a viral reservoir of HIV in vivo.

Fig. 1

HIV co-receptor expression on stimulated NK cells. Purified NK cells were stimulated for 14 days with IL-2, IL-12 and PHA and stained with fluorochrome-labeled monoclonal Abs to CD56, CD4, and either CXCR4 or CCR5. Flow cytometry was performed on these samples, gating included live cells expressing CD56. We found that 86% of our gated cells expressed CXCR4 (black population) in our histogram analysis (isotype in gray). CCR5 expression was minimal (black population), at less than 3% compared to our isotype control (gray population). Results are representative of at least 5 independent experiments.

Fig. 2

HIV infection of stimulated NK cells. Purified, stimulated NK cells were either mock infected (top) or challenged with either HIV-IIIB or HIV-Bal. Post-infection day 7 aliquots of cells were permeabilized and stained with fluorochrome-labeled mAbs in the following combination: CD56-PE, KC57-FITC (p24 gag), CD4-APC, and 7AAD staining was performed to exclude dead cells. The dot plots represent cells gated based on size and 7AAD exclusion. The plot at the far right represents a gated subset of p24 expressing cells (gate shown on dot plot to the immediate left, with arrow). Results are representative of at least 5 independent experiments.

Fig. 3

HIV infection of stimulated NK cells. Purified, stimulated NK cells were either mock infected) or infected with HIV. Post-infection day 5 aliquots of cells were stained with fluorochrome-labeled mAbs in the following combination: CD56-PE, KC57-FITC (p24 gag), CD3-PerCP, and CD4-APC. The histograms demonstrate KC57 (p24 gag) staining cells are gated based on size and CD56 staining, all cells were CD3 negative. HIV-IIIB, BZ167, QZ4589 and 96USHIPS9 challenged cells reveal bimodality, signifying a KC57 positive subpopulation, whereas mock infected and HIV-Bal histograms are unimodal.

Fig. 4

Treatment with anti-CCL antibodies results in enhanced CCR5 expression and permits infection with HIV-Bal. Purified NK cells were cultured in the presence of an excess of anti-CCL Igs for 3 days followed by challenge with HIV-Bal. At four days post-infection cells were stained with CCR5-PE, KC57-FITC (p24 gag), CD56-ECD, and CD4-APC. The histograms demonstrate KC57 (p24 gag) and CCR5 staining, anti-CCL Ig treated cells are white and untreated cells are black. Dot plots show CD4 and KC 57 expression in mock infected and HIV-Bal challenged NK cells (in the presence or absence of anti-CCL Igs), the percent of KC57 cells is shown in the top right of each dot plot.

Fig. 5

HIV-IIIB Infection of CD4+ NK cells is mediated by the CXCR4 coreceptor. Purified NK cells were cultured in the presence of AMD3100 or SDF-1α followed by challenge with HIV-IIIB. At four days post-infection cells were stained with CD56-PE, KC57-FITC (p24 gag), and CD4-APC. Dot plots show CD4 and KC 57 expression in mock infected and HIV-IIIB challenged NK cells some of which were treated with AMD3100 or SDF-1α. The percent of KC57 cells is shown in the top right of each dot plot.

Fig. 6

Kinetics of HIV replication in CD4+ NK cells. Purified, stimulated NK cells were infected with HIV-IIIB at an MOI of 0.1. KC 57 expression was quantitated via flow cytometric analysis and cell culture supernatants were removed and assayed for the presence of p24 at the indicated days post infection. Data shown is representative of a duplicate infection, some conditions were cultured in the presence of AZT following a 2 hour viral attachment period. Results are representative of 3 experiments.

Fig. 7

HIV-infected NK cells mediate NK cell cytotoxicity. CD4 expressing NK cells were infected with HIV-IIIB (or mock infected) and at 5 days post-infection viable cells were incubated with ⁵¹Cr-labeled K562, 2F7, Ramos, or Daudi cells for four hours using effector: target ratios ranging from 0.625 - 10: 1. ⁵¹Cr release from target cells into cell culture supernatants was quantitated to measure NK cell cytotoxicity. Total release (targets lysed with 0.5 % triton) and spontaneous release (no effector cells) controls were performed with each assay and % cytotoxicity was calculated by comparing samples to the total release (100%) and spontaneous release (0%) controls. Each condition was assayed in triplicate and averages are shown. HIV-infected NK cells had diminished cytotoxic activity against Daudi cells at E:T ratios 2.5 and below, *P=0.01, +P=0.05, when compared to uninfected samples from the same donor. These data are representative of three independent experiments performed with separate donors.

Fig. 8

HIV infection decreases CD4+ NK cell migration towards IL-16. HIV-IIIB infected or mock infected CD4+ NK cells (day 5 post-infection) at a concentration of 1×10⁶ cells/ml were placed in the upper chamber of a Costar transwell plate and allowed to migrate for four hours in response to 200ng/ml IL-16 or 20ng/ml SDF-1α. Results are the percentage of cells migrating compared with the medium only controls (spontaneous migration, set at 100%). Cell migration was quantified by counting cells in the lower chamber of the chemotaxis chamber at the conclusion of the migration period. Triplicate wells were performed for each condition, and averages were calculated. Data shown is representative of the results obtained following three independent experiments using NK cells derived from different donors. HIV-infection of NK cells was associated with a statistically significant decrease in migration towards the cytokine IL-16 (p=0.05).