Impaired Downregulation of NKG2D Ligands by Nef Proteins from Elite Controllers Sensitizes HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

Abstract

HIV-1 Nef clones isolated from a rare subset of HIV-1-infected elite controllers (EC), with the ability to suppress viral load to undetectable levels in the absence of antiretroviral therapy, are unable to fully downregulate CD4 from the plasma membrane of CD4⁺ T cells. Residual CD4 left at the plasma membrane allows Env-CD4 interaction, which leads to increased exposure of Env CD4-induced epitopes and increases susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated largely by natural killer (NK) cells, which control their activation status through the cumulative signals received through activating and inhibitory receptors. Recently, the activating NKG2D receptor was demonstrated to positively influence ADCC responses. Since HIV-1 Nef has been reported to reduce the expression of NKG2D ligands, we evaluated the relative abilities of Nef from EC and progressors to downmodulate NKG2D ligands. Furthermore, we assessed the impact of EC and progressor Nef on the ADCC susceptibility of HIV-1-infected cells. We observed a significantly increased expression of NKG2D ligands on cells infected with viruses coding for Nef from EC. Importantly, NKG2D ligand expression levels correlated with enhanced susceptibility of HIV-1-infected cells to ADCC. The biological significance of this correlation was corroborated by the demonstration that antibody-mediated blockade of NKG2D significantly reduced ADCC of cells infected with viruses carrying Nef from EC. These results suggest the involvement of NKG2D-NKG2D ligand interactions in the enhanced susceptibility of EC HIV-1-infected cells to ADCC responses.IMPORTANCE Attenuated Nef functions have been reported in HIV-1 isolated from EC. The inability of elite controller Nef to fully remove CD4 from the surface of infected cells enhanced their susceptibility to elimination by ADCC. We now show that downregulation of NKG2D ligands by HIV-1 Nef from EC is inefficient and leaves infected cells susceptible to ADCC. These data suggest a critical role for NKG2D ligands in anti-HIV-1 ADCC responses.

Keywords: ADCC; CD4; HIV-1; NKG2D; Nef; elite controllers; gp120.

FIG 1

Attenuated CD4 downregulation by Nef alleles from elite controllers enhances exposure of ADCC-mediating epitopes. Primary CD4⁺ T cells from healthy donors were infected with a panel of pNL4.3-based viruses: wild type (wt; coding for Nef_SF2), lacking Nef (N−), or encoding Nef from 18 and 19 randomly selected clones from ECs or CPs, respectively. Infected primary CD4 T cells were stained at 48 h postinfection with an anti-CD4 (OKT4) (A and B) or A32 (C and D) antibody, with HIV⁺ sera from 7 different donors (shown in different colors) (E and F), or with the conformation-independent 2G12 antibody (G and H). On the left are histograms depicting representative staining obtained with wt versus Nef-infected cells. On the right, the median fluorescence intensities (MFI) obtained for multiple staining using cells infected with viruses coding for Nef from ECs (red) or CPs (blue) are shown. Error bars indicate means ± standard errors of the means (SEM). Statistical significance was evaluated using unpaired t test. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 2

Attenuated downregulation of NKG2D ligands by Nef alleles from elite controllers. Primary CD4⁺ T cells from healthy donors were infected with a panel of pNL4.3-based viruses: wild type (wt; coding for Nef_SF2), lacking Nef (N−), or encoding Nef from 18 and 19 randomly selected clones from ECs or CPs, respectively. Infected primary CD4 T cells were stained at 48 h postinfection with 5 μg/ml of a recombinant NKG2D-Fc chimera or matched IgG Fc molecules and then fluorescently labeled with an Alexa Fluor 647-conjugated anti-human IgG secondary Ab. (A and B) Histogram (A) and graph (B) depicting representative staining of infected (p24⁺) cells with mock (gray), wt (blue), and N− (in red) virus. (C) Recognition of infected (p24⁺) cells by viruses coding for Nef from ECs (red) or CPs (blue) with NKG2D-Fc. Data shown are the results of four different experiments, and error bars depict the SEM. Statistical significance was tested using paired one-way analyses of variance (ANOVAs) and unpaired t test (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

FIG 3

Attenuated downregulation of NKG2D ligands by Nef alleles from elite controllers enhances the susceptibility of infected cells to ADCC mediated by A32 and HIV⁺ sera. Primary CD4⁺ T cells from 5 healthy individuals infected with a panel of pNL4.3-based viruses, wild type (wt; coding for Nef_SF2), lacking Nef (N−), or encoding Nef from at least 18 randomly selected clones from ECs or CPs, were used at 48 h postinfection as target cells using a previously reported FACS-based ADCC assay (11, 21) to determine their susceptibility to ADCC by autologous PBMCs. ADCC mediated by A32 (A and C) or HIV⁺ sera from two to seven different donors (B and D) is shown. Data shown are the results from at least six different experiments, with means ± SEM. Statistical significance was tested using a paired or unpaired t test (*, P < 0.05; **, P < 0.01, ***, P < 0.001; ****, P < 0.0001; ns, not significant).

FIG 4

Enhanced levels of CD4 and NKG2D ligands at the surface of HIV-1-infected cells correlate with enhanced ADCC. The presence of CD4 and NKG2D ligands at the surface of HIV-1-infected cells enhances the sensitivity of infected cells to ADCC mediated by HIV⁺ sera and A32 monoclonal antibody. CD4 expression (A) and the presence of NKG2D ligands (B) on the surface of cells infected with viruses encoding Nef from at least 18 randomly selected clones from ECs (in red) or CPs (in blue) correlated positively with ADCC killing mediated by HIV⁺ sera and A32 antibody. (C) No statistically significant correlation was observed between cell surface levels of CD4 and NKG2D ligand expression. Statistical analysis was tested utilizing a Spearman rank correlation.

FIG 5

Nef-mediated CD4 and NKG2D ligand downregulation modulates susceptibility of HIV-1-infected cells to ADCC. Nef clones isolated from CPs provide protection to infected cells from ADCC by downregulating CD4 and NKG2D ligands from the surface of infected cells (left panel). Impaired abilities of Nef clones from ECs to reduce surface levels of CD4 and NKG2D ligands expose Env CD4i epitopes targeted by ADCC-mediating Abs and contribute to strengthening the activation of NK cells (right panel).