Activation of NK cells by HIV-specific ADCC antibodies: role for granulocytes in expressing HIV-1 peptide epitopes

Abstract

HIV-specific ADCC antibodies could play a role in providing protective immunity. We have developed a whole blood ADCC assay that measures NK cell activation in response to HIV peptide epitopes. These HIV peptide-specific ADCC responses are associated with escape from immune recognition and slower progression of HIV infection and represent interesting HIV vaccine antigens. However, the mechanism by which these epitopes are expressed and whether or not they induce NK-mediated killing of cells expressing such peptide-antigens is not understood. Herein, we show that fluorescent-tagged ADCC peptide epitopes associate with blood granulocytes. The peptide-associated granulocytes become a specific target for antibody-mediated killing, as shown by enhanced expression of apoptosis marker Annexin and reduction in cell numbers. When HIV Envelope gp140 protein is utilized in the ADCC assay, we detected binding to its ligand, CD4. During the incubation, cells co-expressing gp140 and CD4 reduce in number. We also detected increasing Annexin expression in these cells. These data indicate that blood cells expressing HIV-specific ADCC epitopes are targeted for killing by NK cells in the presence of ADCC antibodies in HIV+ plasma and provide a clearer framework to evaluate these antigens as vaccine candidates.

Keywords: ADCC; Apoptosis; Granulocytes; HIV; NK cells.

Figure 1. Peptide-based NK cell activation ADCC assay. (A) The assay used whole blood samples in which we theorise that granulocytes expressing HIV peptides activate NK cells in the presence of peptide-specific ADCC antibodies in HIV⁺ plasma. The ADCC response is measured by intracellular IFNγ expression (previously shown to correlate with CD107 degranulation¹³) by NK cells in the presence of HIV+ plasma and Vpu peptide ADCC epitope at the end of 5 h incubation at 37°C by flow cytometry. (B) Gating strategy: lymphocytes are identified on forward and side scatter; CD3^-CD56⁺ NK cells are then analyzed for intracellular IFNγ expression. The assay was performed using cells from 9 different HIV negative controls (C1-C9, see Table 1) in the presence of plasma from one HIV+ subject S1; plots are shown for C1 and C9 compared with S1. The value in each plot represents the percentage of CD56+ NK cells expressing IFNγ in the presence of HIV+ plasma following stimulation by Vpu peptide antigen compared with an unstimulated control.

Figure 2. Granulocytes express ADCC epitopes and are targets for killing. (A) Fluorescently labeled Vpu peptide bind to granulocytes. The ADCC epitope Vpu peptide 19 (sequence EMGHHAPWDVDDL) was conjugated with FITC fluorochrome. The fluorescent Vpu peptide induced robust ADCC activity in the presence of HIV+ plasma (top right plot). Based on FSC and SCC criteria, cells expressing fluorescent peptide localized primarily to granulocytes (bottom right plot). (B) Loss of CD66c⁺ granulocytes (bottom right plot) occurs in the presence of HIV+ plasma during the 5 h incubation following stimulation by Vpu peptide compared with a control peptide (sequence KKFGAEVVPC) that has no known ADCC inducing ability (top right plot). (C) Granulocytes are the major cells that undergo apoptosis in the NK cell activation ADCC assay in the presence of HIV+ plasma. Decrease in CD66c⁺ granulocytes (top right graph) but not CD56⁺ NK cells (top left graph) occurred over time. Annexin V staining indicated that CD66c⁺ granulocytes were undergoing apoptosis (bottom right graph) compared with CD3⁺ T cell populations over time. Cell loss and Annexin expression in the presence of Vpu peptide (black bars) was compared with a control peptide (white bars). (D) Across 5 donors, granulocyte numbers reproducibly fell in the presence of HIV+ plasma following Vpu peptide stimulation (circles) compared with control peptide stimulation (triangles; mean and SD shown). (E) Granulocyte loss does not occur in the absence of other leukocytes. Granulocytes numbers decrease in the presence of HIV+ plasma when the ADCC assay is performed in whole blood containing NK cells (2nd bar) but not in the presence of purified granulocytes alone (4th bar). Loss of granulocytes in the presence of Vpu peptide (black bars) was compared with incubation in the presence of a control peptide (white bars).

Figure 3. Higher ADCC Activity is observed in the presence of granulocytes. Autologous granulocytes were added back to PBMC and assessed for their ability to restore ADCC activity. Whole blood (WB), PBMCs (isolated by ficoll gradient, P-G) and PBMCs with autologous granulocytes (isolated by percoll gradient) added back (P+G) were analyzed by flow cytometry. (A) Granulocyte population for the 3 assay conditions circled. (B) Depletion of granulocytes markedly reduces IFNγ expression by CD56 NK-cells in the presence of HIV+ plasma after stimulation with Vpu peptide (top middle plot) compared with whole blood (top left plot); this response is significantly restored by adding back granulocytes (top right plot); a control peptide showed no responses (bottom plots). (C) Across 6 donors, there is a significant decrease in IFNγ expression by NK-cells in the presence of HIV+ plasma during the ADCC assay when granulocytes were depleted (p = 0.005); this response was nearly completely restored when granulocytes were added back to the incubation (p = 0.002; Mann-Whitney) (D) Shows the same data from C normalized to NK cell activation in the whole blood culture.

Figure 4. Cells co-expressing CD4 and HIV-1 gp140 Envelope protein reduce over time during the NK cell activation ADCC assay. (A) HIV-1 gp140 envelope protein antigen stimulation of the whole blood samples in the NK cell activation ADCC assay in the presence of HIV⁺ plasma induces NK cell IFNγ expression. (B) Cells co-expressing CD4 and gp140 reduced in number over time (% baseline) in the presence of HIV positive serum (right graph) but not in the presence of HIV negative serum (left graph). (C) The reduction in the number of cells co-expressing CD4⁺ and gp140 was associated with an increase in Annexin V expression over time in the same population when incubated in the presence of HIV+ plasma (white bars) compared with HIV- plasma (black bars).