Antibody-Dependent Cellular Phagocytosis of HIV-1-Infected Cells Is Efficiently Triggered by IgA Targeting HIV-1 Envelope Subunit gp41

Abstract

Antibodies mediate a broad array of non-neutralizing Fc-mediated functions against HIV-1 including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Accordingly, ADCC and ADCP induced by anti-HIV envelope gp120 IgG have been correlated to the limited success of the HIV-1 phase III vaccine trial RV144. It remains elusive whether ADCP can also be triggered by IgA, the isotype predominant at mucosal surfaces through which HIV-1 is mainly transmitted. Yet, we have previously shown that the HIV envelope subunit gp41-specific broadly neutralizing antibody 2F5 under the IgA isotype (2F5-IgA) triggers ADCC and cooperates with 2F5-IgG to increase HIV-1-infected cell lysis. Here, we now demonstrate that 2F5-IgA, more efficiently than 2F5-IgG, induces ADCP not only of gp41-coated beads but also of primary HIV-1-infected cells in a FcαRI-dependent manner. Both primary monocytes and neutrophils can act as effector cells of 2F5-IgA-mediated ADCP, although with different kinetics with faster neutrophil phagocytosis. However, unlike for ADCC, 2F5-IgA and 2F5-IgG do not cooperate to increase ADCP. Altogether, our results reveal that gp41-specific IgA mediate the efficient phagocytosis of HIV-1-infected cells. Inducing such ADCC and ADCP-prone IgA response by vaccination in addition to anti-HIV envelope IgG, might increase the protection against HIV acquisition at mucosal level.

Keywords: ADCP; FcαRI/CD89; HIV-1 envelope gp41; IgA; monocytes; neutrophils; phagocytosis.

Figure 1

Anti-gp41 2F5-IgA and gp41-specific IgG induce ADCP of P1-coated beads in a FcαRI- and FcαRI-dependent manner. (A) 2F5-IgA (red bar) and gp41-specific IgGs (blue bars) mediate phagocytosis of P1-coated beads: Fluorescent beads were coated with P1, pre-incubated with the antibodies for 30 min at 37°C prior to addition to primary monocytes for phagocytosis for 3hrs at 37°C. gp120-specific 2G12-IgG (dark blue bar) or irrelevant human IgA2/IgG were used as negative controls. The P1-coated bead uptake was analyzed by flow cytometry and HIV-1 specific phagocytic score was calculated as indicated in the Method section. Values represent means of P1-specific phagocytic score obtained after the subtraction of the background phagocytosis levels induced by human IgA/hIgG ± SEM from 3 independent experiments performed in triplicate, ***p < 0.001, and, unpaired Student's t-test. (B) ADCP depends on FcαRI or FcγRI: Monocytes were pre-incubated with either anti-FcαRI (CD89) or anti-FcγRI (CD64) blocking antibody for 30 min at RT before addition to the immune complexes. Phagocytic score was calculated as in (A). Values represent mean inhibition of phagocytic score in the presence of monocytes preincubated with anti-CD89 or anti-CD64 antibody ± SEM. **p < 0.01, ***p < 0.001 unpaired Student's t-test compared to ADCP in the absence of blocking antibodies.

Figure 2

Anti-gp41 2F5-IgA triggers more efficient ADCP of P1-coated beads compared to anti-gp41 2F5-IgG and 4E10-IgG in the ADCP-SHIP assay. Genuine ADCP of beads triggered by gp41-specific IgA and IgG was evaluated by the ADCP-SHIP method, discriminating surface-attached beads from actual intracellular phagocytosed beads. (A) ADCP-SHIP method validated using the monocytic THP-1 as effector cells. Fluorescent neutravidin beads coated with Cy5-labeled fluorescent internalization probe (FIP_Cy5) and gp41 subunit P1 were incubated with anti-gp41 2F5-IgG (blue bar), and 4E10-IgG (light blue bar), anti-gp120 2G12-IgG (dark blue bar) or irrelevant human IgA2 or IgG prior to addition to the THP-1 effector cells for 3 h at 37°C. Cy5 fluorescence of surface-bound beads was quenched by incubation with the complementary quenching probe (QP_C). ADCP was evaluated by flow cytometry and calculated as follows: % of FITC⁺ Cy5⁺ cells with antibody—background % of FITC⁺ Cy5⁺ cells without antibody. ADCP % in the presence of control human IgA/hIgG was subtracted from ADCP % triggered by HIV-1 specific antibody. Values represent means of P1-specific beads ADCP % ± SEM from 3 independent experiments performed in triplicate. NS: p > 0.05, unpaired Student's t-test. (B) Primary monocyte-mediated ADCP is more efficiently triggered by 2F5-IgA than gp41-IgGs. 2F5-IgA (red bar), 2F5-IgG (blue bar) and 4E10-IgG (light blue bar) were assayed by the ADCP-SHIP method using primary monocytes as effector cells. When indicated, monocytes were pre-treated with 10 μg/ml of Cytochalasin D (CytoD) for 30 min at 37°C or left untreated and added to beads opsonized with the IgA/IgG Abs. Values represent means of P1-specific beads ADCP % ± SEM from at least 3 independent experiments performed in triplicate, NS p > 0.05, **p < 0.01, ***p < 0.001, unpaired Student's t-test. (C) Characterization of the efficiency of bead phagocytosis by ADCP-SHIP. The number of beads phagocytosed by each cell triggered by 2F5-IgA/IgG or the control hIgA/hIgG was evaluated from the intensity of Cy5 fluorescence associated to each cell. Light, median, or dark red/blue represent the percentage of cells having phagocytosed 1, 2, or >2 beads (i.e., between 3 and 7). Values represent means of P1-specific ADCP distribution % ± SEM, from 3 independent experiments performed in triplicate, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student's t-test.

Figure 3

2F5-IgA mediates ADCP of HIV-1-infected primary CD4⁺T lymphocytes by monocytes more efficiently than 2F5-IgG and 2G12-IgG. Primary CD4⁺T-cells were infected with JR-CSF clade B HIV-1 for 72 h and incubated with (A) various concentrations of 2F5-IgA or hIgA2, (B) 0.2 μg/ml, or (C) 0.5 μg/ml of 2F5-IgA, 2F5-IgG, 2G12-IgG, or hIgA/IgG for 30 min at 37°C. Primary monocytes were stained with 0.1 μM intracellular CellTracker™ Deep Red Dye, added to opsonized infected target cells and allowed to phagocyte for 3 h at 37°C. Cells were washed, fixed in PFA, permeabilized before intracellular staining with anti-p24 FITC-coupled Ab and analyzed by flow cytometry. ADCP percentage is determined as indicated in the Method section. In (A,B), non-specific ADCP mediated by hIgA2 are shown. In (C), background ADCP % obtained in the presence of hIgA/hIgG was subtracted from ADCP % triggered by HIV-1 specific antibody. Values represent means of HIV-1-specific infected cells ADCP % ± SEM, from 4 independent experiments performed in triplicate, NS p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student's t-test.

Figure 4

2F5-IgA-mediated ADCP of HIV-1-infected primary CD4⁺T lymphocytes by neutrophils. Primary CD4⁺T cells were infected with JR-CSF clade B HIV-1 for 72 h and incubated with various concentrations of 2F5-IgA or hIgA2 for 30 min at 37°C. Primary neutrophils were pre-stained with 0.1 μM intracellular CellTracker™ Deep Red Dye, added to opsonized infected target cells and allowed to phagocytose for indicated times. Cells were washed, fixed, permeabilized, stained intracellularly with anti-p24 FITC Ab and analyzed by flow cytometry. ADCP percentage in each condition is determined as indicated in the Method section. Values represent means of HIV-1-specific infected cells ADCP % ± SEM, from 3 independent experiments performed in triplicate, **p < 0.01, unpaired Student's t-test.