Impact of IgG Isotype on the Induction of Antibody-Dependent Cellular Phagocytosis of HIV by Human Milk Leukocytes

Abstract

Approximately 100,000 mother-to-child transmission (MTCT) events of HIV via human milk feeding occur each year. However, only about 15% of infants milk-fed by untreated HIV+ mothers become infected, suggesting a protective effect of the milk itself. Infants ingest 10⁵-10⁸ maternal leukocytes daily via milk, which remain functional beyond ingestion. Such function may be elicited by maternal milk antibody (Ab). Though IgA is dominant in milk, most HIV-specific milk Abs are of the IgG subclass, highlighting the importance of investigating the function of each IgG isotype in the milk context. Though Ab effector function mediated by the constant (Fc) domain via interaction with Fc Receptors (FcRs), such as Ab-dependent cellular phagocytosis (ADCP), are critical in protecting against HIV infection, ADCP is largely unexplored as it relates to mitigation of MTCT. Presently we report the ADCP activity of milk leukocytes against HIV particles and immune complexes (ICs), using 57 unique samples from 34 women, elicited by IgG1/2/3/4 of monoclonal (m)Ab 246-D. Granulocyte ADCP of HIV was most potent compared to other phagocytes when elicited by IgG1/3/4. IgG1/3 activated granulocytes similarly, exhibiting 1.6x-4.4x greater activity compared to IgG2/4, and a preference for virus compared to ICs. Notably, CD16- monocyte ADCP of a given target were unaffected by isotype, and CD16+ monocytes were poorly stimulated by IgG1. IgG2/4 elicited potent IC ADCP, and in terms of total leukocyte IC ADCP, IgG4 and IgG3 exhibited similar function, with IgG4 eliciting 1.6x-2.1x greater activity compared to IgG1/IgG2, and CD16+ monocytes most stimulated by IgG2. These data contribute to a more comprehensive understanding of Fc-mediated functionality of milk leukocytes, which is critical in order to develop therapeutic approaches to eliminating this route of MTCT, including mucosal administration of mAbs and/or a maternal vaccination aimed to elicit a potent milk Ab response.

Keywords: ADCP; HIV; IgG; human milk; lactation; mother-to-child HIV transmission; phagocytes; phagocytosis.

Figure 1

Gating strategy. Flow cytometry was performed using an LSR Fortessa (BD) with initial gating on FACSDiva software. Analytical gating was performed using FCS Express.

Figure 2

Sample data. (A) Milk volume collected for each sample. Milk was expressed just prior to sample pickup and used within 4h. (B) Baby’s age/time post-partum of study participant. (C) Total cell concentration for each sample. Milk was centrifuged at 600g for 15 min, and cell pellet washed with HBSS at 350g. Cells were counted, and stained as directed (BD). (D) Percent CD45+ cells of total cell population as determined by flow cytometry. (E) Phagocyte subset populations expressed as percent of total CD45+ cells. CD45+ cells and phagocyte subsets were identified as described in Figure 1 . Two-tailed Students’ t-tests were used to compare phagocyte populations. ***0.0005 ≥ p > 0.0001. ****p = < 0.0001.

Figure 3

ADCP activity measured using milk phagocytes is actin and FcR dependent. 300ng/mL GFP-HIV per well was incubated with titrated IgG1 mAb for 2hrs at 37°C. Meanwhile, 50,000 milk cells per well in a separate plate were prepared, with certain wells pre-incubated with 10ug/mL of the actin inhibitor Cytochalasin-D (Sigma) or 50ug/mL of FcR blocking agent FcBlock (BD) for 30min at 37°C. (A) Virus ADCP; (B) Immune complex ADCP: 100ug/mL anti-human IgG(Fc) was added to virus-Ab complexes and incubated for 30 min at 37°C prior to the addition of cells. Plated cells were washed and incubated with virus/mAb complexes. Plates were washed and incubated in Accutase cell detachment solution, then washed and stained. ADCP activity was measured as the percent of GFP+ cells, with ADCP scores calculated as [(MFI of GFP-positive cells) x (% of GFP+ cells of CD45+ population)]. Data shown was measured for CD45+ population. Mean values of duplicate experiments are shown with SEM.

Figure 4

ADCP activity of 246-D-opsonized virus and ICs as compared by effector cell type. (A) IgG1 data. (B) IgG2 data. (C) IgG3 data. (D) IgG4 data. ADCP was performed as described above. ADCP scores at each mAb dilution were plotted using GraphPad Prism and Area under the Curve (AUC) values were calculated for each experiment. AUC scores were normalized by subtracting those obtained for each cell type using the irrelevant anthrax mAb 3865 of the same class. Each data point represents cells from a unique milk sample. Mean AUC are shown. Two-tailed Mann-Whitney tests were used to compare data. ****p < 0.0001; ***0.0009 > p > 0.0001; **0.009 > p > 0.001; *0.05 > p > 0.01.

Figure 5

ADCP activity of each phagocyte subset as compared by IgG isotype. (A) Virus ADCP. (B) Immune complex (IC) ADCP. Experiments and analyses were performed as in Figure 4 . ****p < 0.0001; **0.009 > p > 0.001; *0.05 > p > 0.01.