Phagocytosis of a Model Human Immunodeficiency Virus Target by Human Breast Milk Leukocytes Is Predominantly Granulocyte-Driven When Elicited by Specific Antibody

Abstract

Background: Studies demonstrate a protective effect of antibodies (Abs) in breast milk (BM) against motherto-child transmission (MTCT) of human immunodeficiency virus (HIV). Contribution of the BM cellular component has been overlooked. The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with Abs mediating functions through the constant immunoglobulin region-the crystallizable fragment (Fc). These data support induction of vaccine Abs triggering antiviral activities by leukocytes through Fc receptors (FcRs). Objective: To measure Ab-dependent cellular phagocytosis (ADCP), an essential Fc-mediated response, by BM phagocytes. Materials and Methods: Cells were isolated from five human BM samples obtained at 7-183 days postpartum and analyzed for ADCP. Fluorescent beads coated with HIV envelope (Env) epitopes were used as targets. Sixty-seven to 100 mL of milk was utilized.Results: Total cell concentrations per milliliter were 16,083-222,857, with 1.6-12.3% being CD45 + leukocytes. ADCP activity was measurable using the HIV-specific Ab 830A. Use of the actin inhibitor cytochalasin D and FcR blocker indicated that ADCP was actin dependent and required FcR engagement. ADCP scores were variable, but largely consistent, across the samples studied, exhibiting <4-fold difference from lowest to highest activity for CD45 + cells. Of the CD45 + ADCP, significantly more activity was granulocyte derived (72-95%), while the remaining activity was monocyte driven. Conclusions: The data indicate that BM phagocytes can manifest antiviral activities in the presence of specific Abs and therefore may contribute to reduction of MTCT of HIV.

Impact of IgG isotype on the induction of antibody-dependent cellular phagocytosis of HIV by human milk leukocytes

“…In the present study, granulocytes were the most potently induced phagocyte against the virus target for most IgG isotypes, consistent with our previous findings using bead-based targets (32).…”

“…We have undertaken a study to assess the ADCP capacity of human milk leukocytes against HIV. Our previous pilot study confirmed milk leukocytes as capable of ADCP of HIV Env gp120-coated microbeads, and demonstrated that most activity was granulocyte-driven (31,32).…”

“…This primary milk cell assay was adapted from Ackerman et al (38) and has been described previously using latex beads (32). Milk samples were centrifuged at 600g for 15 min, cell pellet was washed 2x with Hank's balanced salt solution (HBSS) without calcium and magnesium with centrifugation at 350g for 10 min.…”

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

“…We have undertaken a study to assess the ADCP capacity of human milk leukocytes against HIV. Our previous pilot study confirmed milk leukocytes as capable of ADCP of HIV Env gp120-coated microbeads, and demonstrated that most activity was granulocyte-driven ( 40 , 41 ). In the present study we report our analysis of the ADCP activity of milk leukocytes in 57 unique samples from 34 women, elicited by recombinant IgG1/2/3/4 of monoclonal (m)Ab 246-D, a weakly-neutralizing Env gp41 (cluster 1)-specific Ab - the type of Ab commonly elicited by natural HIV infection.…”

“…This primary milk cell assay was adapted from Ackerman et al. ( 47 ) and has been described previously using latex beads ( 41 ). Milk samples were centrifuged at 600 g for 15 min, cell pellet was washed 2x with Hank’s balanced salt solution (HBSS) without calcium and magnesium with centrifugation at 350g for 10 min.…”