Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Summary

Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~10⁵−10⁸ maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

Abstract

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~10⁵−10⁸ maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Introduction

Human breast milk (BM) is comprised of maternal cells that are >90% viable¹. Cell composition is impacted strongly by stage of lactation, health status of mother and infant, and individual variation, which remains poorly understood^1,2,3,4. Given that BM contains ~10³−10⁵ leukocytes/mL, it can be estimated that breastfed infants ingest ~10⁵−10⁸ maternal leukocytes daily⁵. Various in vivo studies have demonstrated that maternal leukocyte....

Protocol

Each participant in this study was recruited and interviewed in accord with the ethical and institutional review board (IRB) approval with the guidance and authorization of Mount Sinai’s Program for the Protection of Human Subjects (PPHS) using an IRB-approved protocol for obtaining breast milk samples.

1. Target Microsphere preparation

1. Select a relevant target antigen.
   NOTE: In this example, the recombinant protein V1V2-2F5K was used, which was designed to mimic the trimeric apex of native HIV envelope³⁰.
2. Perform biotinylation using a commercial ki....

Results

Milk can be kept at room temperature or cooler (though not frozen); however, given that we have observed reduced viability when milk has been kept very cold (data not shown), and that it is simpler to collect, store briefly, and transport at ambient temperatures, it is recommended that samples are not refrigerated in order to reduce sample-to-sample variability. In milk obtained 7−183 days post-partum, cell concentration determined by automated cell counter ranged from 16,083−222,857 cell.......

Discussion

The flow cytometry-based technique for measuring ADCP activity described herein was first described in 2011³⁰ and has since been proven robust and cited in more than 80 studies. The protocol described here adapts this technique for use with primary BM cells for the first time. Previous studies of Fc-mediated functionality by BM cells have been largely limited to measurement of oxidative bursts or histology-based phagocytosis assays using cells isolated from colostrum (0−4 days after birth). .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 µm FluoSpheres NeutrAvidin-Labeled Microspheres	Thermo Fisher	F8776
BD Pharmingen PE Mouse Anti-Human CD45	BD	560975
Bovine serum Albumin	MP Biomedicals	8810025
Corning V-bottom polystyrene 96-well plate	Corning	3894
Cytochalasin D	Sigma	22144-77-0
EZ-Link NHS-LC-LC-Biotin kit	Thermo Fisher	21338
Falcon 15 mL Conical Centrifuge Tubes	Corning	352196
Falcon 50 mL Conical Centrifuge Tubes	Corning	352070
Fixable Viability Stain 450	BD	562247
Formaldehyde solution	Sigma	252549
HBSS without Calcium, Magnesium or Phenol Red	Life Technologies	14175-095
Human BD Fc Block	BD	564219
Human Serum Albumin	MP Biomedicals	2191349
Kimtech Science Kimwipes Delicate Task Wipers	Kimberly-Clark Professional	34120
PBS 1x pH 7.4	Thermo Fisher	10010023
Polystyrene 10 mL Serological Pipettes	Corning	4488
Protein Concentrators PES, 3K MWCO, 0.5 mL	Pierce	88512