Isolation and characterization of HIV-1-infected resting CD4 + T lymphocytes in breast milk

Abstract

An HIV-1 reservoir comprised primarily of latently infected resting CD4 + T lymphocytes that can be stimulated in vivo to produce virus may play a critical role in mother-to-child postnatal transmission of HIV-1 by breastfeeding. Here, we describe an experimental protocol for the detection of resting CD4 + T cell HIV-1 reservoir from breast milk. We adapted a method for the purification of resting CD4 + T lymphocytes in blood to isolate resting CD4 + T cells in breast milk from HIV-1-infected-lactating women ( n = 18) and from controls ( n = 3). Purified resting CD4 + T cells from blood and breast milk samples of HIV-1-infected-lactating women were polyclonally stimulated to characterize and enumerate HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) by an enzyme-linked immunospot (ELISpot) assay. Resting CD4 + T cells represented more than 90% of purified viable breast milk cells. CD4 + T cell polyclonal stimulation combined with the ELISpot assay led to the characterization of a breast milk T cell HIV-1 reservoir greater than the blood reservoir (median 400 and 57.14 HIV-1-Ag-SCs/10 6 resting CD4 + T cells, respectively, p < 0.001). Our strategy could be adapted to other body fluids and be useful for characterizing new HIV-1 reservoirs.