Isolation and characterization of HIV-1-infected resting CD4+ T lymphocytes in breast milk.
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.
Petitjean
Gaël
G
Laboratoire de Virologie, Hôpital Lapeyronie, CHU de Montpellier, 371 Avenue du Doyen Giraud, 34295 Montpellier Cedex 5, France.
Becquart
Pierre
P
Tuaillon
Edouard
E
Al Tabaa
Yassine
Y
Valea
Diane
D
Huguet
Marie-France
MF
Meda
Nicolas
N
Van de Perre
Philippe
P
Vendrell
Jean-Pierre
JP
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology / Q000821:virology; D002470:Cell Survival / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015658:HIV Infections / Q000276:immunology* / Q000473:pathology / Q000635:transmission / Q000821:virology; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D018445:Infectious Disease Transmission, Vertical; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology; D008895:Milk, Human / Q000276:immunology* / Q000821:virology*