CD4+ T cells spontaneously producing human immunodeficiency virus type I in breast milk from women with or without antiretroviral drugs

Abstract

Background: Transmission of human immunodeficiency virus type 1 (HIV-1) through breast-feeding may involve both cell-free and cell-associated virus. This latter viral reservoir remains, however, to be fully explored. CD4+ T cell-associated virus production in breast milk was therefore investigated.

Methods: The ex vivo spontaneous production of HIV-1 antigen and HIV-1 RNA by CD4+ T cells was measured in paired blood and breast milk samples from 15 HIV-1 infected women treated or not with antiretroviral drugs. Spontaneous antigen secreting cells (HIV-1-AgSCs) from breast milk and blood were enumerated by an ELISpot assay, and cell-associated HIV-1 RNA was quantified by real-time PCR in supernatants of CD4+ T cells cultured for 18 hours without addition of polyclonal activators.

Results: Among the CD4+ T cells present in breast milk, memory cells expressing high levels of cell-surface activation markers were predominant. Spontaneous HIV-1-AgSCs were detected and enumerated in the breast milk of all 15 women, with a median number of 13.0 and 9.5 HIV-1- AgSCs/106 CD4+ T cells in aviremic (n = 7) and viremic (n = 8) women, respectively. Cell- associated HIV-1 RNA was detected in cell-free supernatants from 4/7 aviremic and 5/8 viremic individuals at median levels of 190 and 245 copies/ml, respectively.

Conclusions: Activated CD4+ T cells producing HIV-1 are detected in the breast milk of untreated individuals as well as those receiving highly active antiretroviral therapy. This finding strongly suggests that HIV-1 replication occurs in latently infected CD4+ T cells that, upon spontaneous activation, revert to productively infected cells. These cells might be responsible for a residual breast milk transmission despite maternal highly active antiretroviral therapy.

Figure 1

Representative dot plots from breast milk and blood samples of an HIV-1-infected woman (no 8) (A) Gating strategy to explore breast milk CD4⁺ T cells and CD8⁺ T cells. (B) Analysis of CD38 and HLA-DR cell-surface expression on breast milk CD4⁺ T cells (left) and CD8⁺ T cells (right). (C) CD38 and HLA-DR cell surface expression on blood CD4⁺ T cells (left) and CD8⁺ T cells (right) using the same gating strategy. The percentage of cells positive for both HLA-DR and CD38 staining is given in the upper quadrant of each dot plot.

Figure 2

Detection of ex vivo HIV-1 Ag secreting CD4⁺ T lymphocytes in breast milk and blood. HIV-1 infected CD4⁺ T cells able to spontaneously produce HIV-1 Ag were enumerated by an ELISpot assay aimed at detecting p24 secretion. Spontaneous HIV-1-AgSCs were detected in breast milk cell samples from all the women tested. Dotted line indicates the lower limit of quantification of the test (3 HIV-1-AgSCs/10⁶ CD4⁺ T cells). The number of HIV-1-AgSCs showed no significant difference between individuals in whom plasma HIV-1 RNA was detectable or not nor was any difference found between breast milk and blood compartments (Mann Whitney U test, P > 0.05).

Figure 3

Cell-associated HIV-1 RNA from breast milk and blood derived CD4⁺ T cell culture supernatants. HIV-1 RNA was quantitated in cell-free culture supernatant following 18 hours of incubation. Results from breast milk and blood cells were separated according to the detection of plasma HIV-1 RNA. Dotted line indicates the lower limit of quantification of the test (60 HIV-1 RNA copies/ml). The cell-associated HIV-1 RNA levels were similar between aviremic and viremic individuals in breast milk-derived cells but were lower in blood-derived cells from aviremic individuals by comparison with viremic individuals (Mann Whitney U test).

Figure 4

Co-culture of breast milk- and blood-cell viral-culture supernatants with CD4⁺ T cells. The infectivity of virus secreted into culture supernatants was tested after 18 h of incubation by co culturing with phytohemagglutinin-activated CD4⁺ T cells from healthy blood donors. A) HIV-1 RNA quantification in CD4⁺ T co-culture with breast milk cell supernatants. B) HIV-1 RNA quantification in CD4⁺ T co-culture with blood cell supernatants.