Apoptosis in cord blood T lymphocytes from infants of human immunodeficiency virus-infected mothers

Abstract

Apoptosis continues to be controversial in human immunodeficiency virus (HIV)-induced pathogenesis. To investigate whether apoptosis occurs with HIV exposure with or without subsequent infection, levels of apoptosis were measured in cord blood lymphocytes (CBL) from seven newborns delivered to HIV-infected mothers and seven normal, unexposed newborns. Live cells were costained with antibodies to cell surface markers and the DNA dye 7-amino actinomycin D to immunophenotype apoptotic CBL subsets. Apoptosis was measured in fresh and cultured CBL in the presence and absence of CD3 T-cell receptor stimulation. Compared to the CD4+ CBL from HIV-unexposed newborns, CD4+ CBL from six HIV-exposed, noninfected newborns demonstrated significantly greater apoptosis after overnight culture even in the absence of CD3 stimulation. Compared to HIV-unexposed controls, CD8+ CBL from the six HIV-exposed newborns also demonstrated increased levels of apoptosis after overnight culture without stimulation. The one HIV-infected newborn in this study showed the highest levels of CD4+ and CD8+ apoptotic CBL. The data suggest that levels of apoptosis are increased in infants in association with HIV infection. Furthermore, CD4+ and CD8+ cord blood lymphocytes from HIV-exposed infants behaved differently than T lymphocytes from either normal, unexposed infants or an HIV-infected infant. These results suggest that exposure to HIV or HIV-induced factors increases the levels of apoptosis in CBL.

FIG. 1

Measurement of apoptosis in CBL subsets by 7-AAD staining. CBL from an HIV-exposed newborn (A and B) and a control newborn (C and D) were cultured overnight in serum-free medium and immunophenotyped with CD5-FITC/CD4-PE followed by staining with 7-AAD. Gates were set on CD5⁺ CD4⁺ cells (A and C), and forward scatter and 7-AAD fluorescence were displayed on this population (B and D). Cells undergoing apoptosis were defined as 7-AAD^dim+ CD5⁺ CD4⁺, live cells were defined as 7-AAD⁻, and dead cells were defined as 7-AAD^bright+ cells. Percentages of early apoptotic, CD5⁺ CD4⁺ cells were 17 and 7% for the HIV-exposed and nonexposed newborn, respectively.

FIG. 2

Apoptosis in CD4⁺ and CD8⁺ CBL from control and HIV-exposed newborns. Median percent apoptosis is shown for CBL immunophenotyped before in vitro culture (Day 0), after overnight incubation in medium (Day 1-Medium), and after overnight stimulation with CD3 (Day 1-CD3-stimulated). CBL were costained for surface markers and 7-AAD for apoptosis as described in Materials and Methods. Cells undergoing apoptosis were defined as 7- AAD^dim+ CD5⁺ CD4⁺ (A) and as 7-AAD^dim+ CD5⁺ CD8⁺ (B) CBL, respectively, from seven control and six HIV-exposed newborns. The ranges of apoptotic cells were as follows. Ranges for day 0 CD4⁺ control and HIV-exposed infants were 0 to 5.4 and 1.3 to 3.7%, respectively, and those for CD8⁺ control and HIV-exposed infants were 1 to 14.6 and 2.9 to 8.0%, respectively. Ranges for day 1 CD4⁺ control and HIV-exposed infants were 2 to 7.9 and 6 to 24%, respectively, and those for CD8⁺ control and HIV-exposed infants were 2 to 19.6 and 7 to 13.4%, respectively. Ranges for day 1 with CD3 stimulation CD4⁺ control and HIV-exposed infants were 2 to 8.1 and 1.2 to 12%, respectively, and those for CD8⁺ control and CD8⁺ HIV-exposed infants were 2 to 23 and 2.6 to 15.3%, respectively.

FIG. 3

Apoptosis in CD4⁺ and CD8⁺ cord blood T cells of an HIV-infected newborn. Percent apoptosis for both CD5⁺ CD4⁺ and CD5⁺ CD8⁺ cells is shown for CBL phenotyped on day 0, after overnight incubation in medium (Day 1-Medium), and after overnight stimulation with CD3 (Day 1-CD3-stimulated). CBL were costained for surface markers and 7-AAD for apoptosis as described in Materials and Methods. Cells undergoing apoptosis were defined as 7- AAD^dim+ CD5⁺ CD4⁺ or as 7-AAD^dim+ CD5⁺ CD8⁺ CBL, respectively. Note the change in the scale of percent apoptotic cells between Fig. 2 and 3.