Human immunodeficiency virus-specific CD8(+) T cells in human breast milk

Abstract

Breast-feeding infants of human immunodeficiency virus (HIV)-infected women ingest large amounts of HIV, but most escape infection. While the factors affecting transmission risk are poorly understood, HIV-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in controlling HIV levels in blood. We therefore investigated the ability of breast milk cells (BMC) from HIV-infected women from the United States and Zambia to respond to HIV-1 peptides in a gamma interferon enzyme-linked immunospot assay. All (n = 11) HIV-infected women had responses to pools of Gag peptide (range, 105 to 1,400 spot-forming cells/million; mean = 718), 8 of 11 reacted to Pol, 7 reacted to Nef, and 2 of 5 reacted to Env. Conversely, of four HIV-negative women, none responded to any of the tested HIV peptide pools. Depletion and tetramer staining studies demonstrated that CD8(+) T cells mediated these responses, and a chromium-release assay showed that these BMC were capable of lysing target cells in an HIV-specific manner. These data demonstrate the presence of HIV-specific major histocompatibility complex class I-restricted CD8(+) CTLs in breast milk. Their presence suggests a role in limiting transmission and provides a rationale for vaccine strategies to enhance these responses.

Figures

FIG. 1.

HIV-1-specific immune responses to overlapping 20-mer peptide pools from Gag, Pol, Env, and Nef of HIV-1; tetanus toxoid (TT); or CMV lysate as measured by the IFN-γ ELISPOT assay. (A) BMC from HIV-seronegative women. (B) BMC responses from HIV+ women (U.S. cohort). (C) BMC responses from HIV+ women (Zambian cohort). Fifty peptides each, comprising the N terminus (Pol N aa 1 to 510) or the C terminus (Pol C aa 501 to 1003) of Pol were used instead of a pool of 100 peptides (Pol aa 1 to 1003) to stimulate BMC from the Zambian volunteers. Asterisks indicate that for volunteer 1 the responses from Pol N and Pol C were added and plotted as Pol, and the same was done for Env using gp120 and gp41. Responses to CMV and TT were not measured in volunteers 4 and 5. PHA responses were >1,000 SFC/million BMC (data not shown).

FIG. 2.

Depletion of CD8+ T cells markedly diminishes responses to the HIV peptide pools but not that to PHA. BMC or CD8-depleted BMC obtained from volunteer 2 were stimulated with medium or with HIV peptide pools from Gag, Pol, or Nef in an IFN-γ ELISPOT assay.

FIG. 3.

Breast milk-derived CTL are capable of lysing infected targets. Fresh BMC from volunteer 12 were stimulated for 3 days with peptide pools from Gag, Pol, and Nef and mixed with autologous BLCL infected with vSC8 (vaccinia virus control) or vP1291-GDR (vaccinia virus expressing Env, Gag, Pol, and Nef epitopes) at the indicated E:T ratios.

FIG. 4.

Breast milk-derived cells stain with HLA class I tetramer reagents. HIV-1 Gag p17 (aa 17 to 28; RLRPGGKKK) HLA A∗0301-restricted tetramer staining. (A) BMC from an HLA A3+ HIV-seronegative woman. (B) BMC from volunteer 2. (C) PBMC from volunteer 2. For tetramer analysis, cells were gated by using three parameters (forward scatter versus side scatter and CD3+ T cells) in order to focus on the CD3+ T-cell population. The percentage of CD8+ T cells that stained with the tetramer is given in the upper right quadrant of each dot plot.

FIG. 5.

Comparison of HIV-specific responses between PBMC and BMC. PBMC and BMC were obtained from HIV+ volunteer 2 (A), HIV+ volunteer 3 (B), HIV+ volunteer 4 (C), and HIV+ volunteer 5 (D) and stimulated with peptide pools overnight in an IFN-γ ELISPOT assay. Net spots shown, i.e., cells incubated with medium alone as a negative control antigen, were subtracted from the values shown (control values of 0 to 25 SFC/million PBMC and 0 to 175 SFC/million BMC). Note that PBMC contain ca. 80% T cells and BMC contain ca. 10% T cells and values are not normalized for lymphocyte content in each compartment.