T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis

Abstract

Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4(+) T cells are the predominantly infected cells but that terminally differentiated memory CD4(+) T cells contain 10-fold fewer copies of HIV DNA. Memory CD8(+) T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8(+) T cells are not infected preferentially. Naïve CD4(+) T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from naïve CD8(+) T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.

FIG. 1.

Flow cytometric sorting strategy for T cells. PBMC from 16 subjects in the cohort were stimulated with overlapping HIV-peptides stained extracellularly with the antibody combination described in the text and intracellularly for IFN-γ. Lymphocytes were defined with forward and side scatter (I). CD3⁺ T cells were then defined based on expression of CD3 without expression of CD56, CD14, or CD19 (dump) (II). CD4⁺ T cells were then defined based on expression of CD4 without expression of CD8, CD8⁺ T cells were defined based on expression of CD8 without expression of CD4 (III). Naïve CD4⁺ T cells were defined based on dull expression of CD11a, no expression of CD45RO or CD57 with expression of CD27 (IV A). Memory CD4⁺ T cells were defined based on expression of CD45RO with high expression of CD11a. Memory CD4⁺ T cells were then separated based on expression of CD57. Naïve CD8⁺ T cells were defined under the same constraints as naïve CD4⁺ T cells (IV B). Memory CD8⁺ T cells were separated into HIV-specific (production of IFN-γ or tetramer binding) and other memory CD8⁺ T cells.

FIG. 2.

Postsort analysis of naïve CD4⁺ T cells. In order to ensure the purity of sorted populations, each sorted population (when possible) was reanalyzed on the same instrument with the same instrument settings. A representative example is shown. Sorted cells must be defined for lymphocytes because cellular debris results from high-speed sorting. The right four plots are only defined for lymphocytes based on characteristic forward and side scatter. All sorted populations were routinely ≥99.8% pure.

FIG. 3.

CD57⁺ CD4⁺ T cells have less viral DNA than memory CD57⁻ CD4⁺ T cells. PBMC from HIV-infected individuals were stained with the antibody combination detailed in Fig. 2. Memory CD57⁻ and CD57⁺ CD4⁺ T cells were sorted, and quantitative PCR for gag DNA and albumin was performed. Infection of CD57⁺ CD4⁺ T cells was compared to infection of CD57⁻ memory CD4⁺ T cells in a subject-independent fashion (A) and a subject-dependent fashion, with white bars representing CD57⁺ memory CD4⁺ T cells and shaded bars representing CD57⁻ memory CD4⁺ T cells (B). Asterisks mark individual subsets where no gag DNA was amplified, and the values listed are calculated based on half of the lower limit of detection. Corresponding subjects are listed along the x axis. The plasma viral load was compared to the number of infected CD57⁻ memory CD4⁺ T cells (C). While there is a correlation between the number of infected CD57⁺CD4⁺ T cells and the number of infected CD57⁻ memory CD4⁺ T cells (D), the CD57⁺ population contains significantly less HIV than the CD57⁻ memory CD4⁺ T-cell subset (A and B).

FIG. 4.

HIV infection of memory CD8⁺ T cells. The fraction of infected memory CD8⁺ T cells was compared to the number of infected memory CD57⁻ CD4⁺ T cells for all subjects (A) and on an individual subject basis (B), with white bars representing memory CD8⁺ T cells and shaded bars representing CD57⁻ CD4⁺ T cells. Asterisks mark individual subsets where no gag DNA was amplified, and the values listed are calculated based on half of the lower limit of detection. Corresponding subjects are listed along the x axis. Infection of HIV-specific CD8⁺ T cells (based on production of IFN-γ following HIV peptide stimulation) was then compared to infection of other memory CD8⁺ T cells, and no significant differences were observed (C). The infection frequency of HIV-specific CD8⁺ T cells was then compared to the infection frequency of other memory CD8⁺ T cells in a subject-dependent fashion (white bars represent HIV-specific CD8⁺ T cells, and shaded bars represent memory CD8⁺ T cells) (D). Corresponding subjects are listed along the x axis.

FIG. 5.

Naïve CD4⁺ T cells have less viral DNA than memory CD57⁻ CD4⁺ T cells. PBMC from HIV-infected individuals were stained with the antibody combination detailed in Fig. 2. Memory CD57⁻ and naïve CD4⁺ T cells were sorted, and quantitative PCR for gag DNA and albumin was performed on sorted T cells. Infection of naïve CD4⁺ T cells was compared to infection of CD57⁻ memory CD4⁺ T cells in a subject-independent fashion (A) and a subject-dependent fashion (B). White bars represent naïve CD4⁺ T cells, and shaded bars represent CD57⁻ memory CD4⁺ T cells (B). Corresponding subjects are listed along the x axis. The plasma viral load was compared to the number of infected naïve CD4⁺ T cells (C). There was no correlation between the number of infected naïve CD4⁺ T cells and the number of infected CD57⁻ memory CD4⁺ T cells (D).

FIG. 6.

Infection of naïve CD8⁺ T cells and peripheral infection of naïve CD4⁺ T cells. Infection of highly purified naïve CD8⁺ T cells was compared to infection of naïve CD4⁺ T cells, memory CD8⁺ T cells, and CD57⁻ memory CD4⁺ T cells (A). Naïve CD8⁺ T cells are significantly less likely to carry HIV than any other subset studied. Comparison of infection of naïve CD8⁺ T cells (white bars, B) and naïve CD4⁺ T cells (shaded bars, B) demonstrates that naïve CD8⁺ T cells rarely contain detectable viral DNA (asterisks mark individual subsets where no gag DNA was amplified and values are calculated as half the lower limit of detection). Corresponding subjects are listed along the x axis. Naïve CD4⁺ T cells were stained and defined as before with side scatter, forward scatter, CD3, dump, CD4, CD8, CD45RO, CD11a, CD27, and CD57 (Fig. 2) from four subjects in the cohort (13 to 16) and were then separated on the basis of surface CD31 expression (C). Sorted T cells were then assayed for gag DNA by quantitative PCR. The number of infected naïve CD31⁺ CD4⁺ T cells (white bars) was compared to the number of infected naïve CD31⁻ CD4⁺ T cells (shaded bars) (D).

FIG. 7.

T cells that harbor HIV. A pie chart averaged from four subjects in the cohort demonstrates the individual contributions of all T-cell subsets studied to the total pool of infected T cells. The magnitude of infection within each subset and the contribution of each subset to the pool of PBMC were used in the calculation.