Productive human immunodeficiency virus type 1 infection in peripheral blood predominantly takes place in CD4/CD8 double-negative T lymphocytes

Abstract

Human immunodeficiency virus type 1 (HIV-1) transcription is subject to substantial fluctuation during the viral life cycle. Due to the low frequencies of HIV-1-infected cells, and because latently and productively infected cells collocate in vivo, little quantitative knowledge has been attained about the range of in vivo HIV-1 transcription in peripheral blood mononuclear cells (PBMC). By combining cell sorting, terminal dilution of intact cells, and highly sensitive, patient-specific PCR assays, we divided PBMC obtained from HIV-1-infected patients according to their degree of viral transcription activity and their cellular phenotype. Regardless of a patient's treatment status, the bulk of infected cells exhibited a CD4+ phenotype but transcribed HIV-1 provirus at low levels, presumably insufficient for virion production. Furthermore, the expression of activation markers on the surface of these CD4+ T lymphocytes showed little or no association with enhancement of viral transcription. In contrast, HIV-infected T lymphocytes of a CD4-/CD8- phenotype, occurring exclusively in untreated patients, exhibited elevated viral transcription rates. This cell type harbored a substantial proportion of all HIV RNA+ cells and intracellular viral RNAs and the majority of cell-associated virus particles. In conjunction with the observation that the HIV quasispecies in CD4+ and CD4-)/CD8- T cells were phylogenetically closely related, these findings provide evidence that CD4 expression is downmodulated during the transition to productive infection in vivo. The abundance of viral RNA in CD4-/CD8- T cells from viremic patients and the almost complete absence of viral DNA and RNA in this cell type during antiretroviral treatment identify HIV+ CD4-/CD8 T cells as the major cell type harboring productive infection in peripheral blood.

FIG. 1.

HIV-1 RNA⁺ cells in total PBMC. Frequencies of cells positive for usRNA (class I), msRNA (class II), and vRNAex (class III) were calculated by limiting dilution analysis. Open bars depict data from treated patients (group A) and shaded bars frequencies in untreated patients (group B). P values indicate comparison of group A to B (Mann-Whitney test). “Box and whisker” bars depict medians, quartiles, and ranges. The broken line indicates the lowest detection threshold for PBMC expressing HIV-1 RNA.

FIG. 2.

High levels of HIV-1 production in CD4⁻/CD8⁻ T cells from untreated patients. (A) Proportions of HIV-1 nucleic acids in PBMC attributed to CD4⁺ T cells (CD4⁺; gray bars, untreated patients; open bars, on-treatment patients) and CD4⁻/CD8⁻ T cells (DN [double negative]; dark bars, untreated patients; no analysis during ART was possible due to almost-complete depletion of viral DNA and RNA). Error bars show standard errors of the means. +, on-treatment patients; −, untreated patients. (B) HIV RNA expression normalized to HIV DNA content in PBMC, CD4⁺ T cells (CD4⁺), and CD4⁻/CD8⁻ T cells (DN) in untreated and treated patients. HIV RNA expression was calculated as the sum of the average msRNA and usRNA expression at each time point. (C) Frequencies of cells positive for vRNAex (category III) in PBMC, CD4⁺ T cells (CD4⁺), and CD4⁻/CD8⁻ T cells (DN) of untreated patients. The broken line indicates the lowest detection threshold for cells expressing HIV-1 RNA. “Box and whisker” bars depict medians, quartiles, and ranges. Analyses A to C comprised one data point per visit, resulting in duplicate measurements for PBMC and CD4⁺ T cells for all patients except for patients 03, 08, and 11. (B and C) Results of nonparametric comparison of CD4⁺ versus CD4⁻/CD8⁻ T cells using Mann-Whitney testing are indicated by P values.

FIG. 3.

Similar HIV-1 transcriptional activity in resting and activated CD4⁺ T cells from treated and untreated patients. (A) Frequencies of HIV RNA⁺ cells in resting (RST) and activated (ACT) CD4⁺ T cells normalized to 10⁶ cells of the corresponding cell types. (B) Geometric means of HIV RNA contents per cell in RST and ACT CD4⁺ T cells normalized to numbers of HIV RNA⁺ cells. Analyses comprised one data point per visit, resulting in duplicate measurements for PBMC and CD4⁺ T cells for all patients except for patients 03, 08, and 11. Results of nonparametric paired analyses of CD4⁺ versus CD4⁻/CD8⁻ T cells using Wilcoxon signed-rank testing are indicated by P values.

FIG. 4.

Specific HIV contents per cell of CD4⁺ and CD4⁻/CD8⁻ T cells. (A and B) Schematic outline of determination of specific cell contents as exercised by measurements of msRNA (class II expression) in CD4⁺ T cells obtained from patient 10. (A) Primary measurements of msRNA levels in serial five-fold dilutions of purified CD4⁺ T cells are shown in panel A. Triangles depict individual measurements and the vertical gray line shows the calculated 50% end point of specimens positive for msRNA. Italic numbers above the panel show the calculated numbers of HIV RNA⁺ cells in each dilution series. (B) Normalization of measured HIV msRNA copies to numbers of HIV RNA⁺ cells results in specific HIV RNA contents in each sample with detectable msRNA (triangles). (C) Per-cell HIV RNA expression data comprising per-cell expression in resting, activated, and total CD4⁺ T cells (CD4⁺) and CD4⁻/CD8⁻ T cells (DNT [double negative]) from all patients and time points is displayed by single data points (open symbols, on-treatment patients; gray symbols, untreated patients) and “box and whisker” bars showing medians, quartiles, and ranges. Note that viral RNA expression in CD4⁻/CD8⁻ T cells obtained from patients on ART, remaining below the threshold of detection, could not be displayed except for one sample (left panel, class I expression). Results of nonparametric comparisons using Mann-Whitney testing are indicated by P values.

FIG. 5.

Distribution of cellular transcription categories in CD4⁺ and CD4⁻/CD8⁻ T cells. Average class I, class II, and class III contributions to total HIV-1 RNA⁺ cells were calculated for CD4⁺ T cells (CD4⁺) from untreated and treated patients and for CD4⁻/CD8⁻ T cells (DN [double negative]) from untreated patients. Analyses comprised one data point per visit, resulting in duplicate measurements for CD4⁺ T cells for all patients except for patients 03, 08, and 11. Error bars depict standard errors of the means.

FIG. 6.

Uniform distribution of HIV-1 env quasispecies in plasma and CD4⁺ and CD4⁻/CD8⁻ T cells. Neighbor-joining phylogenetic tree of env C2-V3-C3 sequences obtained from patients 05 and 06 constructed using MEGA 3.1. Single molecular clones (mean n = 14; range, 5 to 16) were obtained from plasma RNA, from CD4⁺ T cells (CD4⁺; total RNA and total DNA), and from CD4⁻/CD8⁻ T cells (DN [double negative]; total RNA, vRNAex, and total DNA). Symbols indicate individual clones and numbers above branches show bootstrap values. Mean diversities for each cell type are indicated.