HIV-1-specific CD4+ T lymphocyte turnover and activation increase upon viral rebound

Abstract

HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1-specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.

Figure 1

HLA class II tetramer staining of CD4⁺ T cells. PBMCs were stained directly ex vivo and gated on CD14^–, CD19^–, and Via-Probe–negative cells. (A) Representative plots showing positive tetramer staining in HIV-infected individuals bearing DRB1*0101 stained with the HIV Gag p24.17-DR1 (panels Ox55 and Ox79 before and after enrichment with magnetic beads) and p24.14-DR1 (Ox9 before and after enrichment) PE-conjugated tetramers (PE tet). (B) Representative staining of control samples. Controls stained with the Gag p24-specific tetramers included HIV-negative individuals bearing DRB1*0101 (left, upper and lower panels) and HIV-infected non-DRB1*0101 individuals (middle, upper and lower panels). HIV-infected individuals bearing DRB1*0101 were stained with a DRB1*0101 control tetramer complexed to an irrelevant HCV peptide (right, upper and lower panels). The percentage of cells falling into the upper-right quadrant is indicated for each panel.

Figure 2

Validation of the linear recovery efficiency of the magnetic bead enrichment of tetramer-positive (tet⁺) CD4⁺ T cells. Uninfected PBMCs were spiked with a p24.17 epitope-specific CD4⁺ T cell clone, stained with the p24.17-DR1 tetramer, and subjected to magnetic bead enrichment. The observed frequencies (freq.) of tetramer⁺ CD4⁺ cells versus the expected frequencies of tetramer⁺ CD4⁺ cells are plotted. The FACS plots are shown in Supplemental Figure 1.

Figure 3

Effect of ART cessation on the physical numbers of Gag p24-specific CD4⁺ T cells. PBMCs from 8 patients were stained with an HLA class II tetramer before, during, and after ART. (A) Longitudinal sampling of plasma viral load (pVL; top plot) and frequencies of p24.17-DR1 tetramer⁺ cells out of total CD4⁺ T cells (bottom plot). ART cessation is represented by the vertical dashed line, and the data are normalized for therapy cessation (defined as week 0). (B) The frequencies of p24.17-DR1 tetramer-positive CD4⁺ T cells are shown for 5 different clinical states: before antiretroviral therapy (preART), during the viral downslope while on ART (ART), once an undetectable viral load had been maintained for at least 1 month (pVL<50), upon viral rebound (Rebound), and during viral setpoint (Setpoint).

Figure 4

Phenotypic analysis of HIV-specific memory CD4⁺ T cells using HLA class II tetramers. (A) Representative phenotypic staining with anti-CD27, anti-CD28, or anti-CD62L antibodies from subject Ox73. FACS plots are gated on CD4⁺ T lymphocytes, and the percentage of tetramer⁺ CD4⁺ T cells expressing the respective phenotypic marker is indicated in the upper right quadrant of each plot. (B) Phenotypic profile of HIV-specific CD4⁺ T cells in 9 viremic individuals. Each bar represents the mean percentage of tetramer⁺ CD4⁺ cells that express the indicated surface marker. Error bars represent SEM. (C) CCR7 expression on CD4⁺ T cell subsets in viremic HIV infection. CD45RA⁺ and CD45RA^– CD4⁺ cells were gated on to distinguish between the naive and memory subsets, respectively. (D) The HIV-1–specific, tetramer⁺ CD4⁺ T cell subset was significantly more activated than the memory CD4⁺ T cell population. CD38 expression was measured in 9 viremic patients. Cells that fell into the CD45RA^–CD4⁺ or tetramer⁺ CD4⁺ gates were analyzed. The means are represented by horizontal lines and the statistical differences between them were calculated with a paired Student’s t test.

Figure 5

Expression of the intracellular proliferation marker Ki67. (A) Representative costaining of CD4⁺ T cells with the p24.17-DR1 tetramer and Ki67 in 2 viremic patients. The percentage of tetramer⁺ cells expressing Ki67 is indicated in the upper-right quadrant. (B) Ki67 expression in CD4⁺ T cell subsets in viremic infection. CD45RA⁺ and CD45RA^– CD4⁺ cells were gated on to distinguish between the naive and memory subsets, respectively. The means are represented by horizontal lines and the statistical differences between them were calculated with a paired Student’s t test. (C) Correlation between plasma viral load and Ki67 expression in HIV-specific tetramer⁺ CD4⁺ cells.

Figure 6

Effect of plasma viral load on CCR7 expression on HIV-specific CD4⁺ T cells. (A) Representative FACS plots showing CCR7 staining on tetramer-positive CD4⁺ T cells from patient Ox98 while on ART and off ART. Plots are gated on CD4⁺ lymphocytes. The percentages of tetramer⁺ cells expressing CCR7 are indicated in the upper-right quadrant. (B) Comparison of percentage of CCR7 expression on tetramer⁺ CD4⁺ T cells in patients on ART (n = 7) and off ART (n = 9). The means are represented by horizontal lines and the statistical differences between them were calculated with the Mann-Whitney U test. (C) Correlation between plasma viral load and percentage of CCR7 expression on tetramer-positive CD4⁺ T cells.

Figure 7

HIV-specific tetramer⁺ CD4⁺ cells in untreated chronic patients resemble those in viremic patients who received short-course ART at acute infection. (A) Longitudinal detection of tetramer⁺ CD4⁺ cells up to 63 weeks and 85 weeks after the first experience of seroconversion symptoms in patients Ox22 and Ox79, respectively. The frequency of tetramer⁺ CD4⁺ cells for each time point is indicated in the upper-right quadrant of each plot. (B) Phenotypic analysis of tetramer⁺ CD4⁺ cells from patient Ox79. These analyses were performed at time points 52 weeks or more after the first experience of seroconversion symptoms. The percentage of tetramer⁺ CD4⁺ cells expressing the relevant marker is indicated in parentheses in each plot.