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. 2004 Nov;78(22):12638-46.
doi: 10.1128/JVI.78.22.12638-12646.2004.

Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels

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Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels

Eli Boritz et al. J Virol. 2004 Nov.

Abstract

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells has been associated with HIV-1 viremia and declining CD4+ T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4+ T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8+ cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4+ T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-gamma)-producing CD4+ T cells. Among the 20 viremic, treatment-naive subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4+ T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-gamma-producing CD4+ T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4+ T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.

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Figures

FIG. 1.
FIG. 1.
Frequencies of HIV-1 p24-specific, IFN-γ SFCs in CD8-depleted, p24-expanded PBMC of viremic, ART-naive subjects. PBMC were depleted of CD8+ cells and cultured with recombinant p24 antigen and the indicated compounds for 7 days. Expanded cells were then stimulated in IFN-γ ELISPOT assays with pooled p24 peptides or medium alone as a negative control, and the net p24-specific response was calculated by subtracting the medium-stimulated response from the p24-stimulated response. Factors tested included monoclonal CD28 antibody (CD28; open bars), human CD40 ligand trimer (CD40LT; hatched bars), recombinant human interleukin-2 (IL-2; filled bars), recombinant human interleukin-12 (IL-12; open bars), and the nucleoside reverse transcriptase inhibitor didanosine (ddI; hatched bars). All seven subjects were studied using all the indicated factors except for subject UH136, with whom IL-2 and IL-12 were not tested.
FIG. 2.
FIG. 2.
Increased frequencies of HIV-1 p24-specific, IFN-γ SFCs from CD8-depleted PBMC of viremic, ART-naïve subjects. Fresh CD8-depleted PBMC (Fresh) or CD8-depleted PBMC expanded with recombinant p24 antigen for 7 days (Expanded) were stimulated in IFN-γ ELISPOT assays with pooled p24 peptides or medium alone as a negative control. Net p24-specific responses were determined by subtracting the median medium-induced SFCs/million input cells from the median p24-induced SFCs/million input cells. Subjects showing detectable p24-specific responses in the fresh assay are grouped on the left side of the graph.
FIG. 3.
FIG. 3.
HIV-1 p24-induced CD4+ T-cell proliferation in whole and CD8-depleted PBMC of viremic, ART-naïve subjects. CFSE-labeled PBMC, either whole (PBMC) or CD8 depleted (CD8d), were stimulated with recombinant p24 antigen or baculovirus control protein and analyzed by flow cytometry after 7 days of culture. (A) CFSE fluorescence of CD4+ T cells from representative p24-stimulated cultures. Results for three HIV-1-infected subjects (subjects UH195, UH178, and UH189) and one negative-control subject (subject N4) are shown in density plots gated on resting and blasting CD3+ lymphocytes. CD4 events are omitted for simplicity. (B) Net p24-induced CD4+ T-cell proliferative responses from whole or CD8-depleted PBMC for each ART-naïve subject tested in both assays. The percentages of p24- or control-stimulated, CD3+-CD4+ cells with reduced CFSE fluorescence were determined by flow cytometry, and the net p24-induced percentages were calculated by subtracting the control-stimulated percentages from the p24-stimulated percentages. The horizontal line above the x axis indicates the minimum positive response.
FIG. 4.
FIG. 4.
HIV-1 p24-specific, IFN-γ-producing CD4+ T cells from CD8-depleted, p24-expanded PBMC of viremic, ART-naïve subjects that divided in culture. CD8-depleted, CFSE-labeled PBMC expanded with p24 antigen as described for Fig. 3 were divided into two cultures that were then stimulated with medium alone or with pooled p24 peptides, permeabilized, stained for IFN-γ, and analyzed by flow cytometry. Density plots were gated on resting and blasting CD4+ lymphocytes. Results are shown for four ART-naïve subjects. The percentage of CD4+ cells with both reduced CFSE fluorescence and intracellular IFN-γ is shown in the upper left corner of each plot.
FIG. 5.
FIG. 5.
HIV-1 p24-induced CD8+ T-cell proliferation in whole PBMC is associated with p24-induced CD4+ T-cell proliferation in whole PBMC. (A) CFSE fluorescence of CD4+ (left panels) and CD8+ (right panels) T cells from p24-stimulated PBMC of two viremic, ART-naïve subjects. Results for one subject with CD4+ T-cell proliferation in whole PBMC (subject UH185) and for one without (subject UH202) are shown. Density plots were gated on resting and blasting CD3+ lymphocytes. (B) Net p24-induced proliferative responses of CD4+ T cells (CD4) and CD8+ T cells (CD8) from whole PBMC for each ART-naïve subject tested. The percentages of p24- or control-stimulated CD3+-CD4+ or CD3+-CD8+ cells with reduced CFSE fluorescence were determined by flow cytometry, and the net p24-induced percentages were calculated by subtracting the control-stimulated percentages from the p24-stimulated percentages. The results for seven subjects with positive CD4+ T-cell proliferation results are grouped on the left. (C) HIV-1 p24-induced CD4+ T-cell prolif-eration from whole PBMC predicts stronger p24-induced CD8+ T-cell proliferation from whole PBMC. CD8+ T-cell proliferative responses in whole PBMC from subjects with positive (+) or negative (−) p24-induced CD4+ T-cell proliferation in whole PBMC were compared using the Mann-Whitney nonparametric test. The median response for each group is shown as a horizontal bar.
FIG. 6.
FIG. 6.
Plasma HIV-1 RNA levels of >100,000 copies/ml predict lack of proliferation-dependent, p24-induced CD4+ T-cell responses from CD8-depleted PBMC. Subjects were divided into two groups: one consisted of subjects with viral loads greater than 100,000 copies/ml (>105), and the other consisted of subjects with viral loads less than or equal to this value (≤105). HIV-1 p24-induced CD4+ T-cell responses in expanded IFN-γ ELISPOT assays (A) or CFSE proliferation assays from CD8-depleted PBMC (B) were then compared between the two groups by the Mann-Whitney nonparametric test. The median value for each group is shown as a horizontal bar.

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