Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection

Abstract

Human immunodeficiency virus (HIV)-specific T-cell responses are thought to play a key role in viral load decline during primary infection and in determining the subsequent viral load set point. The requirements for this effect are unknown, partly because comprehensive analysis of total HIV-specific CD4(+) and CD8(+) T-cell responses to all HIV-encoded epitopes has not been accomplished. To assess these responses, we used cytokine flow cytometry and overlapping peptide pools encompassing all products of the HIV-1 genome to study total HIV-specific T-cell responses in 23 highly active antiretroviral therapy naïve HIV-infected patients. HIV-specific CD8(+) T-cell responses were detectable in all patients, ranging between 1.6 and 18.4% of total CD8(+) T cells. HIV-specific CD4(+) T-cell responses were present in 21 of 23 patients, although the responses were lower (0.2 to 2.94%). Contrary to previous reports, a positive correlation was identified between the plasma viral load and the total HIV-, Env-, and Nef-specific CD8(+) T-cell frequency. No correlation was found either between viral load and total or Gag-specific CD4(+) T-cell response or between the frequency of HIV-specific CD4(+) and CD8(+) T cells. These results suggest that overall frequencies of HIV-specific T cells are not the sole determinant of immune-mediated protection in HIV-infection.

FIG. 1

Simultaneous measurement of HIV-specific CD8⁺ and CD4⁺ T-cell responses. PBMC from patient 22 were stimulated with overlapping 15-mer peptides from Gag and stained as described in Materials and Methods. Cells were gated initially on small, viable, resting lymphocytes and then gated for CD3⁺ IFN-γ⁺ cells (top left panel). CD3⁺ IFN-γ⁺ lymphocytes were then gated based on CD8 and IFN-γ expression (bottom left panel). Quantification of CD8⁻-mediated HIV-specific responses (top right panel) was determined by gating on CD8⁺CD69⁺ IFN-γ⁺ cells. Quantification of CD4-mediated HIV-specific responses (bottom right panel) was determined by gating on CD8⁻CD69⁺ IFN-γ⁺ cells. HIV Gag-specific cells are highlighted in red, and the percent response from each cell type is indicated.

FIG. 2

Total HIV-specific CD4⁺ (A) and CD8⁺ (B) T-cell responses in samples from 23 untreated HIV-infected patients. PBMC isolated from patients 1 to 23 (see Table 1) were incubated with peptide mixtures containing 15-mers overlapping by 11 amino acids derived from HIV Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, and Vpu in the presence of costimulatory antibodies as described in Materials and Methods. The bars in panel A represent the percentage of CD3⁺CD8⁻CD69⁺ IFN-γ⁺ cells that responded to each peptide mixture with background (CD28-CD49d alone) IFN-γ production from the CD3⁺CD8⁺ population removed. The bars in panel B represent the percent of CD3⁺CD8⁺CD69⁺ IFN-γ⁺ cells that responded to each peptide mixture with background IFN-γ production removed. Background IFN-γ production from CD3⁺CD8⁺ cells was ≤0.1% in all patients except for patients 15 (0.15%), 17 (0.37%), and 22 (0.2%). Background IFN-γ production in CD3⁺CD8⁻ cells was ≤0.1% in all patients except for patients 10 (0.16%), 15 (0.16%), and 21 (0.17%).

FIG. 3

Correlation between HIV-specific CD4⁺ and CD8⁺ T-cell frequency. Total HIV-specific (A) and Gag-specific (B) CD4⁺ and CD8⁺ T-cell frequencies were determined with the indicated overlapping 15-mer peptide mixtures. P values, as determined by the Spearman rank correlation, are as follows: (A) P = 0.8; (B) P = 0.27. The solid line represents a regression line.

FIG. 4

Correlation between viral load and HIV-specific CD4⁺ T-cell frequency. Plasma viral load (RNA copies/ml) was determined in each patient from the same time point in which HIV-specific CD4⁺ T-cell responses were quantified. P values shown in each figure are determined by the Spearman rank correlation. The solid line represents a regression line.

FIG. 5

Correlation between viral load and HIV-specific CD8⁺ T-cell frequency. Plasma viral load (RNA copies/ml) was determined in each patient from the same time point that HIV-specific CD8⁺ T-cell responses were quantified. P values shown in each figure are determined by the Spearman rank correlation. The solid line represents a regression line.