Human immunodeficiency virus type 1 controllers but not noncontrollers maintain CD4 T cells coexpressing three cytokines

Abstract

Here, we evaluate the cytokine coexpression profiles of human immunodeficiency virus (HIV)-specific CD4 T cells for the expression of the cytokines gamma interferon (IFN-gamma), interleukin-2, and tumor necrosis factor alpha. In controllers, CD4 T cells producing three or two cytokines (triple producers and double producers, respectively) represented >50% of the total response. In contrast, in noncontrollers approximately 75% of responding cells produced only one cytokine (single producers), mostly IFN-gamma. Cells producing three cytokines were functionally superior to those producing single cytokines and showed an inverse correlation (P < 0.001) with viral load. These results demonstrate a strong association between the maintenance of highly functional CD4 T cells producing three cytokines and control of HIV-1.

FIG. 1.

Cytokine coexpression profile of HIV Gag-specific CD4 T cells in controllers, noncontrollers, and HAART-treated HIV-infected individuals. (A) The quality of response (cytokine subsets as a percentage of total cytokine-positive CD4 T cells). PBMC were stimulated with a pool of HIV-1 clade B consensus Gag-specific peptides (15-mers overlapping by 11; NIH AIDS Research and Reference Reagent Program catalog no. 8117). The ICS assay was performed as described previously (17). Approximately 500,000 lymphocytes were acquired on the LSRII (BD Immunocytometry Systems) and analyzed using FlowJo software (Treestar, Inc., San Carlos, CA). Lymphocytes were identified based on their scatter pattern, and CD3⁺, CD8⁻, and CD4⁺ cells were all considered as CD4 T cells. These CD4 T cells were then gated for respective cytokine-positive cells. Boolean combination gating was then performed (see http://www.flowjo.com/v8/html/boolcomb.html for an example) to calculate the frequencies of the seven different combinations of cytokines using the Flowjo software. After subtracting the background, these seven different subsets were expressed as a percentage of total cytokine-positive cells and plotted for each antigen per individual. Responses that were greater than 0.07% of total CD4 T cells were considered for analysis. This criterion was defined based on the fact that we were dividing the total response into seven subsets and our detection limit was 0.01%, as described before (17). The percentage of cytokine subsets as a percentage of total cytokine-positive CD4 T cells was then plotted using Graphpad Prism. (B) Magnitude of response (absolute number of cytokine-positive cells per ml of blood). (C) Pie charts representing the quality of response. Mean frequencies of the indicated cytokine subsets are shown. TP, triple producers; DP, double producers; SP, single producers; I, IFN-γ; L, IL-2; and T, TNF-α.

FIG. 2.

Triple producers express higher levels of cytokines and CD40L than single producers. (A) Shown are a representative fluorescence-activated cell sorting plot giving the fluorescence intensity for IFN-γ and IL-2 for triple and single producers and a summary of MFI data for HIV Gag-specific CD4 response in four HIV-1 controllers. PBMC were stimulated with HIV-1 Gag peptide pools, and virus-specific CD4 T-cell responses were measured using an ICS assay. (See Fig. 1 for analysis.) The MFI of each cytokine for different cytokine coexpression subsets was analyzed. TP, triple producers; SP, single producers; I, IFN-γ; L, IL-2; and T, TNF-α. (B) Costimulatory potential of HIV-specific cytokine coexpression subsets. Shown are a representative plot giving the CD40L expression for triple and single producers and a summary of the HIV Gag-specific CD4 responses in four HIV-1 controllers, three noncontrollers, and two HAART recipients. Cytokine-coexpressing subsets were defined as described in the legend to Fig. 1. These subsets were then analyzed for expression of CD40L (CD154). Representative flow charts show the overlay of HIV-specific triple producers or IFN-γ single producers (black) on total CD4 T cells (gray). Numbers on the graphs represent the frequency of CD40L-positive cells as a percentage of the respective cytokine coexpression subset. *, significantly lower than triple producers (P < 0.05).

FIG. 3.

Correlation between plasma viral load and cytokine coexpression subsets. Correlation between the quality (proportion of cytokine coexpression subset as a percentage of total cytokine-positive cells [left panel]) or magnitude (absolute number of cytokine-positive cells per ml of blood [right panel]) of cytokine-coexpressing cell subsets and plasma viremia for triple producers (A), double producers (B), and single producers (C) in untreated HIV-1-infected individuals.