Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment

Abstract

The human immunodeficiency virus (HIV)-mediated immune response may be beneficial or harmful, depending on the balance between expansion of HIV-specific T cells and the level of generalized immune activation. The current study utilizes multicolor cytokine flow cytometry to study HIV-specific T cells and T-cell activation in 179 chronically infected individuals at various stages of HIV disease, including those with low-level viremia in the absence of therapy ("controllers"), low-level drug-resistant viremia in the presence of therapy (partial controllers on antiretroviral therapy [PCAT]), and high-level viremia ("noncontrollers"). Compared to noncontrollers, controllers exhibited higher frequencies of HIV-specific interleukin-2-positive gamma interferon-positive (IL-2(+) IFN-gamma(+)) CD4(+) T cells. The presence of HIV-specific CD4(+) IL-2(+) T cells was associated with low levels of proliferating T cells within the less-differentiated T-cell subpopulations (defined by CD45RA, CCR7, CD27, and CD28). Despite prior history of progressive disease, PCAT patients exhibited many immunologic characteristics seen in controllers, including high frequencies of IL-2(+) IFN-gamma(+) CD4(+) T cells. Measures of immune activation were lower in all CD8(+) T-cell subsets in controllers and PCAT compared to noncontrollers. Thus, control of HIV replication is associated with high levels of HIV-specific IL-2(+) and IFN-gamma(+) CD4(+) T cells and low levels of T-cell activation. This immunologic state is one where the host responds to HIV by expanding but not exhausting HIV-specific T cells while maintaining a relatively quiescent immune system. Despite a history of advanced HIV disease, a subset of individuals with multidrug-resistant HIV exhibit an immunologic profile comparable to that of controllers, suggesting that functional immunity can be reconstituted with partially suppressive highly active antiretroviral therapy.

FIG. 1.

Representative flow analysis and gating strategy using an eight-color flow cytometry panel. PBMCs were stimulated and stained as described in Materials and Methods. (A) Initially, dead cells are excluded from further analysis by gating only on EMA-negative cells. CD3⁺ cells are identified as T cells. T cells are further characterized using forward and side scatter and elimination of doublets and debris (not shown). CD8⁺ and CD4⁺ T cells are then identified based on the presence or absence of a CD8 surface marker. (B) The ability of cells within the CD4⁺ and CD8⁺ subpopulations to produce IFN-γ is then measured. Gating is determined using a sample that has been unstimulated. The final analysis of IFN-γ⁺ cells is determined by subtracting the cytokine production in the unstimulated sample from that of the Gag-stimulated sample. C. Phenotypic analysis can then be performed on the CD8⁺ T-cell population using four markers simultaneously. Initially, gates are drawn to determine the CD27 and CD45RA expression on the cells. Then, each of these four subsets is further characterized based on expression of CCR7 and CD28. The placement of gates is based on “fluorescence minus one” controls. In this manner 16 different phenotypes of CD8⁺ T lymphocytes can be analyzed.

FIG. 2.

Proportion of CD4⁺ and CD8⁺ T cells secreting IFN-γ and/or IL-2 in response to HIV-1 Gag peptide pools. (A) The percentage of cytokine-secreting CD4⁺ T cells (CD3⁺ CD4⁺) in response to Gag peptide pools is shown for four patient groups. * signifies a P value of <0.05 for any cytokine profile comparing the reference group to noncontrollers. (B) The percentage of cytokine-secreting CD8⁺ T cells (CD3⁺ CD4⁻) in response to Gag peptide pools is shown for four patient groups. * signifies a P value of <0.05 for any cytokine profile comparing the reference group to noncontrollers. (C) The percentage of cytokine-secreting CD4⁺ and CD8⁺ T cells in antiretroviral-untreated patients with undetectable HIV RNA levels (“elite suppressors”) and antiretroviral-treated subjects with undetectable HIV RNA levels. All differences between subject groups were significant (P < 0.006 for each pairwise comparison). There was no difference between the groups in terms of IFN-γ⁻ IL-2⁺ CD4⁺ or CD8⁺ T cells (data not shown).

FIG. 3.

Phenotypic characteristics of Gag-specific cytokine-producing CD4⁺ and CD8⁺ T cells. Bar graphs represent the proportion Gag-specific T cells secreting either IFN-γ (A) or IL-2 (B). Only those T-cell subpopulations with a positive cytokine response (>0.03% of CD4⁺ or CD8⁺ T cells secreting cytokine in response to HIV-1 Gag peptide pool) are shown. ** represents a P value of <0.05 between PCAT patients and noncontrollers based on the Mann-Whitney nonparametric test.

FIG. 4.

Measurement of Ki67 expression among CD4⁺ and CD8⁺ T-cell phenotypic subsets. The cohort was divided based on the presence or absence of detectable Gag-specific IL-2-secreting CD4⁺ T cells. The levels of Ki67 expression within each T-cell subset are shown.

FIG. 5.

Levels of CD8⁺ T-cell activation (as defined by expression of CD38) in HIV-uninfected volunteers and four distinct patient groups. Naïve and memory T cells were defined based on expression of CD45RA and CD27. Compared to either controllers or PCAT subjects, noncontrollers had higher levels of immune activation in all memory T-cell subpopulations (P < 0.05 for each pairwise comparison between the noncontrollers and the other group).