Discordance between frequency of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-producing CD4(+) T cells and HIV-1-specific lymphoproliferation in HIV-1-infected subjects with active viral replication

Abstract

One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4(+) T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4(+) Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4(+) T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4(+) T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4(+) T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4(+) T-cell proliferation.

FIG. 1.

Lymphoproliferative responses to HIV-1 recombinant antigens (p24, p66, and gp160) and Candida antigen in subjects with HAART suppression and HIV-1-seronegative subjects. PBMC from HIV-1-infected and seronegative control subjects were cultured in the presence of antigen or control protein for 6 days. Lymphoproliferative responses to these antigens were determined by measuring [³H]thymidine incorporation, and the results were expressed as SIs. Each donor is depicted as a separate point, and a solid line represents the median value for each group. The median and mean SIs and numbers of responders for whom SI was ≥5 for each protein are shown at the bottom. Statistical significance was determined by the Mann-Whitney test.

FIG. 2.

Responder IFN-γ frequencies and lymphoproliferation of HIV-1 p24-, p66-, and CMV-specific CD4⁺ T cells in subjects receiving HAART with suppressed viral loads (HS), subjects failing HAART (HF), HAART-naive subjects (HN), and seronegative control subjects (HIV−). (A, C, and E) Responder frequencies of IFNγ⁺ CD69⁺ (percentage of CD4 T cells) CD4⁺ T cells for HIV-1 p24, p66, and CMV antigens based on intracellular cytokine staining, respectively. (B, D, and F) Lymphoproliferative responses (SIs) to HIV-1 p24, p66, and CMV antigen in HS, HF, HN, and seronegative control subjects, respectively. PBMC from HS, HF, HN, and seronegative subjects were stimulated with the respective antigen and assessed for intracellular IFN-g production within the CD4 1 CD69 1 T-cell population by flow cytometry and for lymphoproliferation by measuring [ 3 H]thymidine incorporation. Responder frequencies were corrected by subtracting the background from PBMC stimulated with control antigens (baculovirus for HIV-1 antigens or CMV-infected cell control lysate). Responses of individual subjects are depicted as separate points. A positive lymphoproliferative response is defined as a SI of $5 (dashed line). Where applicable the median and mean values and number of responders for each protein are shown. Statistical significance was determined by using the Kruskal-Wallis and Dunn multiple-comparison tests.

FIG. 3.

Plasma viral loads of HIV-1-infected subjects that responded (responders, R) or failed to respond (nonresponders, NR) to HIV-1 p24 (A) or p66 (B) proteins in a standard LPA and correlations of HIV-1 p24 (C) and HIV-1 p66 (D) SIs and viral loads for all patients. Responders were defined as subjects who had SIs of ≥5 for HIV-1 proteins. PBMC from HIV-infected subjects were pulsed with either HIV-1 p24 or p66 or baculovirus control protein, and proliferation was measured using a 6-day [³H]thymidine incorporation proliferation assay. Plasma viral loads were determined by using the Roche HIV-1 RNA Monitor kit. Each subject is depicted as a separate point, and the bar represents the median for each group. Statistical significance was determined by using the Mann-Whitney T test (A and B) or the Spearman correlation test (C and D).

FIG. 4.

Correlations between p24-specific proliferation and absolute numbers of p24-specific IFN-γ-producing CD4⁺ T cells in peripheral blood of HIV-1-infected subjects. (A) HIV-1 p24-specific lymphoproliferative responses in net counts per minute (CPM). Net values of counts per minute from LPAs were calculated by subtracting the mean CPM from wells containing baculovirus-stimulated PBMC from the mean counts per minute of PBMC stimulated with HIV-1 p24 protein. A statistically significant difference between the net counts per minute for both cohorts with active viral replication (HF and HN) versus the HS group was found (P = 0.001). (B) Absolute number of HIV-1 p24-specific, IFN-γ⁺ CD69⁺ CD4⁺ T cells per milliliter of blood in each HIV-1-infected cohort. This number was obtained by multiplying the p24-specific IFN-γ⁺ CD69⁺ CD4⁺ T-cell frequency by the peripheral blood CD4 count per milliliter. (C and D) Correlations between the number of HIV-1 p24-specific IFN-γ-producing CD4⁺ T cells per milliliter of blood in subjects with HAART suppression (C) and subjects with active viral replication (D). The correlation coefficients for the subjects with HAART suppression and subjects with active viral replication were 0.62 and 0.40, respectively. HF, HN, HS, and HIV− are as defined for Fig. 2.

FIG. 5.

Expansion of HIV-1 p24-specific IFN-γ⁺ CD4⁺ T cells in vitro. CD4⁺ T cells specific for HIV-1 p24 were detected in fresh PBMC by intracellular IFN-γ staining and flow cytometry. PBMC were labeled with CFSE and stimulated in culture with HIV-1 p24 antigen or baculovirus control protein for 8 days. Cultured cells from each condition were then restimulated with p24 or control antigen in a 6-h assay, and the frequency of IFN-γ producing CD4⁺ T cells was determined as described in Materials and Methods. Density plots are of gated CD4⁺ T cells and the percentages of CFSE-low, IFN-γ⁺ cells are shown in the top left corner or each plot. Density plots and clinical characteristic are shown for representative subjects UH52 and UH32.