IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells.

BACKGROUND: Diminished IL-2 production and lack of effector differentiation have been reported for HIV-specific T cells. In this study, we examined the prevalence of these phenomena using 8-color cytokine flow cytometry, and tested the hypothesis that these two findings were causally related. We analyzed cytokine profiles and memory/effector phenotypes of HIV-specific and CMV-specific T cells using short-term in vitro stimulation with HIV or CMV peptide pools. Nineteen HIV-positive subjects with progressive disease and twenty healthy, HIV-negative subjects were examined. RESULTS: Among HIV-infected subjects, there were significantly fewer CD8+ IL-2+ T cells responding to HIV compared to CMV, with no significant difference in CD4+ IL-2+ T cells. The majority of CMV-specific T cells in both HIV-negative and HIV-positive subjects appeared to be terminally differentiated effector cells (CD8+ CD27- CD28- CD45RA+ or CD8+ CD27- CD28- CD45RA-). In HIV-positive subjects, the most common phenotype of HIV-specific T cells was intermediate in differentiation (CD8+ CD27+ CD28- CD45RA-). These differences were statistically significant, both as absolute cell frequencies and as percentages. There was a significant correlation between the absolute number of HIV-specific CD8+ IL-2+ T cells and HIV-specific CD8+ CD27- CD28- CD45RA+ terminal effector cells. CONCLUSION: IL-2 production from antigen-specific CD8+ T cells correlates with effector cell differentiation of those cells.

Optimal preparation of rhesus macaque blood for cytokine flow cytometric analysis.

BACKGROUND: The rhesus macaque is a common substitute for human subjects in many disease models, including simian immunodeficiency virus, the non-human primate equivalent of the human immunodeficiency virus. Monoclonal antibodies and fluorochromes optimized for use in macaques were included in samples examined for immune responses with the use of intracellular cytokine flow cytometry (CFC). METHODS: Sample preparation was optimized based on the following comparisons: activation of peripheral blood mononuclear cells (PBMCs) versus whole blood; separation of PBMCs using BD Vacutainer cell preparation tubes versus Ficoll; and activation of samples on the day they were collected versus holding samples overnight. RESULTS: When activated with the simian immunodeficiency virus type mac239 and Gag peptide mix or with the superantigen Staphylococcal enterotoxin B, separated PBMCs produced greater CD4 and CD8 fluorescence intensities and a larger percentage of CD69+ cytokine-positive cells than did whole blood samples. PBMCs separated by cell preparation tubes produced absolute T-lymphocyte counts equivalent to that with Ficoll separation, and CFC results with both methods were similar. When subjected to overnight shipping conditions, whole blood and PBMCs sometimes showed a reduction in mean fluorescence intensity and percentage of CD69+ cytokine-positive T lymphocytes. CONCLUSIONS: Due to this reduction in responses, it is preferable to activate samples on the day of blood collection. Samples can be surface stained and frozen in BD FACS Lysing Solution, to be thawed at a later date; this preserves their ability to display a cytokine response. Thus optimal CFC results are achieved by separating macaque PBMCs from whole blood, activating samples on day of collection, and, if necessary, freezing samples after surface staining for future analysis.

Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.

We report here the standardized conditions for stimulation of macaque whole blood samples with various protein or peptide antigens, and production of significant intracellular levels of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in CD4+ as well as CD8+ T lymphocytes. We observed significantly higher levels of TNF-alpha compared with IFN-gamma in both CD4+ and CD8+ T lymphocytes from all the macaque whole blood samples stimulated with staphylococcal enterotoxin B (SEB) as an antigen. Similarly, when whole blood samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine were stimulated with either the peptide cocktail or recombinant gp160, we observed production of significant levels of TNF-alpha by both CD4+ and CD8+ T lymphocytes. These results strongly support the utility of the whole blood cytokine flow cytometry methodology for determining antigen-specific immune responses of macaques in vaccine studies.