Tuberculin-specific T cells are reduced in active pulmonary tuberculosis compared to LTBI or status post BCG vaccination.

Functional characteristics of tuberculosis (TB)-specific CD4 T cells were studied in clinically active pulmonary TB (n = 21) and high TB exposure including LTBI (n = 17). Following tuberculin stimulation, activated CD4 T cells were identified by flow-cytometry (CD154 up-regulation, degranulation, interferon γ [IFN-γ], tumor necrosis factor α [TNF-α], and interleukin 2 [IL-2\ production). Interestingly, CD154 up-regulation accounted for ∼80% of activated CD4 T cells in the active TB group but just 40% in the controls, whereas IFN-γ accounted for only ∼50% of activated cells in each group. The frequencies of CD4 T cells displaying at least 1 activation marker discriminated better between the groups than those displaying degranulation or IFN-γ production alone.

Standardization and optimization of multiparameter intracellular cytokine staining.

Intracellular cytokine staining (ICS) is a common method for rapid quantitation of cytokine-producing antigen-specific T cells. T cell production of IFNgamma in particular, and more recently IL-2 as well, is often taken as a measure of vaccine immunogenicity in experimental vaccine trials. As more fluorochromes become available for use in ICS and other applications detecting intracellular markers, the selection of optimal fluorochrome combinations becomes correspondingly more complicated. Additionally, as more sophisticated flow cytometers become available, more attention is being paid to potential result variability from one instrument to another. This review summarizes an oral presentation given at MASIR 2008, January 30-Feb 1, 2008, in La Plagne, France. We focus on issues associated with multiparameter (>four color) flow cytometry, including matching antibody specificities with available fluorochromes and techniques to optimize fluorochrome combinations. We examine issues specific to intracellular staining as well as broader topics such as instrument setup, experimental controls, sample management, and analysis of multiparameter data sets. Particular emphasis is placed on the use of lyophilized cells, antibodies, beads, peptides, etc. (collectively known as "lyoplates"), which can decrease experiment-to-experiment variability as well as processing time. Most clinical trials compile results from multiple testing sites, using data that was acquired on-site in each location. We present data from two different ongoing multi-laboratory standardization studies, one involving 15 laboratories and one involving nine. We identify issues of variability and, where possible, offer solutions.

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.

Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

The phenotypic distribution and functional profile of tuberculin-specific CD4 T-cells characterizes different stages of TB infection.

BACKGROUND: Recent publications have suggested that altered proportions of functional CD4 T-cell subsets correlate with active pulmonary TB. Also, CD27-expression on tuberculin-activated IFN-γ(+) CD4 T-cells is known to differ significantly between patients with active pulmonary TB and healthy TB-unexposed BCG vaccinees. Here, we explore links between CD4 T-cell phenotype, multiple functional subsets, and control of TB. METHODS: We examined ex-vivo overnight tuberculin activated CD4 T-cells in regards to CD27-expression and the activation markers, CD154 upregulation, IFN-γ, TNF-α, IL-2, and degranulation in 44 individuals, including cases of clinically active pulmonary TB, and hospital staff with prolonged TB exposure, some of whom had latent TB. RESULTS: Active pulmonary TB generally showed an excess of TNF-α(+) subsets over IFN-γ(+) subsets, paralleled by decreased CD27 expression on activated IFN-γ(+) or CD154(+) CD4 T-cells. The single subset distinguishing best between active pulmonary TB and high TB exposure was CD154(+) /TNF-α(+) / IFN-γ(-) /IL-2(-) /degranulation(-) (AUROC 0.90). The ratio between the frequencies of TNF-α(+) /IFN-γ(+) CD4 T-cells was an effective alternative parameter (AUROC 0.87). CONCLUSIONS: Functional subsets and phenotype of tuberculin induced CD4 T-cells differ between stages of TB infection. Predominance of TNF-α(+) CD4 T-cells in active infection suggests an increased effort of the immune system to contain disease.

Loss of receptor on tuberculin-reactive T-cells marks active pulmonary tuberculosis.

BACKGROUND: Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells. METHODOLOGY/PRINCIPAL FINDINGS: Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naïve/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between. CONCLUSIONS/SIGNIFICANCE: Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

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.

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.