72 research outputs found

    Performance of plate-based cytokine flow cytometry with automated data analysis

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    BACKGROUND: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. RESULTS: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. CONCLUSION: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses

    High CD8+ T Cell Activation Marks a Less Differentiated HIV-1 Specific CD8+ T Cell Response that Is Not Altered by Suppression of Viral Replication

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    The relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.We performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag-specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28-) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27-CD28-), low activation phenotype. Participants with the highest fraction of late memory (CD27-CD28-) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28-) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.A more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile

    Standardization of cytokine flow cytometry assays

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    BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4(+)cytokine(+ )cells and CD8(+)cytokine(+ )cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays

    Activation levels on total and antigen specific T cells by differentiation stage.

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    <p>In the first column the proportion of total T cells (CD8+ and CD4+) expressing CD38 and HLA-DR activation markers by T cell maturation category are displayed and defined as early memory (CD27+CD28+) EM, intermediate memory (CD27+CD28−) IM, and late memory (CD27−CD28−) LM. In columns 2 the CD38 MFI level is shown by maturation stage for CD8+ T cell IFN-γ responses to both HIV-1 Gag (red) and CMV pp65 (green). And in column 3 the CD38 MFI is shown for each maturation stage of CD4+ T cell IFN-γ responses to HIV-1 Gag (red) and CMV pp65 (green). Row 1 displays measurements for visit 1, prior to antiretroviral therapy. Row 2 displays measurements for visit 2, during a virologically suppressive anti-retroviral regimen, and row 3 displays measurements for visit 3, after patients had halted an anti-retroviral regimen and viremia had rebounded. Black bars indicate differences between activation levels by response categories (Wilcoxon 2 Sample Test). Red dots notes a significant change from baseline (visit 1) values (Sign Rank Test).</p

    T cell activation declines during anti-retroviral therapy but maturation profile of Gag specific CD8+ T cells does not change

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    <p>HIV-1 RNA levels, total CD8+ T cell activation levels, HIV-1 specific CD8+ T cell activation levels and the proportion of HIV-1 Gag specific IM and LM CD8+ T cells at visit 1 (Pre-ART), visit 2 (On ART) and visit 3 (Post-ART). P-values for changes in viral load, Total and HIV-1 specific CD8+ T cell activation are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004408#pone-0004408-t001" target="_blank">Table 1</a>. HIV-1 RNA, total CD8+ T cell activation levels and HIV-1 gag specific CD8+ T cell activation levels all declined after initiation of ART. Black bars display p-values for a test of change in HIV-1 Gag specific CD8+ T cell IM and LM fractions from visit 1 to visit 2, and visit 3. Neither the Gag specific IFN-γ+ CD8+ IM or LM pool changed significantly from pre-therapy levels after therapy was initiated (On ART), or later halted (Post-ART).</p
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