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

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

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

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102104/1/emboj7601065.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102104/2/emboj7601065-sup-0001.pd

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

<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

<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

A More Differentiated Gag Specific CD8+ T Cell Response is Protective in Early HIV-1 Infection.

<p>Spearman Correlation results shown. Correlation performed on data from pre-treatment, Visit 1. Patients with higher CD4+ T cell counts during early infection and prior to anti-retroviral therapy tended to have higher HIV-1 Gag specific IFN-γ late memory (CD27−CD28−) CD8+ T cells</p

Expression of Maturation and Activation markers on Total CD4+ and CD8+ T cells.

<p>(A): before initiation of ART (i); after treatment (ii); and post-ART (iii). Activation is also shown on CD27 and CD28 subsets from CD8+ T cells post-ART (iv). Expression of Maturation and Activation markers on HIV Gag- and CMV pp65-specific T cells (B). CD27 and CD28 expression and CD38 mean fluorescent intensity (MFI) is shown on IFN-γ+ CD8+T cells. (GM = Geometric Mean).</p