Selective binding of CD4 and CD8 T-cells to antigen presenting cells for enrichment of CMV and HIV specific T-lymphocytes

Pira, Giuseppina Li; Ivaldi, Federico; Manca, Fabrizio

doi:10.1016/j.jim.2012.01.001

Cited by 2 publications

(1 citation statement)

References 30 publications

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“…It is also likely that PBMC resuspension allows a more efficient collection of all PBMC in the proliferation assay. Likewise, thorough dispersion of PBMC after collection of miniaturized ELISPOT assay plates before transferring to 96-well plates for cytokine capture and immunoenzymatic development may disrupt clusters of specific T cells surrounding a single APC (15) and thus reveal individual spot-forming cells rather than spot-forming units. This case, as an artifact, may underestimate the actual frequency of specific T cells in standard ELISPOT assays and thus this may be an additional advantage for the two-step procedure in 384-and 1,536-well plates.…”

Section: Discussionmentioning

confidence: 99%

Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV

Pira

Ivaldi²,

Starc

et al. 2014

Clin Vaccine Immunol

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Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory.

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Section: Discussionmentioning

confidence: 99%