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Structured Treatment Interruptions Workshop Summary

January 31, 2001

Appendix 1
Assays used to detect HIV-1-specific CTL

Kaul and Rowland-Jones
  CTL Culture Elispot Tetramer Staining
What it does Bulk PBMC are co-cultured with HIV-1 antigens, which clonally expands the antigen-specific CTL in the sample. Anti-cytokine antibodies on a plate reveal individual cytokine-producing cells as single spots that are counted by eye or by computer. HLA/peptide complexes are organized into a stable tetrameric structure that specifically binds to T-cells expressing the appropriate T-cell receptor for the HLA/antigen complex.
What it measures When the expanded CTL lyse Cr51 pulsed target cells, radiation is released indicating the presence of functional CTL. INF-gamma release by CTL in response to antigen correlates well with CTL frequencies measured with lysis assays. Tetramer staining directly marks antigen-specific CD8 T-cells that can then be counted by flow cytometry.
Advantages Directly indicates lysis by CTL. Allows screening for response to multiple epitopes in a single assay. Allows virus-specific analysis. Allows sorting of antigen-specific cells for subsequent cloning. Allows analysis of co-expressed surface antigens.
Disadvantages Only detects CTL able to expand in vitro; undercounts exhausted, resting and terminally differentiated CTL. Not a direct measure of CTL function; uses IFN-gamma release as a marker for lytic capacity. Not an assay of CTL function. Requires precise knowledge of both epitope and MHC type; not suitable for screening.
Relation to environment in vivo Extended artificial stimulation in vitro means the frequency and phenotype of CTL are removed from conditions under which lysis occurs in vivo. Detects antigen-specific cells without in vitro expansion. Cells are stained directly ex vivo.
Quantitative? Quantitation is possible with limiting dilution analysis (LDA). Undercounts compared to other techniques. Indirect counting. Undercounts compared to tetramer staining. Direct counting of CTL
Sensitivity LDA can be a highly sensitive technique for detecting low frequency CTL. More sensitive than tetramer staining. Least sensitive technique for detecting low frequency CTL.
Time required Several days Overnight Hours

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