Profile of a Serial Killer: Cellular and Molecular Approaches to Study Individual Cytotoxic T-Cells following Therapeutic Vaccination
Figure 1
Cellular and molecular approaches for the characterization of cytotoxic T-cells following therapeutic vaccination. Each step (1 to 6) in the strategy bears the same name and section number as described in the body of text. (1) Patients undergoing therapeutic vaccination are monitored closely throughout the clinical study, and blood and tissue samples are collected at numerous time points. (2) Antigen-specific CD8 T-cell populations are first visualized by the use of fluorescent peptide-MHC multimers then analyzed for their phenotype and subset composition using multiparameter flow cytometry. T-cell populations of interest can subsequently be isolated for further in vitro [3, 5] or ex vivo [4, 5] studies. (3) In vitro generated T-cell clones can be subjected to a series of assays to determine their functionality, including target cell lysis (51C release assay) and cytokine production (ELISpot). (4) Ex vivo sorted single cells are lysed and cDNA purified for gene-expression and TCR repertoire analysis. (5) Single cell samples or individual T-cell clones can be subjected to spectratyping, for the study of TCR repertoire diversity, selection, and clonotype composition. The unique signature of each T-cell can be identified, and its frequency determined. Furthermore, individual T-cell clonotypes can be followed across different T-cell compartments, and over time following therapeutic vaccination. (6) Conclusions drawn from this complete analysis of phenotype and functionality of vaccine-induced immune responses in melanoma patients can be taken back to the bedside. These results can be translated into improved therapeutic vaccination regimens aiming for more powerful immune activation and more efficient and specific antitumor responses.
