Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays
Figure 3
Validation of FMT (representative example of a melanoma patient). (a) Sequential gating strategy. Representative example dot plots and FACSDIVA analysis. Forward-scatter (FSC) area versus FSC height parameters were used to exclude cell doublets; cells were gated by forward and side scatter for lymphocytes; gated populations are plotted as CD8 (horizontal axis) versus tetramer staining (vertical axis). Direct ex vivo analysis cytokine production (IFNα, TNFγ, IL-2) and degranulation CD107a/LAMP-1) within CD8+ population or CD8+/MART-1 tetramer gated T-cells after stimulation with Melan-A/MART-1 peptide. (b) Histogram plots representing the individual functional combinations as a proportion of the total responding cells after stimulation with Melan-A/MART-1 peptide. Mutual exclusion, red bars: Percentage of cells expressing a certain combination of parameters (+) and not expressing the parameter indicated as (−). No mutual exclusion, blue bars: percentage of cells expressing a certain combination of parameters (+) independently from the expression of the parameter indicated with (−). The pie slices indicate the average proportion of the response producing 1, 2, 3, or 4 functions (regarding in this case “no mutual exclusion” variables). Each slice indicates one of the functions.
