Intracellular cytokine and phenotype analysis of T cells from patient HR.

After 2 in vitro stimulations by DCs pulsed with Id protein, the antigen specificity and T-cell phenotype of HR's T cells were analyzed simultaneously by FastImmune assay. Effector cells were coincubated with stimulators at a ratio of 5 to 1 at 37°C. Autologous tumor cells (solid bars) or autologous PBMC (open bars) were used as stimulators. After a 2-hour coincubation, Brefeldin A was added to block secretion of cytokines. Three hours later, cells were washed and fixed with 1% paraformaldehyde. Fixed cells were permeabilized and triple stained with direct conjugated antibodies of CD69-phosphatidylethanolamine, CD4– or CD8–peridinin chlorophyll protein, and IFN-γ–fluorescein isothiocyanate, conjugated. Cell samples were then acquired by a fluorescence-activated cell sorter (FACScalibur) cytometer. Either CD4⁺ (right panel) or CD8⁺ (left panel) T cells were gated on, and their expression of CD69 and intracellular cytokine was examined. Percentages of activated double-positive cells are shown.