Figure 4
Transgene-specific tumor-infiltrating CD8+ T cells were of relatively low functional avidity. C57BL/6 mice received intradermal injections of 2.5 × 105 B16-F10 melanoma cells. Four days later, they were vaccinated intramuscularly with 1 × 108 IU of an adenovirus expressing human dopachrome tautomerase (hDCT). Ten days after immunization, blood and saline-perfused spleens and tumors were harvested for flow cytometric analysis of CD8+ T cells specific for the immunodominant epitope from DCT (DCT180–188). (a) The relative functional avidity of T cell receptors was compared between the three tissues through flow cytometric assessment of intracellular staining of IFN-γ after re-stimulating T cells with decreasing concentrations of DCT180–188. The frequencies of activated T cells were normalized to those exposed to the highest concentration of the cognate peptide (1 μg/mL). (b) To complement the functional avidity assay, T cells from the blood (left graph) and tumor-derived T cells (right graph) were quantified in two ways: (1) by intracellular cytokine staining of IFN-γ following re-stimulation with 1 μg/mL of the immunodominant epitope from DCT (DCT180–188), or (2) they were directly stained with DCT180–188-loaded tetramers. The scientific literature has shown that tetramer staining tends to be preferentially compromised by low concentrations of T cell receptors on lymphocytes as compared to the intracellular cytokine staining method and can, therefore, be used as an additional indirect assessment of functional avidity. (c) Representative dot plots from blood (upper graphs) and tumors (lower graphs) following intracellular cytokine staining (left graphs) or tetramer staining (right graphs). Data from three experiments were pooled (n = 12/group). Means and standard errors are shown. Results were assessed by (a) two-way analysis of variance with Tukey’s multiple comparisons test (*p < 0.05) or (b) unpaired two-tailed t-tests.
