Figure 2.

RHAMM-specific CD8⁺ T-cell frequencies in healthy donors. Cells were stimulated in an MLPC for 7 days with different peptides and tested for their reactivity in ELISPOT assays for IFN-γ (a) and granzyme B (b) release. Stimulation of cells without any peptide was used as negative control (no peptide), whereas T cells stimulated with CMV and IMP peptides served as positive controls. RHAMM-specific T cells could be detected in two healthy donors (HD 155 and HD 669) by IFN-γ ELISPOT, (c)–(g) RHAMM-specific T-cell frequencies were determined by flow cytometry in HD 155. Reported frequencies correspond to gated CD3⁺CD8⁺ T cells (upper numbers), and from all cells in the lymphocyte gate (lower numbers). (c) Fluorescence minus one (FMO) was used as negative control to assess the intrinsic fluorescence of the cells. (d) As a further negative control, cells were cultured in the absence of any peptide and stained with tetramers specific for the irrelevant antigen G250. As positive controls, CD8⁺ T cells were stimulated with either (e) CMVpp65 peptide or (f) IMP-derived peptide, (g) CD8⁺ T cells were stimulated with RHAMM peptide, (e)–(g) CD8⁺ T cells were stained with respective tetramers.