Fig. 3.

Expansion of T lymphocytes specific to TGF-α or TGF-α-derived peptides. a Schematic representation of the TGF-α precursor and localization of synthesized overlapping peptides. b Resected tumors from kidney cancers were homogenized, and T cells from infiltrating single cell suspensions were expanded with PHA for 10–15 days prior to be tested for TGF-α-specificity. The symbol + means production of IFN-γ above background. Absence means no TGF-α-specific T cell detected. c PBMC from kidney cancer patients were co-cultured in multiple duplicated wells with peptide or TGF-α-pulsed autologous CD40-B cells as in Sect. “Materials and methods”. The specificity of expanded cells in b and c was evaluated by co-culture with autologous-pulsed CD40-activated B cells in IFN-γ (ELISPOT) and GM-CSF (ELISA). In ELISPOT, a T cell line was scored positive when >10 SFC and more than twice the number of SFC were observed in the well with the relevant peptide or protein, when compared with negative controls (irrelevant peptide from TGF-α). In GM-CSF secretion assays, a line was scored positive when secretion was >50 pg/ml and was twice the amount obtained with relevant controls. SFC spot-forming cells. c Numbers indicate number of wells positive over number of tested wells