Figure 5.

TCR gene analysis in DEPDC1-LP2- or MPHOSPH1-LP1-specific Th cells. (A–D) The DEPDC-LP2- or MPHOSPH1-LP1-specific T-cell repertoires were analyzed by deep cDNA sequencing of T-cell receptor α and β genes using next-generation sequencing. TCR-α is presented in the top panels, and TCR-β is presented in the lower panels. The Y-axis indicates the number of Th cell stimulations with DCs pulsed with the cognate peptide. Cloning of DEPDC1-LP2-specific bulk Th cells was performed after five stimulations of bulk Th cells with irradiated PBMCs pulsed with the cognate peptide. Cloning of MPHOSPH1-LP1-specific bulk Th cells was performed after four stimulations of bulk Th cells with irradiated PBMCs pulsed with the cognate peptide. For both, the total number of stimulation was nine times. The X-axis indicates the top 10 most frequent V-(D)-J-CDR3 sequences (details are shown in the right boxes), the Z-axis indicates the frequency of individual V-J-CDR3 sequences. (E) The TCR-negative murine T-cell line TG40 expressing full-length TCR-α and -β genes isolated from DEPDC1-LP2- and MPHOSPH1-LP1-specific T-cell clones specifically responded to HLA-DR expressing L cells that were pulsed with cognate peptides, as revealed by the cell-surface expression of CD69 and CD137.