Figure 4

Identifying dominant HLA-A*0201-restricted cyclin D1 T cell epitopes. (A) Binding capacity for cyclin D1 peptides to HLA-A*0201 molecules on T2 cells. T2 cells were cultured with predicted cyclin D1 peptides which have high binding affinity to HLAAA* 0201 (list in Table 3), in RPMI1604 without FCS for 18 h, and subsequently stained for cell surface expression of MHC class I. Up-regulation of HLA-A2 expression after binding with specific peptides was represented as median fluorescence intensity (MFI). Sequences of positive peptides are shown. (B) IFN-DCs primed by the individual cyclin D1 peptide as (A) were co-cultured with enriched CD8⁺ T cells for 10 days. Then, cells were boosted with the same peptide pulsed T2 cells at 37°C for 36 h, thereafter, supernatant was harvested, and the cytokines production were tested by Luminex®. Sequences of positive peptides are shown. (C) Enriched CD8⁺ T cells from a healthy donor ND239 (HLA-A*0201) were stimulated with autologous IFΝ-DCs pulsed with MCL lymphoma cell dying bodies (Granta 519) and treated with LPS for 6 h. Ten days later, induced specific CTLs were tested in a standard 4-h ⁵¹Cr release assay. Target cells used were cyclin D1 peptide P_58–67 KIVATWMLEV-pulsed T2 cells, P_57–67 RKIVATWMLEV-pulsed T2 cells, P_99–109 QLLGATCMFV-pulsed T2 cells, non-pulsed T2 cells, cyclin D1⁺HLA-A*0201⁺ MCL lymphoma cell line Granta 519, and K562 as natural killing activity controls.