Figure 6.

Preparation of peptide extracts from B16 cells and their fractionation by reversed-phase high performance liquid chromatography (RP-HPLC) and immunogenicity. (A) Peptides were extracted from B16 cells and desalted using the C18 Sep-Pak column, as described in “Materials and method.” The peptides were eluted with 3 ml of 60% acetonitrile (ACN) in water, of which 300 μl was vacuum-dried and then dissolved in 10% DMSO in PBS. The dissolved B16 cell elute was reacted with 6 × 10⁶ splenocytes from mice that had been immunized twice at 2-week intervals with Trp2 peptides plus CpG-ODN. Additionally, 10 μg of Trp2 peptides was dissolved in 3 ml of citrate-phosphate buffer, followed by passage through a C18 Sep-Pak column as described above. This sample was eluted with 3 ml of 60% ACN in water, of which 300 μl was dried and reacted with 6 × 10⁶ splenocytes from Trp2 peptide+CpG-ODN-immunized mice. For the IFN-γ control, 5 μg of Trp2 and E7 peptides were tested. (B) Negative control (E7 peptide), B16 cell elute, and 1 × 10⁶ of B16 and TC-1 cells were reacted with 6 × 10⁶ splenocytes from B16-bearing mice that had been treated with anti-PD-L1. After 2 days of incubation, the cell supernatants were collected for the IFN-γ assay. (C) Dried peptides from 1 ml of elute were dissolved in DMSO and fractionated by RP-HPLC, as described in “Materials and method.” The fractions displaying a high peak at 214 nm were selected and vacuum-dried. Blue line; peaks at 280 nm. (D) The dried fraction samples were re-suspended in 10% DMSO in water and stimulated for 3 days with 6 × 10⁶ splenocytes from B16 tumor-bearing mice. The cell supernatants were measured for IFN-γ levels. Arrows indicate the fraction number displaying the highest levels of IFN-γ production among the tested samples. *p < .05 compared with naïve control. ^#p < .05 compared with E7 peptide