Skip to main content
. 2017 Mar 20;6(5):e1306617. doi: 10.1080/2162402X.2017.1306617

Figure 6.

Figure 6.

Successful identification of an immunogenic neo-epitope encoded by mouse sarcoma CMS7. (A) After subcutaneous inoculation with CMS7 cells, mice were randomly divided into two groups. One group was left untreated, and the other was administered intraperitoneally with anti-CTLA-4, anti-PD-1 and anti-GITR mAbs on days 7, 9, and 11. Both mouse groups were sacrificed at day 21 and splenic cell suspensions were prepared from pooled spleens (n = 3 per experiment). Splenic cells from untreated (upper) or treated mice (lower) were incubated with each panel of neo-epitope peptides for 5 h and the fold increase in CXCL9 mRNA levels compared with DMSO was quantified. One representative data set out of three independent experiments is shown. (B) Splenic cells from untreated (upper) or treated mice (lower) were incubated with mutated Snd1 peptide, its wild-counterpart or DMSO as a control for 5 h and the fold increase in CXCL9 mRNA levels compared with DMSO was quantified. (C) Splenic cells from untreated (upper) or treated mice (lower) were incubated with mutated Snd1 peptide, its wild-counterpart or DMSO as a control for 15 min at room temperature and subsequently with GolgiPlug for 4 h. Following stimulation, cells were stained for cell surface CD8+ and intracellular IFNγ. Representative dot plots gated on CD8+ splenic T cells are shown. The number indicates the percentage of CD8+ IFNγ+ T cells. These experiments were repeated three times with similar results. (D) CD8+ splenic T cells were obtained from antibody-treated mice pooled spleens by positive enrichment using the MACS system, and were stimulated in vitro with mutated Snd1-pulsed CD8− splenic cells. Cultured CD8+ splenic T cells were subjected to ELISPOT assays 10 d later. The target cells were CD8− splenic cells pulsed with mutated Snd1 peptide, or its wild counterpart. Splenic cells pulsed with DMSO were used as control targets. (E) Age-matched female BALB/c mice were inoculated with CMS7 at day 0 following two injections (at days −14 and −7) with mSnd1 peptide and poly (I:C) formulated in PBS or poly (I:C) alone using prophylactic schedules. The tumor size was monitored three times a week. Each group consisted of five mice. Mice without any immunization and mice treated by the co-administration of antibodies (anti-CTLA-4/PD-1/GITR Abs) at days 7, 9, 11 served as a negative control group and a positive control group, respectively.