Figure 5.

Antigen presentation of neoepitope Tnpo3 and immune response and tumor protection elicited by it. (A) Mice were immunized with mutant Tnpo3 peptide. Draining LNs, harvested 1 wk after immunization, were briefly stimulated ex vivo without (No pep) or with WT or mutant Tnpo3 peptides (left), or with a weekly in vitro stimulation with 1 µM mutant Tnpo3 peptide (right). After 5 d, cells were tested for the responsiveness to mutant Tnpo3-pulsed cells (Tnpo3) or Meth A cells (Meth A) by ELISpot. IFN-γ⁺ CD44⁺ CD8⁺ T cells were counted. (B) Mice were immunized twice with ovalbumin peptide (SIINFEKL) or Tnpo3 mutant peptide. 6 d after the second immunization, splenocytes from both groups were stained with K^d/SYMLQALCI tetramer. Tetramer positive cells were counted in CD8⁺ gate. (C) Mice were immunized with irradiated Meth A cells. (left) 6 d later, inguinal LN cells were stimulated overnight without peptide, irrelevant Prpf31 peptide or Tnpo3 peptide. % activated effector CD8⁺ cells is shown, as assessed by flow cytometry. (right) Splenocytes were stimulated in vitro in multiple rounds with 1 µM of indicated peptides for a total of 19 d. Irrelevant peptide from Prpf31 was used as a control. 5 d after stimulation, cells were tested for the responsiveness to indicated peptides by flow cytometry. Typically, for each sample, 150,000 lymphocytes, or at least 19,000 CD8⁺CD4⁻ cells, were acquired. See in Fig. S3 for FACS gating strategy and representative primary data. (D) Mice were injected with 200,000 Meth A cells on the right flank. 21 d later, tumor-draining LNs and contralateral LNs were harvested and stained with anti-CD8 antibody and Tnpo3 and Nfkb1 tetramers K^d/SYMLQALCI and K^d/GYSVLHLAI, respectively (left and middle). Splenocytes were used to purify CD8⁺ cells to assess the responsiveness to mutant Tnpo3-pulsed cells (Tnpo3) or Meth A cells (Meth A) by ELISpot assay with no peptide (No pep) stimulation as negative control (right). (E) Naive mice or Tnpo3 mutant peptide-immunized mice were challenged with Meth A cells and treated with anti-CD25 antibody or anti-CTLA-4 antibody as indicated. AUC for each group is plotted (Duan et al., 2012), and complete tumor growth curves for all the mice in all groups are shown. Between four and six mice per group were used in each experiment, and each experiment was repeated between three and five times. *, P < 0.05; **, P < 0.01; ***, P < 0.001.