Fig. 6.

Combinatorial treatment with Lm-ANXA2 and anti-PD-1 blockade antibody increases the ANXA2 epitope-specific T cell response in the tumor microenvironment. a CD8+ T cells were isolated and purified from livers on day 28 after hemispleen implantation of KPC tumor cells. Tumor-bearing mice were treated with Cy, either Empty Lm or Lm-ANXA2, and either anti-PD-1 antibody or IgG as in prior study schemes. IFNγ ELISA assays were performed on supernatants collected from CD8+ T cells cocultured with Kb T2 cells exposed to Peptide Group #3. There is a significant difference between combination treatments of Lm-ANXA2 and anti-PD-1 blockade versus Lm-ANXA2 and IgG (p = 0.0389). b Mice were treated with Lm-ANXA2 on days 1 and 8 and were sacrificed on Day 10. Splenocytes were processed and CD8 cells isolated by negative selection. Kb T2 cells were pulsed with individual 15-mer peptides from ANXA2 Peptide Group #3 as well as positive and negative control peptides. IFNγ ELISA was run on supernatant 18 h following the co-culture of T2 and CD8 T cells. Two epitopes with H5 and E6 peptides were identified with highest expression of IFNγ thus most likely to contain the MHC class I restricted epitopes on ANXA2 protein. c IFNγ ELISPOT assays were performed on supernatants collected from CD8+ T cells co-cultured with Kb T2 cells stimulated with individual 8-mer peptide sequences of the active 15-mers H5 and E6, each overlapping by one amino acid. The sequences H5–4: YDAGVKRK and E6–8: VFDRYKSY appear to be the CD8+ T cell ANXA2 epitopes of interest. Each experimental group consisted of 5 mice, pooled, and analyzed individually in triplicates. Data represent mean + SEM from one representative experiment that was repeated once. * p < 0.05, *** p < 0.001