Anti-tumor specific CD8+T-cell functions in tumor-bearing mice following different treatments. A, Splenocytes harvested from mice treated with scFvMTBHsp70 fusion protein, equimolar mixture of MTBHsp70 plus P4 scFv, or saline (n = 10 per group) were re-stimulated with Her2/neu peptide or MSLN Ld1 peptide. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Dunnett’s multiple comparison tests. B, Representative flow data are presented. C, In vivo CD8+ T-cell depletion study. FVB/NJ mice were injected i.p. with anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a, and were treated with scFvMTBHsp70 or saline as described in the methods. CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70 treated BR5FVB1 tumor-bearing animals compared to non depleted actively treated (n = 10 per group, representative of two independent experiments; Med. sur. = 32.5 days vs. 48 days) animals. After CD8+ T cells depletion, scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites), compared with saline (Med. sur. = 32.5 days vs. 31.5 days; p = 0.5938). P values were determined using log-rank test. *,p< 0.05; **,p < 0.01, ***,p < 0.001.