Figure 5. CD8⁺ T cells were essential for the antitumor effect by SBRT plus mFX (A-F).

Orthotopic pancreatic tumors were established and treated with mFX and SBRT as in figure. 1. On day 11 (1 day after SBRT), mice were sacrificed, peripheral blood and tumors collected, and tumor-infiltrating CD8⁺ T cells (A), IFNγ⁺CD8⁺ T cells (B), PD1⁺CD8⁺ T cells (C), CTLA4⁺CD8⁺ T cells (D), peripheral IFNγ⁺CD8⁺ T cells (E), and effector CD8⁺ T cells (F) were determined by flow cytometry. (G-H) Mice were injected with 2×10⁵ KCKO-OVA cells in tail of pancreas, and treated with mFX on day 5 and SBRT on day 7, 8, 9 and 10 after tumor implantation. Mice were sacrificed on day 11 and SIINFEKL/H-2K^b dextramer binding CD8⁺ T cells in tumor were determined by flow cytometry. (G) Representative flow cytometry plots of SIINFEKL/H-2K^b dextramer positive CD8⁺ T cells. (H) Quantitative analysis of G. Data in (A-F) and (H) are expressed as mean ± SEM from 5 mice/group and analyzed by one-way ANOVA with Dunnett post-test. *, p<0.05, treated group compared with no treatment group; #, p<0.05, mFX+SBRT or SBRT treated group compared to mFX treated group. (I) Orthotopic pancreatic tumors were established and treated with mFX and SBRT as in figure 1. Left panel, tumor growth was determined by IVIS imaging. Data shown are mean ± SEM from 5 mice/group. *p<0.05, mFX+SBRT-IgG compared with mFX+SBRT-CD8 depletion, using one-way ANOVA with Dunnett post-test. Right panel, survival was observed. *p<0.05, mFX+SBRT-IgG compared with mFX+SBRT-CD8 depletion group by log-rank (Mantel-Cox) test. (J) Orthotopic pancreatic tumor were established in WT or Rag1 KO mice and treated with mFX and SBRT as in Fig. 1. Tumor growth was determined by IVIS imaging and the value of IVIS in wide type mice on day 5 set as 1. Data shown are mean of fold change of IVIS value ± SEM from 5 mice/group. *p<0.05, WT-mFX+SBRT compared with Rag1KO-mFX+SBRT using one-way ANOVA with Dunnett post-test.