Figure 5.

B/P/R-ISV is superior to ISV with single agent BCG naPRRa, promoting priming and recruitment of TA-specific T-cells. (A) Schema of murine preclinical lymphoma ISV model for ex vivo analysis. GFP-A20 lymphoma tumors were inoculated in the right flank, and tumor-bearing mice were treated intratumorally with Flt3L. Tumors were then locally irradiated (XRT), and B/P/R or saline was injected intratumorally. Mice were sacrificed on day 11 post-ISV and tumors and TdLN were harvested. (B, C) Tumors were used for analysis by fluorescence microscopy, images were analyzed and CD8 pixel intensity quantified using Fiji ImageJ software. (D–F) Tumors and TdLN were processed and analyzed by spectral flow cytometry. Statistical significance was calculated by t test. n=4–5 mice per group. (G) Schema of murine preclinical lymphoma ISV model for ex vivo T-cell neoantigen reactivity analysis. Tumor and TdLN cell suspensions isolated from tumor-bearing naPRRa-ISV treated animals were cocultured with mutant Lrrk1 peptide-pulsed DCs. T-cells were analyzed by flow cytometry after 24 hours. (H) Contour and before-after plots showing IFN-γ production by CD44⁺ PD1⁺ CD8+ T-cells. Statistical significance was calculated by t test. n=6 mice. DC, dendritic cells; ISV, in situ vaccination.