Fig. 2
Injection route dictates anti-cancer activity of AII therapy. A Schematic outline of the three investigated conditions of animal experiments. B On day 27 tumors and organs were excised and analyzed by IHC. Panels show representative images of tumors, livers and spleens stained for CD45, demonstrating higher CD45 levels in all tissues for both hIL15 NOG, i.v. and NOG, MFP conditions compared to NOG, i.v. Primary tumor expansion for this animal experiment is shown in Fig. S3. n = 5 (NOG, untreated), n = 5 (NOG, i.v.), n = 3 (NOG, MFP), n = 3 (hIL15 NOG, i.v.) C-E Quantification of the immune cell density (CD45+) in tumors, livers and spleens, respectively. F Same as in B, but co-stained for CD3 (red) and Ki67 (brown). White arrows indicate proliferating (double positive) cells. G TCR alpha and beta chain analysis comparing the frequencies of the 50 most prevalent chains in tumors with their frequency prior to administration, showing selective expansion of these clones. H Cancer killing assay as in Fig. 1d showing no added benefit of blocking PD1 or PDL1 (10 μg/mL). I Flow cytometry analysis of the PD1 level of CD4+ and CD8+ AII cells in culture and in tumors, demonstrating increased PD1 expression in tumors. Numbers indicate geometric mean fluorescence intensity. J Same as in B but stained for PDL1 showing increased PDL1 expression in tumors for both hIL15 NOG, i.v. and NOG, MFP conditions. K Quantification of PDL1 tumor expression. Statistical difference was determined by the Mann Whitney test C, D, E or the paired t-test G or Student’s t-test K. *0.05 > P ≥ 0.01, ***0.001 > P. Black, white and grey scale bar 250, 100 and 25 μm, respectively. i.v.; intravenous