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. 2020 Nov 5;8(2):e001250. doi: 10.1136/jitc-2020-001250

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© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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NAM+GEM increased the influx of immune cells and activates T cells while reducing TAM and MDSC. Immune cells (CD45+) were isolated from whole tumors and analyzed by flow cytometry for the presence of CD4 and CD8 T cells (A), TAM (B) and MDSC (C). In addition, blood was also analyzed for MDSC (D). Subsequently, tumor tissues were analyzed by IHC for the presence of CD4 and CD8 T cells (E, G), and quantified (F, H). 5 fields in each group were analyzed and the number of CD4 and CD8 T cells was calculated per mm². n=5 mice per group. The results were averaged and analyzed by Mann-Whitney U test. *p<0.05, **p<0.001 is significant. Granzyme B (I) and perforin (K) were analyzed by IHC, and quantified (J, L). Ten fields in each treatment group were analyzed and the number of perforin and granzyme B-producing cells was calculated per mm². n=5 mice per group. The results were averaged and analyzed by Mann-Whitney U test. *p<0.05, **p<0.001 is significant. finally, spleen cells from NAM+GEM-treated and control mice were restimulated with survivin in an ELISPOT assay (M). CD4 and CD8 T cells were depleted by magnetic beads technology. The spleens of five mice were pooled in each treatment group. The spots of six wells were averaged and analyzed by Mann-Whitney U test. **P<0.01 is significant. GEM, gemcitabine; IHC, immunohistochemistry; MDSC, myeloid-derived suppressor cells; NAM, nicotinamide; TAM, tumor-associated macrophage.