Figure 7. N1IC in T cells overcomes the tolerogenic effect induced by MDSC.

(A) MDSC were isolated from tumors and spleens of mice bearing s.c. 3LL cells for 17 days using anti-Gr1 kits. Then, total RNA was isolated and tested for Notch ligands by Quantitative PCR. Results represent mean ± SD from 4 independent animals and tested in triplicate. ***, P < 0.001. (B) Activated CD3⁺ T cells were co-cultured at different ratios with tumor-infiltrating MDSC for 48 hours. Then, T cells were negatively isolated using anti-CD11b beads and whole protein extracts harvested and used for detection of Notch-1 and -2 isoforms by western blot. A representative experiment of 3 repeats is shown. (C) Activated T cells co-cultured with MDSC at a 1:1/2 ratio were treated with L-NMMA (500 μM), D-NMMA (500 μM), and NN (200 μM) for 48 hours. Then, extracts were isolated and used as in (B). Representative results are from 3 similar experiments. (D) CD8⁺ T cells from CD45.2⁺ N1IC or N1IC^f/f mice were adoptively transferred into CD45.1⁺ congeneic recipients. Following transfer, mice were vaccinated with a mix of dendritic cells (DC) and/or MDSC pulsed with siinfekl, as described in the Methods, and the draining lymph nodes harvested, activated with siinfekl, and tested for the production of IFMγ using ELISpot. Results represent mean ± SD from 2 similar independent experiments. Ns= Non-statistical significance, P < 0.001; ***, P < 0.001.