Figure 1. NOS2 is essential for effective ACT.

(A) Representative images of CD11b⁺ cells sorted from spleens or tumors of EG7 tumor-bearing WT mice stained for NOS2, ARG1 and DAPI. Scale bar, 25 μm.

(B, C) CD11b⁺ cells sorted from spleens or tumors of EG7 tumor-bearing WT mice were quantified for the percentage of ARG1⁺, NOS2⁺, and ARG1⁺NOS2⁺ cells (B) or for fluorescence intensity of NOS2 expression (C, Median [50^th percentile] represented as a line inside the box. Lines at the bottom and top of the box represent, respectively, the 25^th and the 75^th quartile, the whiskers represent 10^th and 90^th percentile. Outliers beyond the whiskers are individually plotted as black dots; *** p ≤ 0.001, unpaired Student t-test analysis). n=357 cells from spleen or from tumor were evaluated.

(D) CD11b⁺ cells from tumors of WT, ARG KO, NOS KO or ARG NOS KO mice were sorted and co-cultured with OVA-specific, CFSE-labeled, CD8⁺CD45.1⁺ T lymphocytes in the presence of OVA peptide. Percent of proliferating T cells within a given number of cell divisions (from 0 to 7 as indicated) is shown in bar graph format relative to positive control (mixed lymphocyte peptide culture [MLPC], lymphocytes stimulated with OVA peptide) or negative controls without OVA (NS, not stimulated).

(E, F) WT and EG7 tumor-bearing WT and NOS KO mice were adoptively transferred with OVA-specific CD8⁺CD45.1⁺ T lymphocytes. Cells derived from spleens (E) or tumors (F) were stimulated with OVA peptide and percent of CD45.1⁺CD8⁺ cells and IFN-γ⁺ cells shown in CD45.1⁺CD8⁺ gate. Mean ± s.d.; n=4, representative of 3 independent experiments. ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05, by using One Way ANOVA.

(G) Survival percentages of EG7 tumor bearing WT and KO mice either untreated (n=5, for each group) or treated with ACT (WT n=20; ARG KO n= 15; NOS KO n=18; ARG NOS KO n=17). *p ≤ 0.05, logrank test.

See also figure S1.