Fig. 6. TGR enhances tumor-specific CD8⁺ T_E function in vivo.

(a) Ova-specific CD8⁺ T cells (OT-I) from CD45.2 female mice were activated by SIINFEKL peptide and IL-2 (100 U/mL), expanded for 72h, then exposed to 20h to 10mM (Control, black) or 1mM (TGR, orange) glucose. CD45.1 female recipient mice were injected with 1×10⁶ E.G7-OVA lymphoma cells, tumors were established for 8 days, and 3 ×10⁶ OT-I⁺ T_E were injected per tumor bearing mouse. (b) Average tumor diameter of control (black n=9) or TGR (orange n=10) T_E recipient animals. (c-e) Blood was collected 16 days post tumor injection and the percent and function of donor (CD45.2) CD8⁺ T cells was assessed by (c) congenic markers and (d-e) IFN-γ expression after re-stimulation with PMA/ionomycin + brefeldin-A. (f) Blood was collected day 28 post tumor injection to assess percent of donor (CD45.2) CD8⁺ T cells. (c-f) Statistical significance was calculated on n=9 and n=10 animals by 2-tailed Student’s t-test. * p<0.05; *** p<0.001. (g) T_E cells were generated as described in (a). CD45.1 female recipient mice were injected subcutaneously with 1×10⁶ B16-OVA melanoma cells and tumors established for 5 days. 5 ×10⁶ control (black) or TGR (orange) OT-I⁺ T_E were injected intravenously per tumor bearing mouse. Mice receiving no T_E were used as control. 3, 6 and 9 days after tumor inoculation, mice received 200 μg anti-PD-1 antibody or lgG2a isotype control in 100 μl PBS intraperitoneally. (h-i) Lines depict average tumor diameter of grouped mice. Statistical significance was calculated by multiple 2-tailed Student’s t tests comparing control T_E and TGR T_E over the timecourse on n=5 animals. * p<0.05; ** p<0.01. (j-k) 21 days after tumor inoculation mice were humanely euthanized and the tumor mass was excised. Single cell suspensions were analyzed by flow cytometry. The number of donor (CD45.2) CD8⁺ T cells per mg tissue (j) and the percent of donor (CD45.2) CD8⁺ T cells (k) was quantified. Statistical significance was calculated by 2-tailed Student’s t test. * p<0.05; ** p<0.01. Tumor size measurements were performed by blinded researchers. All error bars show SEM.