GITR Agonism Enhances Cellular Metabolism to Support CD8⁺ T-cell Proliferation and Effector Cytokine Production in a Mouse Tumor Model

Mouse anti-GITR agonism by DTA-1 enhances CD8⁺ T-cell activation and metabolism despite optimal anti-CD3/anti-CD28 stimulation. A, Cell size and (B) viability in IgG2a control versus DTA-1–treated CD8⁺ T cells; N = 3. C, FACS plots of activation markers. D, ELISA IFNγ concentrations; N = 3. E, Representative NF-κB pathway Western blots from two separate experiments. F, NF-κB pathway gene expression; N = 3; ns, not significant. G, ECAR (first two panels) and OCR at baseline and after addition of 100 μmol/L etomoxir (last two panels). H, 2-NBDG uptake, intracellular lipid droplet staining by BODIPY, and C12 and C16 fatty acid uptake. I, Gene-expression heat map depicting DTA-1 regulation of proliferation and activation-associated genes and (J) metabolic gene transcripts. Individual color blocks represent an average of normalized gene expression from 3 individual experiments. FACS plots are representative of at least three individual experiments. Data are shown as mean ± SD. *, P ≤ 0.05 using Student t test for comparing two groups or ANOVA for multiple groups.

DTA-1–induced cellular proliferation requires increased glycolytic and mitochondrial metabolism, whereas increased IFNγ is glycolysis dependent. A, OCR, (B) ECAR, and (C) 2-NBDG uptake of cells treated with Veh, 2-deoxyglucose (2-DG), or etomoxir (Eto). D, Proliferating cells were gated into cells undergoing 1–3 cell divisions or 4+ cell divisions. Graph represents N = 3 for 2-DG and N = 4 for other groups. E, IFNγ ELISA levels for 2-DG and Eto-treated cells. Cells treated with the ATP synthase inhibitor oligomycin (Oligo) and their (F) OCR, (G) ECAR, (H) 2-NBDG uptake, (I) percent proliferating cells, and (J) IFNγ concentration. K, Representative plot of cellular proliferation with cells treated with Veh and oligo. N = 3 for oligo experiments. Data are shown as mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.05 compared with all other IgG2a treatment groups; ‡, P ≤ 0.05 compared with all other DTA-1 treatment groups, as measured by ANOVA; ns, not significant.

DTA-1 upregulates MAPK signaling and can rescue CD8⁺ T cells from MEK inhibition, in part due to increased PI3K/AKT/mTOR signaling. A, Representative MAPK pathway Western blots from two separate experiments. B, OCR, (C) ECAR, and (D) 2-NBDG uptake of cells incubated with DMSO vehicle (Veh), the p38 inhibitor SB203580 (SB), or the MEK inhibitor PD98059 (PD). E, p70S6k Western blot representative of two experiments. F, 2-NBDG uptake (N = 3), (G) basal OCR (N = 4), and (H) basal ECAR (N = 4) of cells incubated with Veh, PD, the PI3Kδ inhibitor SW30 (SW), or the PD/SW combination (Com). I, CellTrace plots representative of three separate experiments. J, IFNγ levels (left; multiple t tests using Holm–Sidak method). The large difference in concentrations between control and treatment groups required the log transformation of data to compare results between treatment groups (right, N = 3). For F, average percent change is depicted in red. Data are shown as mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.05 compared with all other IgG2a treatment groups; ‡, P ≤ 0.05 compared with all other DTA-1 treatment groups, as measured by ANOVA; ns, not significant.

Checkpoint blockade therapy and anti-GITR therapy combine to overcome inhibition of CD8+ T-cell activation by PD-L1 signaling in vitro. A, Cell viability at 72 Hours. *, P ≤ 0.05 versus all other groups via ANOVA. **, P ≤ 0.05 versus all other PD-L1–inhibited groups via ANOVA. B, OCR and C, ECAR, in CD8⁺ T cells, 4 technical replicates representative of N = 3 separate experiments. D, 2-NBDG uptake and (E) cellular proliferation of PD-L1–inhibited CD8⁺ T cells; representative of N = 3. F, IFNγ concentrations by ELISA; N = 5 individual experiments. Data are shown as mean ± SD. *, P ≤ 0.05 by ANOVA.

DTA-1 treatment in a syngeneic mouse tumor model enhances CD8+ T-cell activation and proliferation in vivo. A, Tumor mass and (B) DLN mass on day 8 after treatment; individual masses from N = 4 separate experiments (8–13 mice per experiment), *, P ≤ 0.05 by Student t test. C, Gene-expression heat map depicting DTA-1 regulation of proliferation-associated genes in DLN. Individual color blocks represent an average of normalized gene expression from 4 individual experiments. D, Granzyme and IFNγ gene transcript levels; *, P ≤ 0.05 by ANOVA. F, Klrg1 gene transcript levels; N = 4; *, P ≤ 0.05 by Student t test. E, Effector/memory staining from DLN. Representative plot from N = 4 separate experiments. Data are shown as mean ± SD.