Figure 8: Activated T cells deprive GM-DCs of nutrients to alter GM-DC signalling and function.

(a) Schematic detailing the mTORC1/HIF1α/iNOS signalling circuit. (b–j) GM-DCs were pulsed with SIINFEKL peptide for 6 h+/−LPS (100 ng ml⁻¹), washed and purified OT-I T cells were added at different T:DC ratios for 18 h, as indicated. (b) NBDG uptake into GM-DCs and CD8 T cells and (c) levels of phosphorylated S6 ribosomal protein (p-S6) were measured by flow cytometry. (d–f) p-S6 levels were measured by confocal microscopy. GM-DCs were stained with cell tracker violet and T cells with carboxyfluorescein succinimidyl ester (CFSE). Cocultures were treated with rapamycin (Rapa, 20 nM) for the final hour to provide a pS6-negative control (c–e). Representative images (d,f) and pooled data (e) are shown. (g) Nitrite production was measured by the Greiss reaction and (h) IFNγ production by intracellular flow cytometry. (i,j) IFNγ expression in activated CD8 T cells was measured in cocultures using Nos^−/− GM-DCs. Shown is (i) IFNγ MFI and (j) IFNγ-positive CD8 T cells. (k–m) To analyse DC–T-cell interactions in vivo, GM-DCs were pulsed with LPS and SIINFEKL peptide for 1 h, washed, stained with cell tracker violet and injected subcutaneously into the lateral tarsal region of C57/B6 host mice. Four hours later, CFSE-stained purified CD8 OT-I T cells were injected intravenously into the same mice ranging from 5 × 10⁶ to 2.5 × 10⁵ T cells per mouse. Host mice were killed 18 h later and popliteal and inguinal lymph nodes were isolated. The ratio of injected T cells to GM-DCs was determined and pS6 levels were analysed by flow cytometry in the transferred GM-DCs (k,l) and CD8 OT-I T cells (m). Data are mean±s.e.m. or representative of three to five separate experiments (b–j). Data are mean±s.e.m. or representative of results for at least 10 separate lymph nodes in each group, from 16 host mice, data were obtained from two separate experiments (k–m). Data were analysed using a one-way analysis of variance with Tukey’s post test (b–j) or Student’s t-test (k,m) (*P<0.05, **P<0.01). MFI, mean fluorescent intensity.