Figure 2. SphK1−/− T Cells Secrete Both IFN-γ and IL-17 upon TCR Stimulation.
(A–C) pMel and pMel-SphK−/− T cells activated for 3 days with cognate antigen gp100 were antigen-restimulated overnight, and the supernatant was used for ELISA-based determination of secreted cytokine: (A) IFN-γ, (B) IL-17, (C) RORγt and IRF4 levels.
(D) Splenic T cells from IL-17-GFP reporter or SphK1−/−-IL-17-GFP mice were cultured in Th17 polarizing condition for 3 days, restimulated with anti-CD3 (2 µg/mL) and anti-CD28 antibody (5 µg/mL) for 4 h, and the percentage of IL-17-GFP-positive cells were determined by flow cytometry.
(E) Three-day TCR-activated WT and SphK1−/− T cells were analyzed for expression of stemness-associated genes using mRNA. Data are representative of three independent experiments in (A)–(E).
(F) C57BL/6 mice (n = 10 mice/group) were inoculated subcutaneously (s.c.) with 0.3 × 106 B16-F10 melanoma cells for 9 days, after which mice were either kept untreated or adoptively transferred, either with 1 × 106 pMel or pMel-SphK1−/− T cells. Tumor growth was measured using digital calipers twice weekly. Left panel: estimated growth curves for each experimental condition. Right panel: survival curve (p value pMel versus pMel SphK1−/− is 1e–05).
(G) Upper panel: C57BL/6 mice (n = 5 mice/group) were inoculated (s.c.) with 0.25 × 106 B16-F10 melanoma cells for 14 days, then 1 × 106 3 days gp100-activated pMel T cells, pMel SphK1−/− T cells, or pMel T cells treated with PF543 (1 µM) were adoptively transferred. After 15 days, lymphocytes were retrieved from the excised tumor to determine the percentage of TCR transgenic T cell. Lower panel: retrieved lymphocytes (as in upper panel) were stimulated overnight with gp100 peptide antigen before staining with fluorochrome-conjugated antibodies to determine intracellular IFN-γ levels.
(H) Upper panel: representative flow cytometric analysis of donor cells retrieved from the lung, liver, spleen, and lymph nodes 48 h after adoptive transfer of pMel or pMel-SphK−/− T cells into Rag1−/− mice (n = 3). Lower panel: composite data from different mice.
(I) C57BL/6 mice (n = 10–15/group) were inoculated (s.c.) with 0.3 × 106 B16-F10 melanoma cells for 9 days before injecting cyclophosphamide (CTX). After 24 h of CTX injection, tumor-bearing C57BL/6 were either kept untreated or adoptively transferred with 1 × 106 3 days gp100-activated pMel T cells, or pMel T cells treated with PF543 (1 µM). In the indicated groups, following ACT, recipient mice were injected intraperitoneally (i.p.) either with PF543 or anti-PD1 Ab (200 µg/mouse), or both PF543 and anti-PD1 Ab. Both PF543 and anti-PD1 Ab were given three times in a week. Upper panel: tumor growth measured using digital calipers. Lower panel: survival curve. Error bars represent mean ± SD in (A)–(I). *p < 0.05, **p < 0.01, ***p < 0.001.
See also Figure S2.