Figure 3. SphK1^−/− T Cells Exhibit Improved Migration and Foxo1 Activity.

(A) Three day-activated pMel or pMel-SphK1^−/− T cells were adoptively transferred into Rag1^−/− mice (n = 3). 25 days of post-transfer B16-F10 cells were s.c. injected. Upper panel: representative flow cytometric analysis done to determine the percentage of TCR transgenic T cells retrieved from spleen, lymph nodes, blood, liver, and lung after 5 days of tumor injection. Lower panel: cumulative data from different mice.

(B) Cell surface expression of KLRG1 and CD62L was determined on the retrieved cells from (A).

(C) Flow cytometry analysis of phosphorylated Foxo1 (S256) in WT, SphK1^−/−, and SphK1^−/− T cells treated with S1P (5µM) after 3 days of TCR activation.

(D) Confocal images of the indicated cell stained with Foxo1 (in green) and nuclei stained with DAPI (in blue). Lower panels: magnified images (red boxes) of the upper panels. Scale bars represent 10 µm in upper panels and 5 µm in lower panels.

(E) Foxo1 activity in WT, SphK1^−/−, and SphK1^−/− cells T cells treated with S1P (5 µM) in 3 days TCR-activated T cells, determined using the commercially available kit as per vendor’s protocol.

(F) Flow cytometry analysis of S1PR1 on 3 days TCR-activated WT and SphK1^−/− T cells.

(G and H) SphK1^−/− T cells were activated in presence of AS1842856 and cell surface expression of CD62L/CD44 (G), and S1PR1 was determined using flow cytometry (H). The numerical values within the FACS overlay plots indicate MFI, and the adjacent bar diagrams represent cumulative data from multiple repeat experiments. Data are representative of at least three independent experiments and error bars represent mean ± SD in (B)–(H). *p < 0.05, **p < 0.01.

See also Figure S3.