Figure 1. Activated SphK1^−/− T Cells Preserve Tcm Phenotype.

(A) Tumor infiltrating lymphocytes (TILs) and splenocytes were isolated from healthy or B16-F10 murine melanoma bearing C57BL/6 mice, and flow cytometry-based determination of intracellular SphK1 levels in CD8⁺ T cells was performed.

(B) CFSE dilution was analyzed by flow cytometry to determine the antigen-specific proliferation in activated pMel and pMel-SphK^−/− T cells.

(C–E) Antigen-activated pMel and pMel-SphK^−/− splenic T cells were used for determining the flow cytometry-based cell surface expression of (C) CD25 and CD69, (D) CD62L and CD44, and (E) Sca1, CD127, and CD122.

(F) Schematic diagram showing the strategy to generate memory T cells in vitro that were used for data in (G).

(G) Cells obtained using strategy in (F) with different cytokines post day 3 (IL-2 at 100 IU/mL, and IL-15 at 100 ng/mL, respectively) were analyzed for determining the flow cytometry-based cell surface expression of CD62L and intracellular expression of SphK1.

(H) Day 3 TCR-activated WT T cells were transfected with either WT vector or empty vector for SphK1 enzyme. Cells were subsequently cultured for another 3 days, either in presence of IL-2 (100 IU/mL), or IL-15 (100 ng/mL). After 3 days, expression of CD62L and SphK1 was evaluated in CD8⁺ T cells by flow cytometry.

(I) Day 3 TCR-activated WT T cells were further cultured for 3 days, either in presence of IL-2 (100 IU/mL) or in presence of TGF-β (5 ng/mL) and IL-2 (100 IU/mL). Cells were then used to determine the intracellular expression of SphK1. Data were analyzed using FlowJo software. The numerical values within the fluorescence-activated cell sorting (FACS) overlay plots indicate MFI, and the adjacent bar diagrams represent cumulative data from at least three repeat experiments. Error bars represent mean ± SD; *p < 0.05.

See also Figure S1.