Figure 2.

PKM2 Tetramerization Blocks T Cell Activation In Vitro

Murine CD4⁺CD62⁺ T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM.

(A and B) Cells were collected after 24 h of stimulation.

(A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments).

(B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments).

(C–F) Cells were collected after 3 days of stimulation.

(C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments).

(D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments).

(E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments).

(F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001 compared to CTRL condition, by one-way ANOVA with Dunnett's post-hoc test.