Fig 2: PKM2 Tetramerization Blocks T Cell Activation In VitroMurine 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-a production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-a production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-a-producing cells and TNF-a 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.

Fig 3: Molecular compound screening identifies JQ1 that suppresses PD-1 expression of Jurkat cell and T cell.A Flow diagram of the experimental design. B Plot of the ratio of PD-1 expression on Jurkat cells treated with the indicated compounds as detailed in Supplemental Table 5 (n = 3). C Plot of the ratio of PD-1 expression on Jurkat cells stimulated with 150 ng/mL PHA and the same compounds as that shown in Supplemental Table 5 (n = 3). D Jurkat cells were treated with 0.5 μM JQ1 with and without PHA (150 ng/mL) stimulation, and PD-1 expression was determined by FACS at the indicated time points (n = 4). E Jurkat cells were treated with 0.5 μM JQ1 with and without PHA (150 ng/mL) stimulation, and PD-1 expression was determined by qRT-PCR at the indicated time points (n = 3). F Jurkat cells were treated with indicated doses of JQ1 for 24 h in the presence or absence of PHA (150 ng/mL), PD-1 expression was determined by qRT-PCR (n = 5). G Jurkat cells were treated with indicated doses of JQ1 for 24 h in the presence or absence of PHA (150 ng/mL), PD-1 expression was determined by FACS (n = 4). H CD8 + T cells from healthy donors were treated with 0.5 μM JQ1 for 24 h with and without P/I (PMA: 20 ng/L, ionomycin: 0.5 μM) stimulation, and PD-1 expression was determined by FACS (n = 3). I CD4 + T cells from healthy donors were treated with 0.5 μM JQ1 for 24 h in the presence and absence of PHA (500 ng/ml), P/I (PMA: 20 ng/L, ionomycin: 0.5 μM), or anti-CD3/CD28 (anti-CD3: 200 ng/ml, anti-CD28: 200 ng/ml), and PD-1 expression was determined by FACS (n = 4). J CD8 + T cells from healthy donors were treated with 0.5 μM JQ1 for 24 h with and without P/I (PMA: 20 ng/L, ionomycin: 0.5 μM) stimulation, and CD69 expression was determined by FACS (n = 3). K CD4 + T cells and CD8 + T cells were treated with 0.5 μM JQ1 for 24 h, and the secretions of IL-2 were evaluated by ELISA (n = 4). L CD4 + T cells and CD8 + T cells were treated with 0.5 μM JQ1 for 24 h, and the secretions of IFN-γ were evaluated by ELISA (n = 4). M CD4 + T cells and CD8 + T cells were treated with 0.5 μM JQ1 for 24 h, and the secretions of TNF-α were evaluated by ELISA (n = 4). Data in (B–M) were expressed as mean ± SD. n = 3 or more independent biological replicates, presented as individual points. P value < 0.05 was considered to be significant (D–G: two-way ANOVA, B, C, H–M: one-way ANOVA with Bonferroni post hoc test).

Fig 4: BET inhibition reverses the exhausted phenotype of T cells from AML patients.A Flow diagram of the experimental design. B CD4 + T cells (first row) and CD8 + T cells (second row) from bone marrow (BM) of newly diagnosed AML patients were treated with 0.5 μM JQ1 for 24 h, and the percentages of PD-1-positive (first column), Tim-3-positive (second column) and PD-1 and Tim-3 double-positive (third column) cells were determined by FACS. C CD3 + T cells from patients were treated with 0.5 μM JQ1 for 24 h, and the percentages of IL-2, IFN-γ, and TNF-α from CD3 + T cells were evaluated by intracellular flow cytometry. D CD3 + T cells from AML patients (n = 4 samples) were treated with 0.5 μM JQ1 for 24 h, and the percentages of the CD4 + (upper), and CD8 + (lower) subpopulations were shown with differences in the phenotypic distribution of naive (CCR7 + CD45RA + , second column), TCM (CCR7 + CD45RA − , third column), TEM (CCR7 − CD45RA − , fourth column), and TEMRA (CCR7 − CD45RA + , right) T-cell subsets. E © Concatenated files representing AML samples in the DMSO group (upper) and JQ1 (lower) groups projected in tSEN plots after gating on lymphocyte cells. Distribution of naive, TCM, TEM, and TEMRA CD4 + T cells were shown as dark red, dark yellow, dark green, and dark purple, respectively, and the distribution of naive, TCM, TEM, and TEMRA CD8 + T cells were shown as light red, light yellow, light green, and light purple, respectively. F CD3 + T cells from AML patients (n = 4 samples) were treated with 0.5 μM JQ1 for 24 h, and the percentages of PD-1 positive (upper row) and Tim-3 positive (lower row) cells in naive (left), TCM (second column), TEM (third column), and TEMRA (right) CD4 + T cells were determined by FACS. Data are expressed as mean ± SD. P value < 0.05 was considered to be significant (two-tailed paired Student’s t tests).