Fig 2: CD206- and FRß-targeting T cell engagers activate endogenous ascites T cells to kill ascites macrophages. a-e Total unpurified ascites cells from five different patients were cultured for five days with 50 nM BiTEs/TriTEs in medium only or 50% ascites supernatant from the same patient sample. a, b Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, as well as a LIVE/DEAD fixable stain, then analysed by flow cytometry. a % Live residual CD11b+CD64+ cells were calculated relative to “Mock”-treated samples. b Fold-changes in geometric MFI values of CD64, CD80 and CD86 on live CD11b+CD64+ ascites cells were calculated relative to “Mock”-treated samples for each patient sample. c Activation of endogenous CD4+ and CD8+ ascites T cells was assessed by flow cytometric measurement of CD25 expression. d IFN-? levels in the culture supernatants were determined by ELISA. e Numbers of CD4+ and CD8+ cells were determined through addition of counting beads to samples immediately prior to antibody staining. Fold-changes in CD4+ and CD8+ cell count were calculated relative to “Mock”-treated samples. Data show the grand mean ± SD of five individual patient means (calculated from biological triplicate). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (a, c-e) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)

Fig 3: The change in mRNA expression and Treg population by BCG vaccination; direct data on the TSDR region of the Foxp3 gene and quantification of Foxp3 Treg cells. (a) The change in transcripts in CD4 + T cell by BCG was evaluated with mRNA seq. While no prominent changes were shown at year 1, most of Treg genes including FOXP3, TNFRSF18, CD25, IKZF4, CTLA4, FAS and CD45 were significantly upregulated at year 2 and year 3. Meanwhile, IKZF2, CD62L and IL2 showed upregulated mRNA expressions without significant. (b) The left heatmap indicate overall change in mRNA expression after BCG vaccination in each Treg gene. Red and green indicate up- and down-regulation in mRNA, respectively. (c) The average change in methylation (%) in Treg specific demethylated region (TSDR) was shown at baseline, 8 and 58 weeks after BCG vaccination (T1D, n = 4). While the ratio was 83.3 ± 5.5% at baseline, 80.8 ± 3.4% at 8 weeks and 65.5 ± 14.4% at 58 weeks (at 58 weeks, vs. baseline, p = 0.052; vs. 8 weeks, p = 0.08; one-tailed, paired t-test). (d) The representative gating images in flow cytometry are shown. Images of unstained, from one patient at baseline and 58 weeks after BCG vaccination are displayed in right, middle and right, respectively. (e) The percentage of Treg population was analyzed by flowcytometry. The individual data points for Treg frequency pre- and post-therapy were shown (n = 4). While the average ratio was 4.4 ± 1.1% at baseline, it was 6.3 ± 1.4% at 58 weeks (p = 0.01, two-tailed, paired t-test).

Fig 4: Most Treg signature genes gradually demethylate over a three year time course after in vivo BCG vaccinations. Methylation changes in eleven well-known Treg signature genes were quantified yearly after multi-dose BCG vaccine therapy (n = 13 type 1 diabetic subjects). (a) The methylation values are current methylation values at year 1, year 2 or year 3 compared to baseline expressed as average change in beta values. Positive beta values indicate increased methylation and thus decreased gene expression; negative methylation values indicate demethylation and increased gene expression. Significant decreases in methylation after BCG were seen in eight of 11 genes: FOXP3, CD25, IKZF2, IKZF4, CTLA4, TNFR2, CD62L, CD45 and IL2 (p value < 0.05: *; p value < 0.01: **; p value < 0.001: ***). Red bars represent statistically significant change in methylation. (b) The left heatmap shows average difference in beta values for individual Illumina CG Identifiers (CGs) of the Treg signature genes at yearly intervals. Increased blue coloration in the heat map means increased demethylation of the CpG sites within a Treg signature gene. The right heatmap indicates the p-values in the corresponding CpG sites; Blank and red indicate without or with significance, respectively, and degree of red color reflects the strength of significance. In the both heatmaps each individual CpG gene site is represented as a single bar.

Fig 5: Pharmacological inhibition of PDK1 reduces leukemia growth in vitro and in vivo in humanized niche xenograft mouse models.a Western blot analysis of PDH-subunits with/without 10 µM DAP (24 h) in AML cells (a representative of two independent replicates). b (%) DAPI- viable AML cells upon 10, 20 µM DAP (24 h) (HL60 and MOLM13: n = 4; K562: n = 3; THP1: n = 5, each dot shows means of technical triplicates). c as in b, but shows AML primary CD34+ cells (n = 16, dots show technical triplicates) with DAP (3 days) on MS5-stroma. Data was normalized to the controls in panel b, c (mean ± SEM). d PDK1 protein expression (log10) in FLT3-ITD+ (n = 5) and FLT3-wt (n = 6) AML primary CD34+ cells from panel c (mean ± SD, p = 0.030). e Heatmap demonstrating DAP sensitivity in FLT3-ITD + (n = 9), FLT3-wt (n = 7) patient samples from panel c. f Western blot analysis of P-PDH Ser293 in PBMSC (n = 1), in selected AMLs (n = 4) from panel c (a representative of two independent replicates). g ECAR in CD34+ AML patient samples (n = 4 patients, dots show technical replicates) from panel c, f. Equal viable cell numbers were screened. h Fold-change in Annexin V+/DAPI+ cells CD25+/FLT3-ITD+ and CD25-/FLT3-wt subclones with/without increasing concentrations of DAP (48 h) (data was normalized to controls, n = 3 technical replicates shown as representative of the two biological repeats). i Percentage DAPI-/CD34+ NBM (n = 1, technical triplicates), CB cells (n = 6, each dot shows mean of technical triplicates) and PBSCs (sorting as indicated) upon 10, 20 µM DAP (3 days) (n = 3, in technical triplicates in each). j Mean fluorescent intensity of DCFH-DA (ROS) after 3 days DAP treatment (10, 20 µM) in AML primary cells (n = 3, measured in technical triplicates) and k, in healthy CB CD34+ (n = 2, measured in technical triplicates), CD34+ PBSC cells (n = 3 for liquid, n = 2 for MS5-stroma co-cultured). l In vivo experimental set-up for (intrascaffold) KBM7-transplanted (n = 20, 3 scaffolds per mouse) and m, for AML primary CD34+-transplanted mice (n = 20, 4 scaffolds per mouse). Black arrows indicate treatment days. Relative fold-changes in the tumor sizes compared to the start of the treatment in control and DAP (15 mg/kg) (mean ± SEM). d, l, m Student’s t-test (two-sided); b, c, d, h, i, j, k one-way ANOVA Kruskal–Wallis test for multiple comparisons.