Fig 2: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces increased CD8 T cell activation in presence of cancer cells. The three anti-PD-L1 variants aPDL1NG (light gray bar), aPDL1WT (dark gray bar), and aPDL1GE (black bar) were tested for their effect on T cell activation (donor A) in allogeneic mixed leukocyte reaction (MLRs) in absence or presence of the cancer cell lines HSC-4 (horizontal stripes), ZR-75-1 (plaid), and Ramos (vertical stripes). MLR without addition of test antibody (medium; white bar) served as negative control. The relative frequencies of CD25+ in CD8 T cells were plotted. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control without presence of cancer wells (*p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001).

Fig 3: Peripheral blood Treg subpopulations in RA. (A) Gating of the total Treg population and low and high CD25 expression populations from the CD3+CD4+ lymphocytes. Flow cytometry plots are representative of one healthy donor and one patient with RA. (B) Various Treg subpopulations in the peripheral blood from healthy donors and patients with RA. Healthy donors, n=22, RA patients, n=21. The data are presented as the median and 25-75% interquartile range. The Mann-Whitney test was used to analyze the data. Lo, low; hi, high; RA, rheumatoid arthritis; Treg, regulatory T cells; ns, not significant.

Fig 4: (a) Temporal upregulation of mHsp90 compared to total Hsp90 levels in T cells stimulated with CD3/CD28 antibodies over a 96-h period. The data were analyzed by 2-way ANOVA with Sidak’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed with 6 biological replicates. (b) Flow cytometric analysis of CD3, CD25, and CD69 T-cell populations relative mHsp90 expression compared to baseline. (c) Upregulation of total Hsp90 in activated T cell populations. Experiments were performed with 3 biological replicates for each timepoint (b, c). (d) Mean (± SD) CD25 expression on T cells 24–96 h post CD3/28 stimulation. Temporal upregulation of mHsp90 (green line) post T cell stimulation. CD25 expression; 6–8 biological replicates per time point. mHsp90 expression 6 biological replicates for each timepoint. (e) mHsp90 and total Hsp90 expression in CD3+ T cells isolated from PBMC’s were stimulated with IL-2 (60 IU/mL) for 24 h. Data represent mean ± SD. The data were analyzed by 2way ANOVA with Sidaks multiple comparison test. Experiments were performed once with 3 biological replicates. (f) Cytokine expression 24 h post Th1 or Th2 T cell stimulation treated with or without HS-131 (1 µM). Data represent mean ± SEM. The data were analyzed by One-way ANOVA with Sidaks multiple comparison test *p < 0.05,**p < 0.01, ***p < 0.001. Experiment represents 3–4 biological replicates per treatment.

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.