Cytokine secretion patterns of TLR-3-DCs, cc-3-DCs and IL10-3-DCs.

<p>DCs generated from peripheral blood of healthy donors were analyzed for their cytokine secretion patterns (n = 9). (A) Mature DCs were cocultured with CD40L expressing mouse fibroblasts for 24 hours, the concentration of IL-12p70 and IL-10 in the supernatants was measured by CBA, and the difference to the basal secretion of the same cell populations without CD40 ligation was calculated. (B) Comparison of the IL-12p70/IL-10 ratio for TLR-3-DCs, cc-3-DCs and IL10-3-DCs.</p

IL-12p70 dependency of NK cell activation by TLR-3-DCs.

<p>(A/B) DCs generated from peripheral blood of 10 healthy donors were cocultured with autologous monocyte-depleted (non-adherent) PBMCs for 24 hours with addition of IL-2. Activation of NK cells was analyzed by intracellular IFN-γ staining of CD3⁻CD56⁺ cells (A; circles represent single experiments, the mean is displayed as horizontal line) and by ELISA measurement of IFN-γ in the supernatant (B; box-and-whisker plots). (C/D) In a similar set of experiments, cocultures of TLR-3-DCs and autologous monocyte-depleted PBMCs were compared with or without addition of IL-12p70 blocking antibody. Activation of NK cells was analyzed by intracellular IFN-γ staining of CD3⁻CD56⁺ cells (C; n = 13) and by ELISA measurement of IFN-γ in the supernatant (D; n = 8). *, p<0.05; **, p<0.01.</p

Effect of costimulation and IL-12p70 blockade on Th1 polarization by TLR-3-DCs.

<p>TLR-3-DCs generated from peripheral blood of 6 healthy donors were cocultured with CD3-selected autologous T cells for 4 days either alone or with addition of various combinations of blocking antibodies. Supernatants were analyzed for secretion of IFN-γ by ELISA. T cells without DC stimulation were used as a negative control. Box-and-whisker plots for the different conditions are shown. *, p<0.05.</p

Costimulatory profiles of TLR-3-DCs and cc-7-DCs.

<p>TLR-3-DCs and cc-7-DCs were generated from peripheral blood of healthy donors, and expression of various costimulatory markers was analyzed by flow cytometry. *, p<0.05; **, p<0.01. (A) Expression of the cell surface antigens on both DC populations (n = 7 for B7-H4, n = 8 for CD276, n = 10 for all other markers). (B) Comparison of the CD86/CD274 ratio for TLR-3-DCs and cc-7-DCs (n = 10).</p

Preferential induction of activated, IFN-γ secreting T cells by TLR-3-DCs.

<p>(A to C) TLR-3-DCs and cc-7-DCs generated from peripheral blood of healthy donors were cocultured with autologous monocyte-depleted (non-adherent) PBMCs for 24 hours, and antigen-independent stimulatory capacity of the DCs on regulatory and activated T cells was quantified using flow cytometry with the markers CD4, CD25, CD127 and FoxP3. *, p<0.05; **, p<0.01. (A) Data from one representative donor. Gating strategies used to determine different T cell subsets. (B) Capacity of TLR-3-DCs and cc-7-DCs to stimulate CD4⁺CD25⁺ T cells and their regulatory (FoxP3⁺CD127^-) and non-regulatory activated (FoxP3^-CD127⁺) subsets. Differences between stimulated and unstimulated cells are shown (n = 10). (C) Ratio of activated and regulatory T cells induced by coculture. (D) TLR-3-DCs, cc-3-DCs and IL10-3-DCs were cocultured with CD3-selected autologous T cells for 4 days. Supernatants were analyzed for secretion of IFN-γ, IL-4 and IL-17A by CBA. Differences between stimulated and unstimulated cells are shown (n = 10).</p

Data_Sheet_1.PDF

<p>Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor–ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4⁺ and CD8⁺ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.</p

FLT3-ITD mutants induce STAT5 and Tyr 591 phosphorylation whereas FLT3 point mutations rely on AKT and MAPK activation.

<p>(A) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours prior to lysis. Blots were probed against STAT5 pTyr694, AKT pSer473 and MAPK pThr202/pTyr204 stripped and subsequently reprobed against total STAT5, AKT and MAPK. One out of three independent representative experiments is shown. (B) Semi-quantitive analysis of (p)STAT5 band intensity was performed to calculate the ratio between pSTAT5 and total STAT5. Values are expressed as mean +/− S.D. of three independent experiments. (*) indicates significance to FLT3-WT expressing cells. The signal intensity of Ba/F3 MIY expressing cells has been subtracted from FLT3-WT and FLT3 mutant values. (C) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours prior to lysis. Equal amount of whole cell lysates were used in a chemiluminescent ELISA assay detecting phosphorylation of FLT3 receptor at Tyr591. Values are expressed as mean +/− S.D. of three independent experiments. Grouped analysis of FLT3 mutants revealed significant differences between FLT3-WT expressing cells and FLT3-WT-like cell lines (p = 0.045) as well as FLT3-WT expressing cells and FLT3-ITD cell lines (p = 0.005). Further the group of FLT3-ITD cell lines showed significant differences compared to FLT3-WT-like mutants (p = 0.008) and FLT3-TKD cell lines (p≤0.001). (*) indicates significance of grouped analyses and individually compared to FLT3-WT expressing cells. The signal intensity of Ba/F3 MIY expressing cells has been subtracted from FLT3-WT and FLT3 mutant values. RLU: Relative light units. (D) 4×10⁴ cells/ml stably transduced with indicated constructs, were cultured in presence or absence of 2 µM MK 2206 and/or 10 ng/ml IL-3. Cells were counted after 72 hours by trypan blue exclusion. (*) indicates significant growth reduction by MK 2206 compared to untreated cells. Proliferation with MK 2206 is shown in relation to untreated cells. Control indicates for Ba/F3 MIY expressing cells treated with 2 µM MK 2206 in the presence of IL-3. Values are expressed as mean +/− S.D. of three independent experiments.</p

Glycosylation pattern and cell surface expression of FLT3 receptor differ in FLT3 mutants.

<p>(A) FLT3 mutants were cultured without IL-3 for 24 hours prior to lysis. One out of three independently representative experiments is shown. (B) To illustrate the expression of the 150/130 kDa forms of FLT3 semi-quantitive analyses of western blot band intensity were performed. Depicted is the ratio of the 150 kDa to the 130 kDa FLT3 form. Values are expressed as mean +/− S.D. of three independent experiments. Ratio of 150 kDa to 130 kDa form of FLT3 was significantly different for each FLT3-TKD (p≤0.009) and each FLT3-ITD (p≤0.002) cell line, but not for FLT3-V592A and -I867S expressing cells compared to FLT3-WT expressing cells. FLT3-WT expressing cells showed a significant differnence to the groups of FLT3-WT-like mutants (p = 0.039), FLT3-TKD mutants (≤0.001) and FLT3-ITD mutants (p≤0.001). Further FLT3-WT like mutants were significantly different compared to the FLT3-TKD group (p≤0.001) and FLT3-TKD mutants were significantly different compared to FLT3-ITD cell lines (p = 0.004). (*) indicates significance of grouped analyses and individually compared to FLT3-WT expressing cells. (C) Ba/F3 cell lines stably expressing the indicated FLT3 constructs were stained with CD-135 antibody and analyzed by flow cytometry. Expression is depicted as difference of geometric mean to isotype control (ΔGmean). Values are expressed as means +/− S.D. of four independent experiments. (*) indicates significance compared to FLT3-WT expressing cells.</p

Multivariable analysis of prognostic factors for OS, RFS and CR.

<p>To evaluate the independent prognostic impact of FLT3-ITD and FLT3-TKD on OS, RFS and CR, all candidate prognostic factors were included in the Cox regression model without selection of variables. The analyses were performed using 535 complete datasets of patients with regard to OS for the candidate prognostic factors. HR: Hazard Ratio; CI: confidence interval; CL: confidence limit.</p

Prognostic impact and genetic stability of FLT3-ITD and FLT3-TKD mutations in AML.

<p>(A) OS in 672 patients was significantly different between <i>FLT3</i>-mutated patients compared to patients with <i>FLT3</i>-WT*. OS in patients with <i>FLT3</i>-TKD mutations was not different compared to <i>FLT3</i>-WT* expressing patients (HR: 0.8, 95% CI: 0.7–1.7). Patients with a <i>FLT3</i>-ITD showed a significantly worse OS compared to those with <i>FLT3</i>-WT* (HR: 1.4, 95% CI: 1.1–1.8). There was no significant difference in the OS of patients with both <i>FLT3</i>-ITD and <i>FLT3</i>-TKD mutation compared to those with <i>FLT3</i>-ITD (HR 1.1, 95% CI: 0.3–3.4). (B) RFS in 443 patients in first complete remission was significantly different between <i>FLT3</i>-mutated patients compared to patients with <i>FLT3</i>-WT*. Patients with <i>FLT3</i>-TKD mutation had no significant superior RFS compared to <i>FLT3</i>-WT* (HR: 0.8, 95% CI: 0.4–1.4). Patients with a <i>FLT3</i>-ITD showed a significantly worse RFS compared to those with <i>FLT3</i>-WT* (HR: 1.7, 95% CI: 1.3–2.2). There was no significant difference in the RFS of patients with both <i>FLT3</i>-ITD and <i>FLT3</i>-TKD mutation compared to those with <i>FLT3</i>-ITD (HR 1.2, 95% CI: 0.3–5.0). (C) 10 of 113 patients positive for <i>FLT3</i>-WT* at diagnosis acquired a <i>FLT3</i> mutation at relapse. 27 of 35 (77%) patients with a <i>FLT3</i>-ITD at initial diagnosis displayed a <i>FLT3</i>-ITD at relapse, whereas the majority of patients with a <i>FLT3</i>-TKD mutation (7/11; 63%) lost this mutation at relapse. (D) <i>FLT3</i>-ITD mRNA levels in 20 patients with a <i>FLT3</i>-ITD at diagnosis and at relapse were calculated. <i>FLT3</i>-ITD mRNA levels were calculated as following: (<i>FLT3</i>-ITD/<i>FLT3</i>-WT)/(<i>FLT3</i>-ITD/<i>FLT3</i>-WT+1). Median <i>FLT3</i>-ITD mRNA level was significantly higher at the time of relapse compared to first diagnosis (0.54 [range 0.37–1.00] versus 0.40 [range 0.08–0.88]; p&lt;0.001, Wilcoxon test).</p