Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant ...clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.
•Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases.•Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.
Regulatory CD4
T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation ...in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4
cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.
Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor ...fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and also to clarify whether the CreERT2 system displays background activity, we generated a knock-in mouse line harboring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26-CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo and partial recombination in peripheral and hematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry, we found low-level background recombination in noninduced bitransgenic ROSA26-CreERT2/EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26-CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay, cancer induction was completely restricted to induced bitransgenic CreERT2/K-Ras(V12) mice, whereas noninduced control animals did not show any sign of cancer, indicating the usefulness of the ROSA-CreERT2 system for regulating conditional gene expression in vivo. The ROSA26-CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.
CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells ...into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.
Treatment of cancers by means of viruses, that specifically replicate in (oncotropism) and kill (oncolysis) neoplastic cells, is increasingly gaining acceptance in the clinic. Among these agents, ...parvoviruses have been shown to possess not only direct oncolytic but also immunomodulating properties, serving as an adjuvant to prime the immune system to react against infected tumors. Here, we aimed to establish whether immunomodulating mechanisms participate in the recently reported therapeutic potential of parvoviruses against pancreatic carcinoma. Using adoptive transfer experiments we discovered that the transfer of splenocytes of donor rats harboring H-1PV-treated orthotopic PDAC tumors could significantly prolong the survival of naïve tumor-bearing recipients, compared to those receiving cells from mock-treated donors. Closer investigation of immunological parameters in infected donor rats revealed that virus-induced interferon gamma production and cellular immune response played an important role in this effect. These data have also preclinical relevance since abortive H-1PV infection of human peripheral blood mononuclear cells or cocultivation of these cells with H-1PV-preinfected pancreatic cancer cells, resulted in enhancement of innate and adaptive immune reactivity. Taken together our data reveal that oncolytic H-1PV modulates the immune system into an anticancer state, and further support the concept of using parvoviruses in the fight against pancreatic cancer.
Background The transcriptional regulation of cytokines released and controlled by memory T cells is not well understood. Defective IFN-γ production in allergic asthma correlates in human beings with ...the risk of wheezing in childhood. Objective To understand the role of the transcription factor nuclear factor of activated T cells 2 (NFATc2) in memory and effector T cells in the airways in experimental allergic asthma. Methods We used murine models of allergic asthma and adoptive cell transfer of fluorescence-activated sorted cells in a disease model. Results Mice lacking NFATc2 developed an increase in airway hyperresponsiveness (AHR), remodeling, and serum IgE levels on ovalbumin sensitization. This phenotype was associated with CD8+ CD122− T cells deficient in IFN-γ production in the airways. The origin of this phenotype in NFATc2(−/−) mice was related to an expanded population of lung CD8+ CD122+ (IL-2Rβ chain) CD127hi (IL-7 receptor R α chain+ ) long-lived memory cells. Adoptive transfer of ovalbumin-specific CD8+ NFATc2(−/−) T cells enhanced the AHR generated by NFATc2(−/−) CD4+ T cells in immunodeficient mice, increased IL-17, and reduced IFN-γ production in the reconstituted mice. Depletion of the memory CD8+ CD122+ IL-7Rhigh T-cell population corrected the defect in IFN-γ production by lung NFATc2(−/−) CD8+ CD122− cells and abrogated the increased AHR observed in NFATc2(−/−) CD8+ T-cell–reconstituted mice with a severe combined immunodeficiency disorder. Conclusion Taken together, our results suggest that NFATc2 expression in long-lived memory CD8+ T cells controls IL-2 and IFN-γ production in lung CD8+ T cells, which then limits TH 17 and TH 2 development in the airways during allergen challenge.
Ammonia (NH3) is a promising fuel, because it is carbon-free and easier to store and transport than hydrogen (H2). However, an ignition enhancer such as H2 might be needed for technical applications, ...because of the rather poor ignition properties of NH3. The combustion of pure NH3 and H2 has been explored widely. However, for mixtures of both gases, mostly only global parameters such as ignition delay times or flame speeds were reported. Studies with extensive experimental species profiles are scarce. Therefore, we experimentally investigated the interactions in the oxidation of different NH3/H2 mixtures in the temperature range of 750–1173 K at 0.97 bar in a plug-flow reactor (PFR), as well as in the temperature range of 1615–2358 K with an average pressure of 3.16 bar in a shock tube. In the PFR, temperature-dependent mole fraction profiles of the main species were obtained via electron ionization molecular-beam mass spectrometry (EI-MBMS). Additionally, for the first time, tunable diode laser absorption spectroscopy (TDLAS) with a scanned-wavelength method was adapted to the PFR for the quantification of nitric oxide (NO). In the shock tube, time-resolved NO profiles were also measured by TDLAS using a fixed-wavelength approach. The experimental results both in PFR and shock tube reveal the reactivity enhancement by H2 on ammonia oxidation. The extensive sets of results were compared with predictions by four NH3-related reaction mechanisms. None of the mechanisms can well predict all experimental results, but the Stagni et al. React. Chem. Eng. 2020, 5, 696–711 and Zhu et al. Combust. Flame 2022, 246, 115389 mechanisms perform best for the PFR and shock tube conditions, respectively. Exploratory kinetic analysis was conducted to identify the effect of H2 addition on ammonia oxidation and NO formation, as well as sensitive reactions in different temperature regimes. The results presented in this study can provide valuable information for further model development and highlight relevant properties of H2-assisted NH3 combustion.
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