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"Volkmar, Michael"
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The dumbbell workout handbook : weight loss : the best workouts for torching fat and burning calories like never before
Developed by best-selling fitness author and strength and conditioning expert Mike Volkmar, this collection of dumbbell workouts will challenge you by promoting total body fitness while burning away unwanted fat. These results-oriented workouts target all muscle groups, from chest and arms to core and legs, and provide a powerful combination of exercises designed to help sculpt and tone your ideal body. The Dumbbell Workout Handbook: Weight Loss is a great way to change up your routine and break through plateaus. Whether you train at home in your garage gym or at the local fitness club, your workouts will never be boring again, guaranteed! So grab some iron and get fit now!
Book
TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS
TET proteins convert 5‐methylcytosine to 5‐hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with
O
‐GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3–OGT co‐localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3–OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step‐wise model, involving TET–OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.
This paper identifies the N‐acetylglucosamine transferase OGT as binding partner for TET2/3 proteins. Their genome‐wide chromatin binding and the characterization of the Set1/COMPASS complex as OGT target implies coordinated gene regulation.
Journal Article
Proimmunogenic impact of MEK inhibition synergizes with agonist anti-CD40 immunostimulatory antibodies in tumor therapy
Cancer types with lower mutational load and a non-permissive tumor microenvironment are intrinsically resistant to immune checkpoint blockade. While the combination of cytostatic drugs and immunostimulatory antibodies constitutes an attractive concept for overcoming this refractoriness, suppression of immune cell function by cytostatic drugs may limit therapeutic efficacy. Here we show that targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) does not impair dendritic cell-mediated T cell priming and activation. Accordingly, combining MEK inhibitors (MEKi) with agonist antibodies (Abs) targeting the immunostimulatory CD40 receptor results in potent synergistic antitumor efficacy. Detailed analysis of the mechanism of action of MEKi shows that this drug exerts multiple pro-immunogenic effects, including the suppression of M2-type macrophages, myeloid derived suppressor cells and T-regulatory cells. The combination of MEK inhibition with agonist anti-CD40 Ab is therefore a promising therapeutic concept, especially for the treatment of mutant Kras-driven tumors such as pancreatic ductal adenocarcinoma.
Immune checkpoint inhibitors have limited efficacy in tumors with lower mutational burden and non-permissive microenvironment. Here, the authors show that combining MEK inhibition with an agonist anti-CD40 immunostimulatory antibody improves antitumor treatment by inducing immunogenic changes in the tumor microenvironment.
Journal Article
Using combined single-cell gene expression, TCR sequencing and cell surface protein barcoding to characterize and track CD4+ T cell clones from murine tissues
Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4 + T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat , illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4 + T cells from murine tissues.
Journal Article
Identification of TRDV-TRAJ V domains in human and mouse T-cell receptor repertoires
Here, we describe the identification of two T-cell receptors (TRs) containing TRDV genes in their TRA chains, the first one in human and the second one in mouse. First, using 5’RACE on a mixed lymphocyte-tumor cell culture (MLTC), we identified TRDV1 5’-untranslated region (UTR) and complete coding sequence rearranged productively to TRAJ24. Single-cell TR RNA sequencing (RNA-seq) of the MLTC, conducted to identify additional clonotypes, revealed that the analysis software detected the hybrid TRDV-TRAJ TRA (TRA) chain but excluded it from the final results. In a separate project, we performed TR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Here, the predominant clonotype contained a TRA chain with a TRDV2-2-TRAJ49 rearrangement. Again, the hybrid TRA chain was not reported in the final results. Transfection of both TR cDNAs resulted in cell surface localization of TR together with CD3, suggesting a productive protein in both cases. Tumor recognition of the Homo sapiens (Homsap) TRDV1-containing TR could be demonstrated by IFN Gamma ELISA ELISpot kit, whereas the Mus musculus (Musmus) TR did not recognize a tumor-derived cell line. To determine whether the TRDV-containing TRA chains we detected were rare events or whether TRDV genes are commonly incorporated into TRA chains, we queried the NCBI Sequence Read Archive for TR single-cell RNA-seq data and analyzed 21 human and 23 murine datasets. We found that especially Homsap TRDV1, Musmus TRDV1, and to some extent Musmus TRDV2-2 are more commonly incorporated into TRA chains than several TRAV genes, making those TRDV genes a relevant contribution to TRA diversity. TRDV-containing TRA chains are currently excluded from the final results of V-(D)-J dataset analyses with the CellRanger software. We provide a work-around to avoid exclusion of those hybrid TRA chains from the final analysis results.
Journal Article
A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences
To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αβTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order β-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.
Journal Article
Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor-Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display
Tumor-infiltrating lymphocytes (TIL) are considered enriched for T cells recognizing shared tumor antigens or mutation-derived neoepitopes. We performed exome sequencing and HLA-A
02:01 epitope prediction from tumor cell lines from two HLA-A2-positive melanoma patients whose TIL displayed strong tumor reactivity. The potential neoepitopes were screened for recognition using autologous TIL by immunological assays and presentation on tumor major histocompatibility complex class I (MHC-I) molecules by Poisson detection mass spectrometry (MS). TIL from the patients recognized 5/181 and 3/49 of the predicted neoepitopes, respectively. MS screening detected 3/181 neoepitopes on tumor MHC-I from the first patient but only one was also among those recognized by TIL. Consequently, TIL enriched for neoepitope specificity failed to recognize tumor cells, despite being activated by peptides. For the second patient, only after IFN-γ treatment of the tumor cells was one of 49 predicted neoepitopes detected by MS, and this coincided with recognition by TIL sorted for the same specificity. Importantly, specific T cells could be expanded from patient and donor peripheral blood mononuclear cells (PBMC) for all neoepitopes recognized by TIL and/or detected on tumor MHC-I. In summary, stimulating the appropriate inflammatory environment within tumors may promote neoepitope MHC presentation while expanding T cells in blood may circumvent lack of specific TIL. The discordance in detection between physical and functional methods revealed here can be rationalized and used to improve neoantigen-targeted T cell immunotherapy.
Journal Article
DNA methylation profiling reveals a predominant immune component in breast cancers
Breast cancer is a molecularly, biologically and clinically heterogeneous group of disorders. Understanding this diversity is essential to improving diagnosis and optimizing treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease are still poorly understood. By means of DNA methylation profiling of 248 breast tissues, we have highlighted the existence of previously unrecognized breast cancer groups that go beyond the currently known ‘expression subtypes’. Interestingly, we showed that DNA methylation profiling can reflect the cell type composition of the tumour microenvironment, and in particular a T lymphocyte infiltration of the tumours. Further, we highlighted a set of immune genes having high prognostic value in specific tumour categories. The immune component uncovered here by DNA methylation profiles provides a new perspective for the importance of the microenvironment in breast cancer, holding implications for better management of breast cancer patients.
Journal Article
Enhanced mutanome analysis towards the induction of neoepitope-reactive T-cell responses for personalized immunotherapy of pancreatic cancer
BackgroundPersonalized immunotherapy of pancreatic ductal adenocarcinoma (PDAC) through T-cell mediated targeting of tumor-specific, mutanome-encoded neoepitopes may offer new opportunities to combat this disease, in particular by countering recurrence after primary tumor resection. However, the sensitive and accurate calling of somatic mutations in PDAC tissue samples is compromised by the low tumor cell content. Moreover, the repertoire of immunogenic neoepitopes in PDAC is limited due to the low mutational load of the majority of these tumors.MethodsWe developed a workflow involving the combined analysis of next-generation DNA and RNA sequencing data from matched pairs of primary tumor samples and patient-derived xenograft models towards the enhanced detection of driver mutations as well as single nucleotide variants encoding potentially immunogenic T-cell neoepitopes. Subsequently, we immunized HLA/human T-cell receptor (TCR) locus-transgenic mice with synthetic peptides representing candidate neoepitopes, and molecularly cloned the genes encoding TCRs targeting these epitopes.ResultApplication of our pipeline resulted in the identification of greater numbers of non-synonymous mutations encoding candidate neoepitopes with increased confidence. Furthermore, we provide proof of concept for the successful isolation of HLA-restricted TCRs from humanized mice immunized with different neoepitopes, several of which would not have been selected based on mutanome analysis of PDAC tissue samples alone. These TCRs mediate specific T-cell reactivity against the tumor cells in which the corresponding mutations were identified.ConclusionEnhanced mutanome analysis and candidate neoepitope selection increase the likelihood of identifying therapeutically relevant neoepitopes, and thereby support the optimization of personalized immunotherapy for PDAC and other poorly immunogenic cancers.
Journal Article