Explore the vast range of titles available.

Song et al . present the first method for global analysis of 5-hydroxymethylcytosine, a recently identified epigenetic modification in mammalian cells. They use a bacteriophage-derived enzyme to tag the hydroxymethyl group with an azide-modified glucose residue that can be used for affinity purification and sequencing of modified genomic DNA fragments. In contrast to 5-methylcytosine (5-mC), which has been studied extensively 1 , 2 , 3 , little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types 4 , 5 . Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC–containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized 4 . We also find a gene expression level–dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual’s genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus. Significance Human B cells secrete highly diverse antibody molecules to recognize and defend against infectious agents. Developing B cells independently rearrange their genomes to produce antibody-encoding sequences. It is uncertain to what degree genetic factors control antibody repertoires and the antibodies elicited by defined antigenic stimuli. Analysis of 134,000 antibody heavy chain sequences from genetically identical twins vaccinated with varicella-zoster vaccine indicates that twins show increased correlation in antibody gene segment usage, junctional features, and mutation rates in their antibody pools but show little similarity in clonal responses to an acute stimulus. Therefore, a shared germ-line genome sequence is correlated with overall convergence of antibody repertoires, but the particular antibody response to a given vaccination is less predictable.

BackgroundA first-in-human, randomized pilot phase II clinical trial combining vaccines targeting overexpressed, non-mutated tumor blood vessel antigens (TBVA) and tyrosine kinase inhibitor dasatinib was conducted in human leukocyte antigen (HLA)-A2+ patients with advanced melanoma.MethodsPatient monocyte-derived type-1-polarized dendritic cells were loaded with HLA-A2-presented peptides derived from TBVA (DLK1, EphA2, HBB, NRP1, RGS5, TEM1) and injected intradermally as a vaccine into the upper extremities every other week. Patients were randomized into one of two treatment arms receiving oral dasatinib (70 mg two times per day) beginning in week 5 (Arm A) or in week 1 (Arm B). Trial endpoints included T cell response to vaccine peptides (interferon-γ enzyme-linked immunosorbent spot), objective clinical response (Response Evaluation Criteria in Solid Tumors V.1.1) and exploratory tumor, blood and serum profiling of immune-associated genes/proteins.ResultsSixteen patients with advanced-stage cutaneous (n=10), mucosal (n=1) or uveal (n=5) melanoma were accrued, 15 of whom had previously progressed on programmed cell death protein 1 (PD-1) blockade. Of 13 evaluable patients, 6 patients developed specific peripheral blood T cell responses against ≥3 vaccine-associated peptides, with further evidence of epitope spreading. All six patients with specific CD8+ T cell response to vaccine-targeted antigens exhibited evidence of T cell receptor (TCR) convergence in association with preferred clinical outcomes (four partial response and two stabilization of disease (SD)). Seven patients failed to respond to vaccination (one SD and six progressive disease). Patients in Arm B (immediate dasatinib) outperformed those in Arm A (delayed dasatinib) for immune response rate (IRR; 66.7% vs 28.6%), objective response rate (ORR) (66.7% vs 0%), overall survival (median 15.45 vs 3.47 months; p=0.0086) and progression-free survival (median 7.87 vs 1.97 months; p=0.063). IRR (80% vs 25%) and ORR (60% vs 12.5%) was greater for females versus male patients. Tumors in patients exhibiting response to treatment displayed (1) evidence of innate and adaptive immune-mediated inflammation and TCR convergence at baseline, (2) on-treatment transcriptional changes associated with reduced hypoxia/acidosis/glycolysis, and (3) increased inflammatory immune cell infiltration and tertiary lymphoid structure neogenesis.ConclusionsCombined vaccination against TBVA plus dasatinib was safe and resulted in coordinating immunologic and/or objective clinical responses in 6/13 (46%) evaluable patients with melanoma, particularly those initiating treatment with both agents.Trial registration numberNCT01876212.

B-cell neoplasms possess clonal B-cell receptor rearrangements (BCR clonotype lineages) that can be identified by sequencing the B-cell repertoire for use in diagnostics, risk stratification, and high-sensitivity monitoring. BCR somatic hypermutation (SHM) can result in clonality detection failure from point mutations in PCR primer binding regions, often necessitating splitting samples into multiple reactions which increases test costs, turnaround times, and sample requirements. We evaluated the Oncomine BCR Pan-Clonality Assay, a novel single-tube PCR reaction that simultaneously amplifies all BCR loci for next-generation DNA sequencing, using neoplastic B-cell lines and clinical research samples from multiple myeloma (MM) patients, a plasma cell neoplasm associated with high SHM levels. The assay showed a linear detection range down to 1 ng of clonal DNA input, sensitivity to 10−6 in a polyclonal background, and high reproducibility. Clonotype lineages were identified in 42/45 (93%) MM samples. Ion Reporter software packaged with the assay permitted straightforward identification of MM subgroups. As expected, SHM was identified in 94% of MM cases, but several unexpected subgroups were identified including biased IGHV3-11 or IGHV4-34 usage in 20% of MM samples, and two cases with very low levels of SHM. Evidence of intraclonal diversity/ongoing SHM was identified in 18% of samples, suggesting a possible germinal center origin for some MM cases. The single-tube Oncomine BCR Pan-Clonality assay efficiently detects BCR clonotype lineages at rates comparable to existing multiple reaction assays and permits their characterization for cell of origin studies and lymphoma classification.

Immune checkpoint inhibitors (ICI) are designed to activate exhausted tumor-reactive T cells thereby leading to tumor regression. Durvalumab, an ICI that binds to the programmed death ligand-1 (PD-L1) molecule, is approved as a consolidation therapy for treatment of patients with stage III, unresectable, non-small cell lung cancer (NSCLC). Immunophenotypic analysis of circulating immune cells revealed increases in circulating proliferating CD4 + and CD8 + T cells earlier after durvalumab treatment. To examine durvalumab’s mechanism of action and identify potential predictive biomarkers, we assessed the circulating T cells phenotypes and TCR genes of 71 NSCLC patients receiving durvalumab enrolled in a Phase I trial (NCT01693562, September 14, 2012). Next-generation sequencing of TCR repertoire was performed on these NSCLC patients’ peripheral blood samples at baseline and day 15. Though patients’ TCR repertoire diversity showed mixed responses to the treatment, patients exhibiting increased diversity on day 15 attained significantly longer overall survival (OS) (median OS was not reached vs 17.2 months for those with decreased diversity, p = 0.015). We applied network analysis to assess convergent T cell clonotypes indicative of an antigen-driven immune response. Patients with larger TCR clusters had improved OS (median OS was not reached vs 13.1 months for patients with smaller TCR clusters, p = 0.013). Early TCR repertoire diversification after durvalumab therapy for NSCLC may be predictive of increased survival and provides a mechanistic basis for durvalumab pharmacodynamic activity.

The progressive restriction of cell fate during lineage differentiation is a poorly understood phenomenon despite its ubiquity in multicellular organisms. We recently used a cell fusion assay to define a mode of epigenetic silencing that we termed ＂occlusion＂, wherein affected genes are silenced by cis-acting chromatin mechanisms irrespective of whether trans-acting transcriptional activators are present. We hypothesized that occlusion of lineage-inappropriate genes could contribute to cell fate restriction. Here, we test this hypothesis by introducing bacterial artificial chro- mosomes （BACs）, which are devoid of chromatin modifications necessary for occlusion, into mouse fibroblasts. We found that BAC transgenes corresponding to occluded endogenous genes are expressed in most cases, whereas BAC transgenes corresponding to silent but non-occluded endogenous genes are not expressed. This indicates that the cel- lular milieu in trans supports the expression of most occluded genes in fibroblasts, and that the silent state of these genes is solely the consequence of occlusion in cis. For the BAC corresponding to the occluded myogenic master regu- lator MyfS, expression of the Myf5 transgene on the BAC triggered fibroblasts to acquire a muscle-like phenotype. These results provide compelling evidence for a critical role of gene occlusion in cell fate restriction.

The transcriptional program that controls oligodendrocyte maturation and central nervous system (CNS) myelination has not been fully characterized. In this study, we use high-throughput RNA sequencing to analyze how the loss of a key transcription factor, zinc finger protein 191 (ZFP191), results in oligodendrocyte development abnormalities and CNS hypomyelination. Using a previously described mutant mouse that is deficient in ZFP191 protein expression (Zfp191 ), we demonstrate that key transcripts are reduced in the whole brain as well as within oligodendrocyte lineage cells cultured in vitro To determine whether the loss of myelin seen in Zfp191 mice contributes indirectly to these perturbations, we also examined the transcriptome of a well-characterized mouse model of hypomyelination, in which the myelin structural protein myelin basic protein (MBP) is deficient. Interestingly, Mbp (shiverer) mice had far fewer transcripts perturbed with the loss of myelin alone. This study demonstrates that the loss of ZFP191 disrupts expression of genes involved in oligodendrocyte maturation and myelination, largely independent from the loss of myelin. Nevertheless, hypomyelination in both mouse mutants results in the perturbation of lipid synthesis pathways, suggesting that oligodendrocytes have a feedback system that allows them to regulate myelin lipid synthesis depending on their myelinating state. The data presented are of potential clinical relevance as the human orthologs of the Zfp191 and MBP genes reside on a region of Chromosome 18 that is deleted in childhood leukodystrophies.

The transcriptional program that controls oligodendrocyte maturation and central nervous system (CNS) myelination has not been fully characterized. In this study, we use high-throughput RNA sequencing to analyze how the loss of a key transcription factor, zinc finger protein 191 (ZFP191), results in oligodendrocyte development abnormalities and CNS hypomyelination. Using a previously described mutant mouse that is deficient in ZFP191 protein expression (Zfp191 null ), we demonstrate that key transcripts are reduced in the whole brain as well as within oligodendrocyte lineage cells cultured in vitro. To determine whether the loss of myelin seen in Zfp191 null mice contributes indirectly to these perturbations, we also examined the transcriptome of a well-characterized mouse model of hypomyelination, in which the myelin structural protein myelin basic protein (MBP) is deficient. Interestingly, Mbp shi (shiverer) mice had far fewer transcripts perturbed with the loss of myelin alone. This study demonstrates that the loss of ZFP191 disrupts expression of genes involved in oligodendrocyte maturation and myelination, largely independent from the loss of myelin. Nevertheless, hypomyelination in both mouse mutants results in the perturbation of lipid synthesis pathways, suggesting that oligodendrocytes have a feedback system that allows them to regulate myelin lipid synthesis depending on their myelinating state. The data presented are of potential clinical relevance as the human orthologs of the Zfp191 and MBP genes reside on a region of Chromosome 18 that is deleted in childhood leukodystrophies.

Pluripotent stem cells can exist in either the naive state representing a developmental blank slate or the downstream primed state poised for differentiation. Currently, known differences between these two states are mostly phenomenological, and none can adequately explain why the two states should differ in developmental priming. Gene occlusion is a mode of epigenetic inactivation that renders genes unresponsive to their cognate transcriptional activators. It plays a crucial role in lineage restriction. Here, we report that a defining feature distinguishing the two pluripotent states lies in the ability of naive but not primed cells to erase occlusion. This “deocclusion” capacity requires Esrrb, a gene expressed only in the naive but not primed state. Notably, Esrrb silencing in the primed state is itself due to occlusion. Collectively, our data argue that the Esrrb-dependent deocclusion capacity in naive cells is key for sustaining naive pluripotency, and the loss of this capacity in the primed state via the occlusion of Esrrb poises cells for differentiation.