Direct in vivo V(H) to J(H) rearrangement violating the 12/23 rule

V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that V(H) to J(H) joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the D(H)-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular V(H) gene family or J(H) element in V(H)J(H) junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo

STAT3 dysregulation in mature T and NK cell lymphomas

T cell lymphomas comprise a distinct class of non-Hodgkin’s lymphomas, which include mature T and natural killer (NK) cell neoplasms. While each malignancy within this group is characterized by unique clinicopathologic features, dysregulation in the Janus tyrosine family of kinases/Signal transducer and activator of transcription (JAK/STAT) signaling pathway, specifically aberrant STAT3 activation, is a common feature among these lymphomas. The mechanisms driving dysregulation vary among T cell lymphoma subtypes and include activating mutations in upstream kinases or STAT3 itself, formation of oncogenic kinases which drive STAT3 activation, loss of negative regulators of STAT3, and the induction of a pro-tumorigenic inflammatory microenvironment. Constitutive STAT3 activation has been associated with the expression of targets able to increase pro-survival signals and provide malignant fitness. Patients with dysregulated STAT3 signaling tend to have inferior clinical outcomes, which underscores the importance of STAT3 signaling in malignant progression. Targeting of STAT3 has shown promising results in pre-clinical studies in T cell lymphoma lines, ex-vivo primary malignant patient cells, and in mouse models of disease. However, targeting this pleotropic pathway in patients has proven difficult. Here we review the recent contributions to our understanding of the role of STAT3 in T cell lymphomagenesis, mechanisms driving STAT3 activation in T cell lymphomas, and current efforts at targeting STAT3 signaling in T cell malignancies

Negative selection, not receptor editing, is a physiological response of autoreactive thymocytes

Antigen receptor editing—a process of secondary rearrangements of antigen receptor genes in autoreactive lymphocytes—is a well-established tolerance mechanism in B cells, whereas its role in T cells remains controversial. Here, we investigated this issue using a novel Tcra knock-in locus, which ensured appropriate timing of TCRα expression and allowed secondary rearrangements. Under these conditions the only response to self-antigen that could be unambiguously identified was negative selection of CD4/CD8 double positive thymocytes. No evidence could be obtained for antigen-induced TCR editing, whereas replacement of the transgenic TCRα chain by ongoing gene rearrangement occurred in some cells irrespective of the presence or absence of self-antigen

Simultaneous deletion of Tet1 and Tet3 increases transcriptome variability in early embryogenesis

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.J.K. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. W.A.P. was supported by the National Science Foundation predoctoral graduate research fellowship while this work was being performed, and subsequently by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. L.C. was the recipient of a Feodor-Lynen fellowship from the Alexander von Humboldt foundation. M.L. is supported by the Max Planck Society within its International Max Planck Research School for Computational Biology and Scientific Computing program (IMPRS-CBSC). A.T. was the recipient of an Irvington postdoctoral fellowship from the Cancer Research Institute. This work was supported by NIH R01 Grants AI044432 and HD065812 (to A.R.) and a Director’s New Innovator Award (DP2-OD-008646-01) (to S.K.).This is the author accepted manuscript. The final version is available from The National Academy of Sciences via http://dx.doi.org/10.1073/pnas.151051011

MicroRNAs in the pathogenesis, diagnosis, prognosis and targeted treatment of cutaneous T-cell lymphomas

Cutaneous T-cell lymphoma (CTCL) represents a heterogeneous group of potentially devastating primary skin malignancies. Despite decades of intense research efforts, the pathogenesis is still not fully understood. In the early stages, both clinical and histopathological diagnosis is often difficult due to the ability of CTCL to masquerade as benign skin inflammatory dermatoses. Due to a lack of reliable biomarkers, it is also difficult to predict which patients will respond to therapy or progress towards severe recalcitrant disease. In this review, we discuss recent discoveries concerning dysregulated microRNA (miR) expression and putative pathological roles of oncogenic and tumor suppressive miRs in CTCL. We also focus on the interplay between miRs, histone deacetylase inhibitors, and oncogenic signaling pathways in malignant T cells as well as the impact of miRs in shaping the inflammatory tumor microenvironment. We highlight the potential use of miRs as diagnostic and prognostic markers, as well as their potential as therapeutic targets. Finally, we propose that the combined use of miR-modulating compounds with epigenetic drugs may provide a novel avenue for boosting the clinical efficacy of existing anti-cancer therapies in CTCL

Random Operator Approach for Word Enumeration in Braid Groups

We investigate analytically the problem of enumeration of nonequivalent primitive words in the braid group B_n for n >> 1 by analysing the random word statistics and the target space on the basis of the locally free group approximation. We develop a "symbolic dynamics" method for exact word enumeration in locally free groups and bring arguments in support of the conjecture that the number of very long primitive words in the braid group is not sensitive to the precise local commutation relations. We consider the connection of these problems with the conventional random operator theory, localization phenomena and statistics of systems with quenched disorder. Also we discuss the relation of the particular problems of random operator theory to the theory of modular functionsComment: 36 pages, LaTeX, 4 separated Postscript figures, submitted to Nucl. Phys. B [PM

STAT5 induces miR-21 expression in cutaneous T cell lymphoma

In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL

High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans

Antibodies' protective, pathological, and therapeutic properties result from their considerable diversity. This diversity is almost limitless in potential, but actual diversity is still poorly understood. Here we use deep sequencing to characterize the diversity of the heavy-chain CDR3 region, the most important contributor to antibody binding specificity, and the constituent V, D, and J segments that comprise it. We find that, during the stepwise D-J and then V-DJ recombination events, the choice of D and J segments exert some bias on each other; however, we find the choice of the V segment is essentially independent of both. V, D, and J segments are utilized with different frequencies, resulting in a highly skewed representation of VDJ combinations in the repertoire. Nevertheless, the pattern of segment usage was almost identical between two different individuals. The pattern of V, D, and J segment usage and recombination was insufficient to explain overlap that was observed between the two individuals' CDR3 repertoires. Finally, we find that while there are a near-infinite number of heavy-chain CDR3s in principle, there are about 3–9 million in the blood of an adult human being