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Wesemann, Duane

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Wesemann

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Duane

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Wesemann, Duane
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    Analyzing Immunoglobulin Repertoires
    (Frontiers Media S.A., 2018) Chaudhary, Neha; Wesemann, Duane
    Somatic assembly of T cell receptor and B cell receptor (BCR) genes produces a vast diversity of lymphocyte antigen recognition capacity. The advent of efficient high-throughput sequencing of lymphocyte antigen receptor genes has recently generated unprecedented opportunities for exploration of adaptive immune responses. With these opportunities have come significant challenges in understanding the analysis techniques that most accurately reflect underlying biological phenomena. In this regard, sample preparation and sequence analysis techniques, which have largely been borrowed and adapted from other fields, continue to evolve. Here, we review current methods and challenges of library preparation, sequencing and statistical analysis of lymphocyte receptor repertoire studies. We discuss the general steps in the process of immune repertoire generation including sample preparation, platforms available for sequencing, processing of sequencing data, measurable features of the immune repertoire, and the statistical tools that can be used for analysis and interpretation of the data. Because BCR analysis harbors additional complexities, such as immunoglobulin (Ig) (i.e., antibody) gene somatic hypermutation and class switch recombination, the emphasis of this review is on Ig/BCR sequence analysis.
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    Microbial colonization influences early B-lineage development in the gut lamina propria
    (2013) Wesemann, Duane; Portuguese, Andrew J.; Meyers, Robin M.; Gallagher, Michael P.; Cluff-Jones, Kendra; Magee, Jennifer M.; Panchakshari, Rohit A.; Rodig, Scott J.; Kepler, Thomas B.; Alt, Frederick
    The RAG1/RAG2 endonuclease ("RAG") initiates the V(D)J recombination reaction that assembles Ig heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B cell antigen-binding receptor ("BCR"). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3–5. This “receptor editing” process, which also can lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen-encounter at the Rag-expressing immature B cell stage helps shape pre-immune BCR repertoires. As the major site of post-natal B cell development, the bone marrow is the principal location of primary Ig repertoire diversification in mice. Here, we report that early B cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP Ig repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B lineage cells that harbor intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B, and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing BM counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B cell development occurs in the intestinal mucosa, where it is regulated by extra-cellular signals from commensal microbes that influence gut Ig repertoires.
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    Publication
    Immature B cells preferentially switch to IgE with increased direct Sμ to Sε recombination
    (The Rockefeller University Press, 2011) Wesemann, Duane; Magee, Jennifer M.; Boboila, Cristian; Calado, Dinis Pedro; Gallagher, Michael P.; Portuguese, Andrew J.; Manis, John; Zhou, Xiaolong; Recher, Mike; Rajewsky, Klaus; Notarangelo, Luigi; Alt, Frederick
    Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cμ (IgM) constant regions (C(H)) exons with downstream C(H) exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cμ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cμ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cμ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cμ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.