Expression of essential B cell development genes in horses with common variable immunodeficiency

Equine Immunology Lab, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Molecular Immunology (Impact Factor: 2.97). 03/2012; 51(2):169-76. DOI: 10.1016/j.molimm.2012.03.018
Source: PubMed


Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p<0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p<0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients.

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Available from: M Julia B Felippe, Jun 26, 2015
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    ABSTRACT: Common variable immunodeficiency (CVID) is a late-onset humoral deficiency characterized by B lymphocyte dysfunction or loss, decreased immunoglobulin production, and recurrent bacterial infections. CVID is the most frequent human primary immunodeficiency but still presents challenges in the understanding of its etiology and treatment. CVID in equine patients manifests with a natural impairment of B lymphocyte differentiation, and is a unique model to identify genetic and epigenetic mechanisms of disease. Bone marrow transcriptome analyses revealed decreased expression of genes indicative of the pro-B cell differentiation stage, importantly PAX5 (p≤0.023). We hypothesized that aberrant epigenetic regulation caused PAX5 gene silencing, resulting in the late-onset and non-familial manifestation of CVID. A significant increase in PAX5 enhancer region methylation was identified in equine CVID patients by genome-wide reduced-representation bisulfite sequencing and bisulfite PCR sequencing (p=0.000). Thus, we demonstrate that integrating transcriptomics and epigenetics in CVID enlightens potential mechanisms of dysfunctional B lymphopoiesis or function. Copyright © 2015. Published by Elsevier Inc.
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    ABSTRACT: Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to an IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1 or IgG4/7 positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5 and CD19 was high compared to the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used herein identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and can be used for classification of equine B cell lymphoproliferative disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    No preview · Article · Aug 2015 · Clinical and vaccine Immunology: CVI