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Immunodominance of the V_H1–46 Antibody Gene Segment in the Primary Repertoire of Human Rotavirus-Specific B Cells Is Reduced in the Memory Compartment through Somatic Mutation of Nondominant Clones¹

Cuixia Tian^*, Grace K. Luskin^*, Kevin M. Dischert^*, James N. Higginbotham^*, Bryan E. Shepherd and James E. Crowe, Jr^2,*,

^* Department of Pediatrics, Department of Biostatistics, and Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232

Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and V_H gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19⁺IgD⁺CD27^–, CD19⁺IgD⁺CD27⁺, or CD19⁺IgD^–CD27⁺) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19⁺IgD⁺CD27⁺ unclass-switched memory B cells and a low frequency of somatic mutations in CD19⁺IgD^–CD27⁺ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the V_H1–46 gene segment was a prominent feature in the V_H gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the V_H gene repertoire of the two memory B cell groups derived from broader usage of V_H gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.

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¹ This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (NIAID; R01 AI-57933). C.T. was a fellow of the NIAID Molecular Basis in Infectious Diseases Training Program (T32 AI-07474). The Vanderbilt Flow Cytometry Core Laboratory was supported by the Vanderbilt Ingram Cancer Center (P30 CA68485). Clinical support was provided by the Vanderbilt General Clinical Research Center (Grant M01 RR-00095 National Center for Research Resources, National Institutes of Health).

² Address correspondence and reprint requests to Dr. James E. Crowe, Jr., Department of Pediatrics, Vanderbilt University Medical Center, T-2220 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2905. E-mail address: james.crowe{at}vanderbilt.edu

³ Abbreviations used in this paper: RV, rotavirus; VLP, virus-like particle; DLP, double-layered particle; VB, virus-specific B cell subset; CB, circulating (randomly selected) B cell subset; FR, framework region.

⁴ The online version of this article contains supplemental material.