Repertoire shift in the humoral response to phosphocholine-keyhole limpet hemocyanin: VH somatic mutation in germinal center B cells impairs T15 Ig function.
Phosphocholine (PC) is a naturally occurring Ag common to many pathogenic microorganisms. Early in the primary response to PC conjugated to keyhole limpet hemocyanin (KLH), T15 Id(+) Abs constitute >90% of the serum Ig in BALB/c mice. During the late primary and memory response to PC-protein, a shift in the repertoire occurs and T15 Id(+) Abs lose dominance. In this study, we use immunohistochemistry and single germinal center microdissection to locate T15 Id(+) cells in the spleen in a primary response to PC-KLH. We demonstrate T15 Id(+) B cells and V(H)1-DFL16.1-JH1 and V kappa 22-J kappa 5 rearrangements in germinal centers early in the immune response; thus loss of T15 dominance is not due to lack of T15 cells within germinal centers. One-hundred thirty one V(H)1 and 57 V kappa 22 rearrangements were cloned and sequenced. Thirty four percent of the V(H)1 clones and 37% of the V kappa 22 clones contained somatic mutations indicating participation in the germinal center response. Six variant T15 H clones were expressed with wild-type T15 L chain in vitro. Two of these Abs were defective in secretion providing the first evidence that mutation occurring in vivo can disrupt Ig assembly and secretion. Of the four secretion-competent Abs, two failed to display binding to PC-protein, while the other two displayed altered carrier recognition. These results indicate that somatic mutation of T15 in vivo can result in the loss of binding and secretion, potentially leading to B cell wastage. The failure of T15 to gain affinity enhancing mutations in the face of these detrimental changes may contribute to repertoire shift.
Wiens
Gregory D
GD
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239, USA. gwiens@ncccwa.ars.usda.gov
Brown
McKay
M
Rittenberg
Marvin B
MB
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis / Q000097:blood / Q000235:genetics* / Q000502:physiology; D016175:B-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D002478:Cells, Cultured; D005260:Female; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D018858:Germinal Center / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006433:Hemocyanins / Q000008:administration & dosage / Q000276:immunology*; D007114:Immunization; D007130:Immunoglobulin Idiotypes / Q000276:immunology* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009194:Myeloma Proteins / Q000276:immunology / Q000378:metabolism; D010767:Phosphorylcholine / Q000008:administration & dosage / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins; D010767:Phosphorylcholine; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin