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.
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239, USA. gwiens@ncccwa.ars.usda.gov
Gregory D
GD
Wiens
McKay
M
Brown
Marvin B
MB
Rittenberg
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