REPERTOIRE SHIFT IN THE HUMORAL RESPONSE TO PHOSPHOCHOLINE-KEYHOLE LIMPET HEMOCYANIN: VH SOMATIC MUTATION IN GERMINAL CENTER B CELLS IMPAIRS T15 IG FUNCTION

Authors: Wiens, Gregory - Greg; BROWN, MCKAY - OREGON HEALTH AND SCI UNI; RITTENBERG, MARVIN - OREGON HEALTH AND SCI. UN

Submitted to: Journal of Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2003
Publication Date: 5/15/2003
Citation: Wiens, G.D., Brown, M., Rittenberg, M.B. 2003. Repertoire shift in the humoral response to phosphocholine-keyhole limpet hemocyanin: vh somatic mutation in germinal center b cells impairs t15 ig function.. Journal of Immunology. 170:5095-5102.

Interpretive Summary: Phosphocholine is present on the surface of the bacterium Streptococcus pneumoniae and many other pathogenic microorganisms. The T15 antibody binds phosphocholine and is highly protective against Streptococcus pneumoniae infection. Large quantities of the T15 antibody are rapidly produced in mice after immunization with phosphocholine conjugated to protein. As the immune response progresses, the composition of the antibody response changes: the T15 antibodies become a minor component and non-protective antibodies dominate the response. This phenomenon is termed repertoire shift. Understanding this process is important for elucidating the mechanisms of antibody generation and maintenance. Here, we followed the location of the T15 antibody producing lymphocytes (B-cells) in the spleens of immunized mice and examined whether the T15 B-cells enter germinal centers. Germinal centers are specialized microenvironments where the B-cell antibody genes undergo mutation and the best B- cells are selected. We found T15 B-cells inside germinal centers on days 7 and 13 of the immune response and many antibody genes contained mutations. The majority of mutations resulted in detrimental changes to the T15 antibody while improvements in the T15 antibody were not observed. Detrimental mutations decreased phosphocholine binding and prevented antibody secretion from cells. Our study provides the first evidence that mutation occurring in germinal centers can disrupt antibody secretion. The failure of T15 to gain affinity enhancing mutations in the face of the detrimental changes may contribute to repertoire shift.

Technical Abstract: 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 Vkappa22-Jkappa5 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 Vkappa22 rearrangements were cloned and sequenced. Thirty four percent of the V(H)1 clones and 37% of the Vkappa22 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.