McKay Brown

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 VH1-DFL16.1-JH1 and Vκ22-Jκ5 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 VH1 and 57 Vκ22 rearrangements were cloned and sequenced. Thirty four percent of the VH1 clones and 37% of the Vκ22 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.

In vitro antibody synthesis in 20 μl hanging drops: Initiation of secondary responses and a simple method of cloning hybridomas

Abstract An in vitro culture system is described in which antibody synthesis is initiated by 10 5 hapten-primed spleen cells in 20 μl hanging drops. Plaque-forming cell (PFC) responses to thymus dependent and thymus independent antigens were obtained in drop cultures comparable to the micro Mishell-Dutton system. Antigens could be administered in soluble form or on antigen-pulsed peritoneal cells. The hanging drop method also provides a reliable yet simple procedure of cloning hybridoma cells while permitting direct visualization of the number of cells in each drop.

Characterization of λ class antibodies from the BALB/c memory response to a [glucosyl-α(1→3) glucosyl]-protein conjugate

Abstract Both dextran B-1355 and nigerosyl-KLH (N-KLH) contain α(1 → 3) diglucosyl moieties and both induce λ class serum antibodies which recognize the α(1 → 3) linkage. However, as shown here, some of the antibodies induced by the thymus-dependent form, N-KLH, have a distinct fine specificity. It is known that B-1355 induces antibodies which resemble the anti-α(1 → 3) myeloma proteins MOPC 104E and J558. The new fine specificity which does not resemble such antibodies is found in the serum of N-KLH primed mice challenged with N-KLH. The two fine specificity types are distinguished by their sensitivity to inhibition by nigerose in ELISA using B-1355 or N-BSA as bound antigen. Twelve hybridomas were produced from N-KLH primed mice boosted with either N-KLH or B-1355. Six of the 12 had the new fine specificity; only two out of the 12 expressed the IdX determinant commonly associated with the B-1355 response and neither of these possessed the new reactivity pattern. Comparative inhibition with the disaccharide nigerose and the tetrasaccharide nigerantetraose indicated that 11 out of the 12 hybridoma proteins have combining sites larger than a disaccharide. Southern and Northern blot analysis of eight hybridomas revealed that all expressed VH genes from the J558 family, but that at least two distinct VH genes from this family were used. The data support the hypothesis that immunization with an α(1 → 3) diglucosyl-protein conjugate alters the composition of the B cell pool capable of producing λ class antibodies from that ordinarily observed following immunization with B-1355.