Clonal analysis of primary B cells responsive to the pathogenic bacterium Salmonella typhimurium.

Abstract

In the present study, a modification of the splenic focus system is used to analyze the S. typhimurium strain TML (TML)-specific B cell repertoire. The results show that the frequency of primary TML-specific splenic B cells in CBA/Ca mice is approximately 1 per 10(5) B cells and less than 30% of these B cells are specific for LPS. In contrast, the frequency of memory TML-specific cells is approximately 1 per 5-8 X 10(3) splenic B cells and greater than 95% of these B cells are specific for LPS. These results suggest that the frequency of primary TML-specific B cells is extremely low and that it expands 15-20-fold after antigen exposure. It is interesting that less than 30% of the primary B cells are specific for the LPS molecule since it is considered to be the major antigenic determinant on Salmonella organisms. Furthermore, the majority of the LPS-specific anti-TML antibody-producing clones are directed against the LPS O antigen region. Conversely, more than half to two-thirds of the memory LPS-specific anti-TML B cell clones are directed against the KDO or lipid A region of the LPS molecule. These results indicate that the preferential expansion of LPS-specific B cell clones observed after immunization resides primarily in the B cell subsets responsive to the KDO/lipid A moieties on the LPS molecule. Finally, unlike B cell responses to chemically defined antigens, TML stimulates very little IgG1 antibody. IgG2 and IgA isotypes appear to play a predominant role in anti-TML antibody responses, although all H chain classes are produced to some extent. Collectively, these findings are consistent with the responses reported for two other natural antigens, HA and PC. Hence, the pattern of stimulation by infectious agents, such as S. typhimurium, appears to be distinct from that of synthetic antigens. Thus, the studies presented herein have begun to provide insights into those subsets of B cells responsive to S. typhimurium and other infectious disease organisms.

Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells

“…2C). This is notable because studies of LPS responses to inactivated Salmonella enterica in mouse models suggest that both O-antigen and lipid A moieties are dominant antigenic determinants of LPS (19, 32). A study of the host-restricted mucosal pathogen Helicobacter pylori demonstrated that human IgA and IgM responses target the lipid A moiety of LPS, while IgG responses primarily target the variable O-specific polysaccharide (33).…”

In Vitro Analysis of the Generation and Propagation of Memory B Cells

“…The cells that could generate memory B cells could thus be inclusive not only of cells with high affinity receptors (as would be required for primary AFC responses) but also a spectrum of cells whose receptor affinities may be low initially, but subsequently increase by somatic mutation and selection. This could account for both tbe higher frequency of responses seen for HSA'" precursors (see Table \) and the incorporation in memory responses of specificities rarely seen in primary responses {Kaplan et al 1985, Duran & Metcalf 1987.…”

Altered expression of the Salmonella typhimurium-specific B-cell repertoire in mice chronically treated with antibodies to immunoglobulin D

“…Eight-to twelve-week-old anti-IgD-treated and control, immunoglobulin-injected BALB/c mice were used as donors in the splenic focus assay. H-2-identical, allotype-different B1O.D2 females, 6 to 8 weeks of age, were obtained from Jackson Laboratory (Bar Harbor, Maine) and used as recipients in the splenic focus assay (5). Chronic anti-IgDand control-treated C3H/HeN mice were used in the serum studies.…”

“…Splenic focus assay. A modification of the limiting-dilution B-cell assay, the splenic focus system, was used to analyze the B-cell repertoire (5,18). Briefly, 15 x 106 to 30 x 106 BALB/c donor splenic lymphocytes were transferred intravenously into antigen-primed, lethally irradiated, H-2-identical, allotype-different B1O.D2 recipients.…”