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Mature B cells class switched to IgD are autoreactive in healthy individuals.

ABSTRACT: Determination of the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that, despite being derived from healthy people, antibodies from B cells that have class switched to IgD via genetic recombination (and thus become class switched to C delta [C delta-CS] cells) are highly reactive to self antigens. Over half of the antibodies from C delta-CS B cells bind autoantigens on human epithelioma cell line 2 (HEp-2) cells or antinuclear antigens, and a quarter bind double-stranded DNA; both groups of antibodies are frequently polyreactive. Intriguingly, some C delta-CS B cells have accumulated basic residues in the antibody variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, while other C delta-CS B cells are naturally autoreactive. Though the total percentage was appreciably less than for C delta-CS cells, a surprising 31% of IgG memory cell antibodies were somewhat autoreactive, and as expected, about 24% of naive cell antibodies were autoreactive. We interpret these findings to indicate either that autoreactive B cells can be induced to class switch to IgD or that autoreactive B cells that use IgD as the B cell receptor are not effectively deleted. Determination of the mechanism by which the majority of C delta-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight into the enigmatic function of the IgD antibody.

SUBMITTER: Koelsch K

PROVIDER: S-EPMC1866247 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ?2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens.IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis ("strep throat") to deadly necrotizing fasciitis (sometimes called "flesh-eating" disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.

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Mature B cells class switched to IgD are autoreactive in healthy individuals.

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