Immune complex-mediated enhancement of secondary antibody responses.

Immunologic memory is a hallmark of the vertebrate immune system. The first antigenic exposure leads to a slow and modest immune response, whereas repeated exposure, even many years later, leads to a rapid and exaggerated response that is two to three orders of magnitude greater than the primary. In the case of humoral immunity, the increased efficacy of recall responses is due to the production of amplified levels of Ag-specific Ab, as well as the accelerated kinetics of their production. Current thinking suggests that this is due to selective activation of long-lived, Ag-specific memory B cells. A downside of restricting secondary responses solely to memory cells is that the repertoire of the memory B cell pool remains static while pathogens continue to evolve. In this study, we propose that during secondary responses, naive Ag-specific B cells participate alongside memory cells. We show that immune complexes formed in vivo between the Ag and pre-existing Abs from the primary response activate these naive B cells, inducing them to respond with accelerated kinetics and increased magnitude. Thus, the continued recruitment of new B cell clones after each antigenic exposure enables the immune system to stay abreast of rapidly changing pathogens.

Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes.

Cryptococcus neoformans is a pathogenic fungus that is relatively amenable to molecular genetic analysis, including gene deletion. However, rates of homologous recombination can be low, so obtaining specific gene deletion transformants is challenging. We have utilized two new technologies, cku deletion strains to improve the efficiency of gene deletions in this organism, and co-transformations. The Ku70-Ku80 heterodimer is predicted to be an essential part of the non-homologous end-joining process in C. neoformans. Here we show that a deletion in one or both of these proteins results in an increase in the rates of homologous recombination. Importantly, we demonstrate that after generation of a strain with a particular deletion of interest, the cku deletion can be removed by mating and segregation. We also utilize co-transformation of wild-type genes and selectable markers on separate linear DNA molecules to complement a deletion event. We show that co-transformation results in the successful restoration of wild-type phenotype, though variations in this phenotype often occur.