Molecular characterization of antigen-specific B1 and B2 B-cells populations in Cryptococcus neoformans infection in mice (43.1)

Abstract

Abstract We examined B1 and B2 B-cell repertoires in Cryptococcus neoformans infection in mice. Flow-cytometry based antigen-specific staining for B-cell populations generated in response to pulmonary infection was performed using labeled un-encapsulated and encapsulated C. neoformans strains. FACS sorting and single-cell nested PCR was employed to characterize the IgH repertoire. An increase in total peritoneal B1a, B1b and B2 cell numbers was observed as early as day 3 post-infection. Frequencies of both acapsular and capsular antigen-specific cells were increased in the B1a subpopulation (18%, 5%) in infected compared to naïve mice (11%, 2%). The heavy chains of antigen-specific B2 and B1b cells recognizing acapsular and capsular cryptococcus belonged predominantly to the VH1 family. Interestingly, increased VH11 (22%) and VH12 (33%) usage was observed in antigen-specific B1a cells recognizing acapsular and encapsulated cryptococcus, respectively, compared to the conventional B1a population. Although all acapsular (VH11+) and capsular (VH12+) B1a cells used the same VH11.2.53 and VH12.1.78 genes, 60% and 80% rearrangements were unique, respectively. VH genes in both cases were essentially germline, however 40% of acapsular specific B1a cells had 4 or more N-nucleotide additions compared to capsular specific B1a cells (20%). These results indicate that naturally occurring phosphatidyl choline reactive B1a cells have broad specificities and can bind to encapsulated cryptococcus.