Molecular basis of B cell responses

Ph D Students
Soma Rohatgi (since Jan 2004)

B cells critically control infections caused by medically important extracellular bacteria. We use the encapsulated bacterial pathogen Streptococcus pneumoniae as a model system to understand B cell responses against particulate antigens. S.pneumoniae has several potentially immunogenic protein (T cell dependent) and polysaccharide (T cell independent) antigens on its cell surface. Antibodies directed against these cell surface determinants confer protective immunity to S.pneumoniae. Splenectomized and asplenic patients have a high incidence of infections by encapsulated bacteria thereby indicating the importance of spleen in combating these infections. The theme of this project is to decipher the molecular and cellular basis of spleen-dependent induction and regulation of B cell responses against particulate antigens.

The objectives of the project are (i) to characterize the spacio-temporal changes taking place in the splenic microarchitecture in response to immunization with heat-killed S.pneumoniae, (ii) to investigate the population genetics and selection of B cells in antigen-specific splenic germinal centers during humoral immune response to S.pneumoniae and (iii) computational analyses of the rearranged immunoglobulin VH and VL genes recovered from antigen-specific splenic germinal centers. These will be studied during primary as well as secondary B cell responses.

Immunohistochemical analysis of splenic sections from mice immunized with S.pneumoniae

During this reporting year, efforts were initiated to study systematically the spacio-temporal changes taking place in the splenic microarchitecture and kinetics of development of antigen-specific splenic germinal centers induced in response to immunization with heat-killed S.pneumoniae. Splenic sections from different strains of mice immunized with heat-killed encapsulated or unencapsulated strain of S.pneumoniae were stained immunohistochemically using antibodies to various cell type and differentiation stage specific markers. The markers used were – B220 for follicular B cells, peanut agglutinin (PNA) for germinal center B cells, IgD for naïve B cells, and proliferating cell nuclear antigen for identifying proliferating cells.

As expected, immunization of CBA/J mice with the extracellular domain of PspA induced PNA positive splenic germinal centers. We were able to detect splenic germinal centers in C57BL/6J mice immunized intravenously with heat-killed particulate antigen (strain R36A). Although germinal centers are typically induced in response to T cell dependent (e.g. proteins) antigens but under rare circumstances T cell independent antigens (e.g. polysaccharides) can also induce germinal centers. Since whole bacteria (which bears both T cell dependent as well as T cell independent antigens on its cell surface) were used as immunogen, we tested whether splenic germinal centers were induced in CBA/N (xid/xid) mice following immunization with R36A. We choose this strain since CBA/N mice fail to make natural antibodies to pneumococcal polysaccharides, including teichoic and lipoteichoic acids because of a Btk (XID) immune response defect. Positive staining of splenic sections with PNA indicated that germinal centers were induced in CBA/N mice in response to R36A. CBA/J and CBA/N mice when immunized intraperitoneally with heat-killed encapsulated strain (ATCC 6302, serotype 2) also generated germinal centers in the spleen. It is highly likely that the observed germinal centers were induced in response to the protein antigen(s) present on the cell surface of S.pneumoniae. Efforts are currently on in the laboratory to optimize the methodologies/reagents for identifying and quantitating antigen-specific germinal centers. We are also investigating the possibility whether any of the S.pneumoniae induced germinal centers are specific for the capsular polysaccharide.

Towards molecular analysis of rearranged VH and VL genes from particulate antigen-specific splenic germinal centers

Mouse germline immunoglobulin databases were used for designing family-specific primers for PCR amplifying the rearranged immunoglobulin VH and VL genes. We designed nested PCR (16 external and 16 internal) primers to cover all the 15 VH families. The total number of germline VH genes considered for designing these primers was 309. Similarly, we designed nested PCR (19 external and 20 internal) primers to cover all the 19 Vk families. The total number of germline Vk genes considered for designing primers was 183. To test the family-specific nature of the designed primers we randomly selected VH5, Vk4/5 and Vk9/10 families for constructing three separate libraries using genomic DNA from spleen as template. DNA sequence analysis of the recombinant clones recovered for the libraries confirmed that the primers were indeed family-specific and they were able to amplify different members of their respective families. In preliminary experiments, we were able to microdissect small groups (20-50) of immunohistochemically stained splenic germinal center cells using a hydraulic micromanipulation system. We were able to successfully amplify the VH5 family-specific rearranged genes for the microdissected cells. We are currently in the process of optimizing microdissection of single B cells followed by PCR amplification of the rearranged VH and Vk genes from the microdissected cells using the complete set of family-specific VH and Vk primers.

Publications

Original peer-reviewed articles

1.   †Mehr R, Edelman H, Sehgal D and Mage RG (2004) Analysis of mutational lineage trees from sites of primary and secondary immunoglobulin gene diversification in rabbits and chickens. J Immunol (in press) (†on deputation/work done elsewhere).