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Analysis of antibody repertoire and V(H) family usage in BALB/c mice
Abstract
A number of factors influence the generation and establishment of primary B cell repertoire in mice. These include the rules of immunoglobulin gene rearrangement and expression, formation of different antibody specificities and their distribution to lymphoid tissues, and the survival of these specificities in the periphery. With roughly a third of the peripheral B cell pool undergoing rapid turn-over every few days, the immune system at any given moment approximates a steady state. The main issues of such a dynamic model are: (i) what are the levels of expression of individual immunoglobulin genes or families? (ii) how equally are the B cell specificities arising in bone marrow distributed to the peripheral lymphoid organs? and (iii) what changes, if any, occur in the repertoire during ontogeny? Addressing these questions by characterizing the emerging repertoire of bone marrow, the immunocompetent but rapidly turning over repertoire of peripheral tissues, and the underlying variable region gene usage in these populations will be the focus of this thesis. To compare the emergence and distribution of new B cell specificities, frequency of B cells specific for three diverse antigens (DNP, PC, and mouse RBC) were estimated by LPS-stimulation and limiting dilution in "newly generated" sIg$\sp+$ cells from bone marrow, spleen and Peyer's patches of BALB/c mice. To monitor changes in repertoire during ontogeny, these comparisons were performed in animals of 4 different age groups (i.e. 2-4 days old, 3-, 12- and 18-months). The frequency of B cells reactive with DNP, PC and RBC were comparable in primary and secondary tissues suggesting that new B cells generated in the bone marrow are distributed equivalently to peripheral lymphoid tissues. While DNP and PC frequencies showed no differences between neonatal, adult and older animals, there was a specific two-fold increase in the generation of B cells reactive with autologous mouse RBCs in old animals (12- and 18-months) compared to younger animals. This indicated a selective increase in auto-reactive B cell frequencies in older animals. A novel assay system based on fluorescent in situ hybridization and flow cytometry was devised to analyze V$\sb{\rm H}$ gene expression. Using this approach, V$\sb{\rm H}$ family usage in large number of individual B cells were analyzed in different tissues and during ontogeny. The expression of V$\sb{\rm H}$ J558 family in neonatal spleen was two-fold lower than adult spleen while the expression V$\sb{\rm H}$ S107 remained the same between these two age groups. However, the expression of these two families showed no differences in different lymphoid tissues or during adult life. Overall, these data suggest that selective changes take place in the antibody repertoire and V$\sb{\rm H}$ family usage during murine ontogeny.
Subject Area
Immunology
Recommended Citation
Ravichandran, Kodimangalam Sethuramasarma, "Analysis of antibody repertoire and V(H) family usage in BALB/c mice" (1992). Doctoral Dissertations Available from Proquest. AAI9233139.
https://scholarworks.umass.edu/dissertations/AAI9233139