Quantitative Analysis of the B Cell Repertoire by Limiting Dilution Analysis and Fluorescent in Situ Hybridization

Abstract

We have made a quantitative and concurrent analysis of B cell frequencies and V H gene family expression to study the influence of tissue type and age on the development and establishment of the primary B cell repertoire. Using LPS-mediated limiting dilution analysis and a panel of antigens we show that the newly generated B cell specificities from bone marrow get distributed without a bias to peripheral tissues such as spleen and Peyer's patches throughout the lifetime of the animal. Comparison of the B cell frequencies in animals of four different age groups (2-4 days old, 3, 12, and 18 months old) reveals that while the neonatal repertoire is comparable to that of adults, there was a selective twofold increase in the generation and distribution of B cells reactive with autologous mouse red blood cells in older mice compared to young ones. By means of a novel technique that employs fluorescent in situ hybridization and flow cytometry, we have also compared the V H gene family usage in large numbers of single B cells from these mice. Analysis of the same cell population (surface Ig +, functional B lineage cells) for expression of 7183, J558, and S107 V H families shows a preferential twofold increase in the use of V H 7183 in neonates compared to adults, while all three families show no significant difference in levels of expression during adult life or between primary and secondary lymphoid tissues. Taken together, our data indicate that specific and selective changes occur in both V H gene usage and antibody frequencies during murine ontogeny.