Functional and molecular characterization of single, (4‐hydroxy‐3‐nitrophenyl)acetyl (NP)‐specific, IgG1+ B cells from antibody‐secreting and memory B cell pathways in the C57BL/6 immune response to NP

Abstract We have used multiparameter flow cytometry to identify a population of IgG 1 + IgM − antigen‐specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4‐hydroxy‐3‐nitrophenyl)acetyl (NP). Characterization of the specificities of IgG 1 antibodies produced by single, sorted IgG 1 + NP + cells in both Elispot assays and in microcultures containing lipo‐polysaccharide, interleukin (IL)‐2, IL‐4 and IL‐5 indicates that the splenic IgG 1 + NP + B cell population includes both IgG 1 anti‐NP antibody‐secreting cells and non‐secreting, IgG 1 + memory B cells. Each functionally discrete population of IgG 1 + B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody‐secreting, IgG 1 + cells were uniquely identified by co‐expression of the matrix receptor, syndecan. The NP‐specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG 1 + NP + λ + B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo .