Repertoire diversity of antibody response to bacterial antigens in aged mice. IV. Study of VH and VL gene utilization in splenic antibody foci by in situ hybridization

Abstract

Mouse Abs against a bacterial epitope, the phosphorylcholine (PC) hapten are encoded by the T15 genes VH1(S107) and V kappa 22. It has been shown that PC-specific hybridomas from aged animals often express IgV gene families other than T15. To determine the extent of this age-dependent molecular shift in the anti-PC response, we examined antibody-forming cells (AFC) in individual young (2 to 4 month) and aged (20 to 24 month) mice by an in situ RNA hybridization. Mice were immunized either with PC coupled to keyhole limpet hemocyanin or with a Streptococcus pneumoniae strain R36a vaccine. Frozen splenic sections were prepared, and the clusters of PC-specific AFC (i.e., antibody foci) were identified by immunocytochemical staining. The adjacent splenic sections were hybridized with digoxigenin-labeled VH1(S107) and V kappa 22 DNA probes and with a C mu DNA probe as a control. The splenic sections were examined for 1) the number of Ab foci hybridized with the T15 probes, and 2) the estimated proportion of VH1+ and V kappa 22+ AFC within each focus. The results were comparable regardless of the form of PC Ag administered. Virtually all Ab foci (> 85%) in young mice hybridized with the T15 probes and were occupied by the VH1+/V kappa 22+ AFC. In aged mice, the fraction of PC-binding Ab foci that hybridized with a given T15 probe varied from 35% to > 85%; T15+ AFC always represented a minor population of the focus (< 50%), the remaining PC-specific AFC being C mu + but T15-. Also, there appeared to be a greater loss of the V kappa 22 expression relative to the VH1(S107). Thus it appears that the T15+, PC-reactive B cells in aged mice responded to the Ag but that they could not dominate the response. The possibility of an intrinsic molecular change in the aging B cells in discussed.