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.

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.