Generation of a fusion partner to sample the repertoire of splenic B cells destined for apoptosis

Abstract

B cells proliferate and diversify in germinal centers in response to antigen. Only a small percentage of these B cells will emerge to form the serum antibody response. Other B cells making lower affinity antibodies, acquiring nonsense mutations, or expressing autoreactivity as a result of somatic mutation undergo an apoptotic cell death and are not efficiently sampled in current analyses of B-cell hybridomas. We have demonstrated that expression of bcl-2 in the NSO myeloma fusion partner leads to a higher yield of viable hybridomas, with a selective increase in hybridomas from B cells that produce autoantibodies and are seldom recovered when spleen cells from non-autoimmune mice are fused to the conventional NSO cell line. Using this fusion partner, we have generated hybridomas from anti-DNA antibody-producing transgenic B cells that are anergic in vivo and destined for apoptosis. These studies provide a strategy to sample the repertoire of B cells that arise in vivo but are not selected to contribute to the expressed antibody response. Furthermore, they demonstrate that restricted expression of bcl-2 in B cells contributes to the maintenance of self-tolerance in secondary lymphoid organs.