B lymphocyte recognition of cytochrome c: higher frequency of cells specific for self versus foreign antigen early in the immune response and V gene usage in the response to self antigen

To study immunoglobulin gene usage in the antibody response of mice to the self antigen (Ag) mouse cytochrome c (cyt), B cell hybridomas were prepared from splenic B cells of immunized BALB/c mice prior to the onset of somatic mutation, i.e. 3 days after injecting ovalbumin (OVA)-primed mice with mouse cyt coupled to OVA. Monoclonal antibodies (mAb) from all of the seven primary hybridomas we obtained were sensitive to a single amino acid substitution from aspartic acid to glutamic acid at position 62 in mouse cyt. This is the specificity of the vast majority of B cells responding to mouse cyt as determined from assays of B cells activated in splenic fragment cultures. Six of the mAb derive from the 19.1.2 J558 V_H gene which is also used in the response to α (1 → 6) dextran and three of these mAb derive from the R9 V_x gene, a member of the V_xOx-1 family. The other mAb derive from distinct, although similar, V_x genes. Attempts to obtain hybridomas secreting primary (unmutated) mAb specific for cyt foreign to mice have been hampered by the much lower frequency of B cells responding early to foreign cyt in comparison to the self Ag. This suggests that, contrary to expectation of tolerance mechanisms, in naive BALB/c mice B lymphocytes specific for a single epitope on self cyt are present in higher frequency than B lymphocytes specific for similar epitopes on foreign cyt. Possible explanations for this result include biased expression in the B cell repertoire of the particular combination of V genes encoding mouse cyt-specific mAb or to positive selection of developing B lymphocytes by endogenous Ag.