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 wer e 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 aci d at position 62 in mouse cyt. This is the specificity of the vast maj ority 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 al pha (1 --> 6) dextran and three of these mAb derive from the R9 V kapp a gene, a member of the V kappa Ox-1 family. The other mAb derive from distinct, although similar, V kappa genes. Attempts to obtain hybrido mas 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 i n higher frequency than B lymphocytes specific for similar epitopes on foreign cyt. Possible explanations for this result include biased exp ression in the B cell repertoire of the particular combination of V ge nes encoding mouse cyt-specific mAb or to positive selection of develo ping B lymphocytes by endogenous Ag.