We shall investigate the genetic basis for the production of human anti DNA antibodies by Epstein-Barr virus (EBV) transformed B cell lines and human human hybrids derived from both normal individuals and patients with systemic lupus erythematosus (SLE). Human human hybrids and EBV cell lines which produce anti DNA antibodies have been developed. The specificities and affinities of some of these lines have been fully characterized and we will complete these analyses. We have prepared a cDNA library from one of the anti DNA antibody producing hybrids and have isolated a fragment containing the heavy chain variable region. This fragment will be sequenced and then used as a probe for further studies. We will examine RNA extracted from the hybrids and B cell lines and, using Northern blots, see whether the same VH gene is used by normals and SLE patients. We will examine what V-J rearrangements are used. Similarly, we will evaluate the light chains used in the hybrid and B cell lines, first, by using probes for the kappa and lambda constant regions and then second, by screening our already prepared cDNA library for the kappa light chain gene and by constructing a cDNA library from a lambda producing hybrid and screening it for the lambda light chain gene. We will try to identify the genes used to make the anti DNA antibodies by comparing them to other published antibody sequences to see if they are similar to genes used for antibodies to known exogenous antigens. We will examine the anti DNA gene repertoire in normals and patients with SLE by extracting unrearranged genomic DNA from neutrophils, size separating it, Southern blotting it and hybridizing with the derived variable region probes. We will correlate our molecular analysis and the amino acid sequence with the specificity and affinity data for the antibodies. We should be able to ascertain what genes are used in the construction of anti DNA antibodies by human hybrids and EBV transformed B cell lines, how they are combined, and whether normal derived anti DNA producing hybrids and B cell lines differ from those derived from patients with SLE.
