The humoral response to xenografts is controlled by a restricted repertoire of immunoglobulin VH genes

BACKGROUND: The early phases of the host immune response to xenografts are dominated by anti-donor antibodies. The immunological pathways responsible for mediating the host humoral responses to xenografts are largely unknown, and this report addresses the nature of the immunoglobulin genes controlling the host antibody response to xenografts. METHODS: cDNA libraries established from rat anti-hamster monoclonal antibodies and splenic lymphocytes from LEW rats rejecting hamster heart xenografts were used to clone, sequence, and identify the immunoglobulin genes responsible for encoding rat xenoantibodies to hamster heart grafts. Libraries for germline variable region heavy chain (VH) genes encoding the anti-hamster xenograft antibodies were established by genomic DNA cloning and analyzed by nucleotide sequencing. The frequency of Ig VH gene usage for controlling the antibody responses to hamster xenografts was examined by colony-filter dot hybridization. The nucleic acid structure of these genes was then compared to their genomic progenitors to identify the number and structural diversity expressed by the Ig VH genes used to mediate the response. RESULTS: Rat monoclonal antibodies selected for their ability to precipitate the rejection of hamster xenografts exclusively use a closely related group of VH genes. The VH genes used by these antibodies are restricted to a single family of germline genes (VHHAR) for which 15 family members have been identified. The frequency of VHHAR gene usage in splenic IgM-producing B cells from LEW rats rapidly expands from 0.8% in naive animals to 13% in recipients 4 days after xenotransplantation. cDNA libraries expressing VHHAR genes were established from splenic lymphocytes derived from naive or xenograft recipients at 4 and 21 days after transplantation. Examination of 20 cDNA clones revealed that the majority (75%) of these clones express VHHAR genes displaying limited somatic mutation. CONCLUSIONS: The use of a closely related group of Ig VH genes in a germline configuration to control the early humoral response to xenografts suggests that this response may represent the utilization of a primitive, T cell-independent pathway of antibody production by the graft recipients.