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We have previously reported that the early phases of the immune response of rats to hamster xenografts are characterized by the production of IgM xenoantibodies encoded by a restricted group of Ig germline VH genes (VHHAR family). In the later phases of the reaction, an IgM to IgG isotype switch occurs and our study examines the structure of the rearranged VHHAR genes used to encode IgG antibodies after this isotype switch. A quantitative polymerase chain reaction was used to investigate the changes in the levels of VHHAR+ IgG mRNA seen after xenotransplantation. cDNA libraries specific for VHHAR+ Ig γ chain were established from total RNA extracted from splenocytes of naive rats and xenograft recipients of hamster hearts at days 4, 8, 21, and 28 posttransplantation. Colony filter hybridization was used to estimate the relative frequency of the use of individual VHHAR+ IgG subclasses. Selected IgG clones from day 21 cDNA libraries were sequenced and analyzed for VH-D-JH gene usage and antibody combining site structure. The level of mRNA for VHHAR+ IgG increased 6-fold in xenograft recipients at day 21 post-transplantation when compared with naive animals. The relative frequency of isotype usage for VHHAR+ IgG1 antibodies alone increased from 22.3% at day 0 to 37.4% at day 21 PTx. Ten IgG clones from the day 21 cDNA libraries have been sequenced for the rear-ranged VH-D-JH genes. Thirty percent (3/10) of these IgG clones used VHHAR genes for the coding of heavy chain variable region with limited numbers of nucleic acid substitutions (>98% identity with their germline progenitors) although others demonstrated increased variation in nucleotide sequences (95-97% identity) when compared with germline VH genes. Analysis of the canonical binding site structure from the predicted amino acid sequences demonstrated that the majority of IgG clones (9/10) displayed a similar pattern of conserved configurations for their combining sites. The change in IgM to IgG antibody production in the later stages of the humoral immune response of rats to hamster xenografts is associated with an IgM to IgG isotype switch and an increased production of antibodies of the IgG1 isotype. Rat anti-hamster IgG xenoantibodies continue to express the VHHAR family of VH genes, many in their original germline configuration, to encode antibody recognition of the hamster target antigens. There are, however, a majority of antibodies for which the VH genes express evidence of increased nucleic acid sequence variation when compared to currently available germline sequences. The source of this variation is not known but may represent the expression of as yet unidentified germline genes and/or the introduction of T cell-driven somatic mutations. Despite the appearance of this variation, the unusual level of conservation in key antigen binding sites within the VH region suggests the variation, independent of its origin, may have a limited influence on the restricted nature of the host antibody response to xenografts. |
