Genetic control of the humoral responses to xenografts. III. Identification of the immunoglobulin V-H genes responsible for encoding rat immunoglobing xenoantibodies to hamster heart grafts

Background. 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 V-H gen es (V(H)HAR family). In the later phases of the reaction, an IgM to IgG iso type switch occurs and our study examines the structure of the rearranged V (H)HAR genes used to encode IgG antibodies after this isotype switch. Methods. A quantitative polymerase chain reaction was used to investigate t he changes in the levels of V(H)HAR(+) IgG mRNA seen after xenotransplantat ion, cDNA libraries specific for V(H)HAR(+) Ig gamma chain were established from total RNA extracted from splenocytes of naive rats and xenograft reci pients of hamster hearts at days 4, 8, 21, and 28 posttransplantation, Colo ny filter hybridization was used to estimate the relative frequency of the use of individual V(H)HAR(+) IgG subclasses. Selected IgG clones from day 2 1 cDNA libraries were sequenced and analyzed for V-H-D-J(H) gene usage and antibody combining site structure. Results. The level of mRNA for V(H)HAR(+) IgG increased 6-fold in xenograft recipients at day 21 posttransplantation when compared with naive animals. The relative frequency of isotype usage for V(H)HAR(+) IgGl antibodies alo ne increased from 22.3% at day 0 to 37.4% at day 21 PTx, Ten IgG clones fro m the day 21 cDNA libraries have been sequenced for the rearranged V-H-D-J( H) genes. Thirty percent (3/10) of these IgG clones used V(H)HAR genes for the coding of heavy chain variable region with limited numbers of nucleic a cid substitutions (>98% identity with their germline progenitors) although others demonstrated increased variation in nucleotide sequences (95-97% ide ntity) when compared with germline V-H genes. Analysis of the canonical bin ding site structure from the pre-dieted amino acid sequences demonstrated t hat the majority of IgG clones (9/10) displayed a similar pattern of conser ved configurations for their combining sites. Conclusions. The change in IgM to IgG antibody production in the later stag es of the humoral immune response of rats to hamster xenografts is associat ed with an IBM to IgG isotype switch and an increased production of antibod ies of the IgG1 isotype, Rat anti-hamster IgG xenoantibodies continue to ex press the V(H)HAR family of V-H genes, many in their original germline conf iguration, to encode antibody recognition of the hamster target antigens. T here are, however, a majority of antibodies for which the V-H genes express evidence of increased nucleic acid sequence variation when compared to cur rently available germline sequences. The source of this variation is not kn own but may represent the expression of as yet unidentified germline genes and/or the introduction of T cell-driven somatic mutations, Despite the app earance of this variation, the unusual level of conservation in key antigen binding sites within the V-H region suggests the variation, independent of its origin, may have a limited influence on the restricted nature of the h ost antibody response to xenografts.