Maturation of xenoantibody gene expression during the humoral immune response of rats to hamster xenografts

Immunoglobulin isotype switching represents an important component of antib ody maturation in the development of humoral immune responses. We have rece ntly conducted a series of studies in a nonimmunosuppressed rodent model to define the kinetics of xenoantibody production and seek evidence for the m aturation of xenoantibody Ig gene expression by xenograft recipients. LEW r ats were transplanted with hamster cardiac xenografts and the grafts were a llowed to remain in situ for prolonged immune stimulation of the host. Anti -hamster antibodies were examined at days 4, 8, 21, 28 and 40 posttransplan tation. cDNA libraries specific for rat mu or gamma heavy chains were const ructed from B lymphocytes of the xenograft recipients at day 4 and day 21 p ost-transplantation. Selected cDNA clones encoding the Ig V(H)HAR family of genes from each group were sequenced and analyzed for the presence of soma tic mutations. We found that the reactivity of xenoantibodies examined with flow cytometry underwent sequential changes in which IgM titers peaked at day 8 post-transplantation (PTx) and returned to low levels after 21 days. IgG titers started to increase at abo Lit one week PTx and peaked at 21-28 days. All the IgG isotypes (IgG1, 2a, 2b and 2c) were differentially involv ed in the IgG responses. Serum passive transfer experiments demonstrated th at IgM antibody fractions separated from sera at day 4 post-transplantation were capable of causing hyperacute rejection (HAR) of hamster xenografts, whereas IgM fractions from days 21-40 failed to cause HAR (N=7, MST=4 days) , a pattern that was consistent with a rise in total xenoreactive IgM level s at days 4-8 and a fall to low levels at 21 days post-transplantation. IgG -containing fractions separated from day 21-40 antisera caused HAR (N=7, MS T=36 min) whereas IgG fractions from day 8 sera failed to induce graft reje ction. Genetic analysis of the rearranged V-H genes from 10 cDNA clones dem onstrated that the Ig mu (n=5) and gamma (n=5) chain clones used the same f amily of V-H genes (V(H)HAR family) to encode their antibody binding activi ty. The majority (80%) of the IgM clones were present in their original ger mline configuration. In contrast, the nucleotide sequences from IgG clones manifested an increase in the numbers of replacement mutations in the CDR r egion of the Ig heavy chain genes, providing evidence for a potential role for somatic mutation in the maturation of IgG xenoantibody responses as the humoral response matures with time posttransplantation.