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

Abstract

Immunoglobulin isotype switching represents an important component of antibody maturation in the development of humoral immune responses. We have recently conducted a series of studies in a nonimmunosuppressed rodent model to define the kinetics of xenoantibody production and seek evidence for the maturation of xenoantibody Ig gene expression by xenograft recipients. LEW rats were transplanted with hamster cardiac xenografts and the grafts were allowed to remain in situ for prolonged immune stimulation of the host. Anti-hamster antibodies were examined at days 4, 8, 21, 28 and 40 post-transplantation. cDNA libraries specific for rat mu or gamma heavy chains were constructed from B lymphocytes of the xenograft recipients at day 4 and day 21 post-transplantation. Selected cDNA clones encoding the Ig V(H)HAR family of genes from each group were sequenced and analyzed for the presence of somatic 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 about one week PTx and peaked at 21-28 days. All the IgG isotypes (IgG1, 2a, 2b and 2c) were differentially involved in the IgG responses. Serum passive transfer experiments demonstrated that 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 levels 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, MST = 36 min) whereas IgG fractions from day 8 sera failed to induce graft rejection. Genetic analysis of the rearranged VH genes from 10 cDNA clones demonstrated that the Ig mu (n = 5) and gamma (n = 5) chain clones used the same family of VH genes (V(H)HAR family) to encode their antibody binding activity. The majority (80%) of the IgM clones were present in their original germline configuration. In contrast, the nucleotide sequences from IgG clones manifested an increase in the numbers of replacement mutations in the CDR region 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 post-transplantation.

Molecular case report: IgVH analysis in acute humoral and cellular liver allograft rejection suggests a selected accumulation of effector B cells and plasma cells

“…This is the first experimental evidence that B cells might be specifically involved in human liver allograft rejection. In xenotransplants, a high number of replacement mutations in the CDRs have been found, providing evidence for the role of somatic mutations in the maturation of IgG xenoantibodies [17]. This is well in line with our findings, since the majority of the somatically highly mutated immunoglobulin genes belonged to the IgG isotype.…”

“…The involvement of antibodies in rejection of organ transplants is increasingly recognized as a relevant pathomechanism, other than in xenografts [17] and kidney and heart [9] transplants. Two recent studies demonstrated depositions of C3c and C4d in humoral [5] and cellular [7] liver rejection, suggesting an involvement of B lymphocytes and plasma cells in both acute humoral and cellular liver allograft rejection to a different extent.…”

“…It has been applied to other organ transplants; however, only xenotransplantations have been examined [17], and no data exist on the IgV-gene repertoires of B-cells in allografts.…”

“…CLR and HLR show different clinical symptoms and can be kept apart in most cases. However, they share histopathological similarities with respect to infiltrating B and T lymphocytes [1,3,4,7,11,13,15,17]. In a recently published study, B lymphocytes, plasma cells, chemokines and complement factor C4d were detected in tissue biopsies of CLR, suggesting that, in addition to the T-cell response, a local B-cell response may be involved in the pathogenesis of CLR [7].…”

Commentary

“…In a hamster‐to‐rat xenograft model, Wu et al. [5] also found that IgG, but not IgM, acquired mutations, although constriction of the mutated repertoire was not explored.…”