Anti-pig antibody response and changes in B cell memory phenotype following wild-type pig-to-monkey corneal xenotransplantation

Abstract

Purpose: As the cornea is avascular, hyperacute rejection has not been reported following pig-to-nonhuman primate corneal xenotransplantation (Tx). However, after pig corneal Tx, few data relating to the development of anti-pig antibodies and no data on the B cell response have been reported. The aim of the present study was to investigate the serum antibody response and changes in B cell phenotype after pig-to-monkey corneal Tx.

Methods: Chinese rhesus macaques received corneal transplants from wild-type (WT) pigs (penetrating keratoplasty [PKP]: n=2, or Descemet's stripping endothelial keratoplasty [DSEK]: n=2). Immunosuppression consisted of local and systemic corticosteroids only. Monitoring included observation for clinical features of rejection. To determine whether sensitization to pig xenoantigens developed, recipient blood was monitored for (i) anti-Gal antibody levels by ELISA, (ii) anti-nonGal antibody levels by flow cytometry using α1,3-galactosyltransferase gene-knockout (GTKO) pig red blood cells, and (iii) CD20+B cell immunologic profiles (naïve [IgD+/CD27-], non-switched [IgM dominant: IgD+/CD27+], and switched [IgG dominant: IgD-/CD27+] memory B cell subsets) by flow cytometry. At 3 months, the monkeys were euthanized and the corneal grafts were examined histologically.

Results: There was minimal cellular infiltration in all recipients’ grafts at 3 months after Tx. There was an increase in both IgM and/or IgG anti-Gal antibodies in all recipients (maximum IgM and IgG were 3±2-fold and 18±15-fold higher than pre-Tx, respectively). Although there was a modest increase in anti-nonGal IgM in PKP (<2-fold compared with pre-Tx), there was no increase in anti-nonGal IgM in DSEK. There was no increase in anti-nonGal IgG in any recipient. With regard to B cell phenotype, the frequency of naïve memory B cells decreased in all monkeys (pre-Tx 57±13%; 3 months post-Tx 38±4%). In contrast, there were increases in non-switched memory B cells (pre-Tx 12±5%; post-Tx 17±2%) and switched memory B cells (pre-Tx 21±8%; post-Tx 34±5%).

Conclusions: Even though expression of Gal is minimal in WT pig corneas, the increases in anti-Gal antibodies and in non-switched and switched memory B cells suggest B cell sensitization. The relative lack of increase in anti-nonGal IgG suggests that GTKO pigs would be a preferable source of corneal grafts.

Keywords: 480 cornea: basic science • 741 transplantation • 529 flow cytometry