Development of antidonor antibody directed toward non-major histocompatibility complex antigens in tolerant animals

Abstract

Background: The clinical significance of antibodies directed against antigens other than major histocompatibility complex (MHC) antigens is poorly understood, and there are few large animal models in which such antibodies can be examined. We studied, both retrospectively and prospectively, the development of antibodies to non-MHC antigens in tolerant miniature swine.

Methods: Our database was assessed for cases of antidonor antibody formation in tolerant animals over the last 20 years. Flow cytometry, absorption assays, and familial analyses for inheritance pattern of the gene(s) potentially responsible for the antibody reactivities were carried out, and an animal determined to be negative for this reactivity was immunized by a skin graft and subcutaneous injections of peripheral blood monocyte cells from an antigen-positive donor.

Results: Sixteen of 469 tolerant animals tested were found to have developed antidonor antibodies. These antibodies were found to be specific for the same, presumably single, non-MHC antigen. Familial analyses indicated that the gene encoding this antigen was expressed in an autosomal-dominant manner in approximately 95% of the herd. In a prospective study, antidonor antibodies with the same specificity as those observed retrospectively were successfully induced in an antigen-negative animal after immunization with peripheral blood monocyte cells.

Conclusion: To our knowledge, this is the first report of the development of antibodies to a highly prevalent, non-MHC antigen present on peripheral blood mononuclear cells and developing in tolerant animals without signs of graft dysfunction. Considering the concern often raised by the appearance of antidonor antibodies in transplant recipients, these data could have important implications for clinical transplantation.

Figure 1

A: Determining the number of antigens involved and gene segregation: In Figure 1A-1, PBMCs from a SLA^dd PAA-2 positive animal were incubated for 30’ with serum from an antibody-producing SLA^dd animal (19312, green curve), or fetal pig serum (FPS, black curve, negative control) or anti-SLA^dd pig serum (red curve, positive control). After a wash, FITC labeled goat-anti swine IgG was added and incubated for 30’. After two more washes, the cells were analyzed by flow cytometry. Serum from 19312 showed presence of antibodies directed to an antigen present on the surface of the MHC-matched (SLA^dd) target cells. Fig 1A-2: The same curves as shown in A-1, except that serum from 19312 was absorbed on PAA-2 positive cells prior to incubation with the target SLA^dd PAA-2 positive cells (blue curve) and the negative control curve of fetal pig serum is not shown (for clarity, since it was essentially identical to that of the absorbed serum). Figure 1B: Representative data of the serum absorption assays. Serum from 20392, a PAA-2 negative animal that developed anti-PAA-2 antibodies after immunization, was absorbed at serial dilutions on cells from several PAA-2 positive animals bearing different MHCs (SLA^dd, red curves, and SLA^gg, green curves). The absorbed serum was then tested back on target cells from animals from the SLA^dd (left panel) and SLA^gg (right panel) lines. Essentially identical serum absorption curves were observed for absorption with either SLA^dd and SLA^gg cells, indicating that PAA-2 is a single antigen, or a set of antigens that segregate together. The blue lines are positive controls of serial dilutions of unabsorbed sera tested on the same target cells. Figure 1C: Pedigree of a family of animals analyzed in this study. By convention, circles indicate female animals and squares male animals. The segregation of PAA-2 positive (filled) vs. negative (open) animals is most consistent with Mendelian inheritance of the gene for PAA-2 in an autosomal dominant manner. Cells from animal 16171 were unfortunately not available for testing. Figure 1D: Antibody production in a naïve PAA-2 negative animal after immunization with a PAA-2 positive skin graft and subcutaneous PBMCs injections: Prior to immunization (grey curve) 20392 had no detectable anti-PAA-2 antibodies to 20105 (an animal known to be PAA-2 positive). Animal 20392 developed antibodies to PAA-2 after immunization with a skin graft and subcutaneous PBMCs injection (green curve). Animal 19312 had known anti-PAA-2 antibodies and was used as a positive control (black curve). The turquoise curve is a negative control in which the target cells were incubated with FPS before staining with the secondary goat-anti swine antibody.