Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys

Abstract

We transplanted kidneys from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

Figure 1

Elicited antibodies to non-Gal antigens lead to AHXR of GalT-KO kidney grafts. (a) CDC against GalT-KO porcine lymphocytes measured from sera collected before transplantation and on the day of death (Endpoint). Sera from 20-02, 64-03, 19-02 and 66-03 showed a marked elevation in CDC titers at the endpoint, when the xenografts developed severe AHXR. Target cells incubated with medium alone, without baboon serum and rabbit complement, served as medium control (M) and cells incubated with rabbit complement alone served as complement control (C). (b) Changes in serum creatinine levels of each recipient after transplantation. (c) Sequential measurement of antibodies to non-Gal antigens in the circulation and CDC to porcine GalT-KO endothelial cells in a representative recipient (64-03): IgG levels (upper panels) and IgM levels (middle panels). Both IgG and IgM levels were measured by FACS using GalT-KO porcine lymphocytes as targets. Cells were incubated with media and then stained with the secondary antibody serving as a control (dashed lines). Values represent geometric mean fluorescence intensity. There was marked elevation of both IgG and IgM antibodies to non-Gal antigens after GalT-KO kidney grafting. Lower panels show CDC against porcine GalT-KO endothelial cells. CDC increased after transplantation, and reached a peak level at the endpoint. (d) Representative gross and pathological changes of a GalT-KO kidney graft with severe AHXR in baboon 20-02: gross finding of the terminal graft (Gross); H&E staining of the terminal graft showing severe AHXR (HE); MSB staining of the terminal graft showing intravascular thrombosis (red stain with arrows, MSB). Immunopathology staining of the terminal graft showed positive platelet deposition (arrow, Platelet), massive IgM deposition (arrow, IgM), massive IgG deposition (arrow, IgG), massive C3 deposition (arrow, C3) and positive C4d deposition (arrow, C4d). (e) Representative photo of reactivity of baboon serum (19-02) against GalT-KO pig endothelial cells in western blots. Lane 1, incubated with baboon serum before transplantation (pre-Tx) and stained for human IgG, IgM and IgA antibodies; Lane 2, incubated with baboon serum at the endpoint stained with human IgG, IgM and IgA antibodies (left panel). Intensity comparison between pre-transplantation and endpoint as determined by western blot (right panel). We performed signal quantification with ImageJ software (US National Institutes of Health).