Both Natural and Induced Anti-Sda Antibodies Play Important Roles in GTKO Pig-to-Rhesus Monkey Xenotransplantation

Abstract

Sda, produced by the B4GALNT2 enzyme, has been recognized as an important xenoantigen for pig-to-nonhuman primate xenotransplantation. However, little is known about Sda expression in pigs and its immunogenicity in xenotransplantation. In this study, peripheral blood mononuclear cells (PBMCs) were isolated from wildtype, GTKO (with high, moderate, and low Sda expression), GTKO/β4GalNT2KO, GTKO/CMAHKO, or GTKO/CMAHKO/β4GalNT2KO pigs. Anti-pig IgM/IgG binding and complement-dependent cytotoxicity (CDC) to pig PBMCs was measured by flow cytometry using pooled rhesus monkey sera (n=20) or human sera (n=20). As compared to wild-type pigs (n=12), GTKO pigs (n=17) had a significantly higher mean level of Sda expression on PBMCs and showed a greater individual difference in expression. Both the overall binding of monkey serum IgM/IgG antibody to GTKO pig PBMCs and CDC against these PBMCs decreased significantly with a progressive reduction in Sda expression, showing a clear dose-effect relationship. Both the monkey serum antibody binding and CDC decreased significantly after the additional deletion of Sda, whereas the binding of human serum antibody and CDC against the GTKO pig PBMCs were markedly reduced after the deletion of Neu5Gc in the pigs. In addition, anti-Sda antibody accounted for > 50% of the induced anti-non-Gal antibody at the time of rejection in two rhesus monkeys that received GTKO/hCD55 pig kidney xenotransplantation, and the anti-Sda antibody showed significant cytotoxic activity against GTKO pig cells. We conclude that both natural and induced anti-Sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation, thus providing further evidence for GTKO/β4GalNT2KO pigs as the preferred organ source for rhesus monkeys as a preclinical model of xenotransplantation.

Keywords: Sda; genetic engineering; pig; rhesus monkey; xenotransplantation.

Figure 1

Expression of Gal, Sda, and Neu5Gc on pig PBMCs. (A) The expression of Gal, Sda, and Neu5Gc on pig PBMCs from wildtype (WT), GTKO, GTKO/β4GalNT2KO, GTKO/CMAHKO, and TKO/hCD55 pigs (Gmean values were shown). White histograms outlined in black represent negative controls that were unstained cells for lectin experiments. (B) The expression of human CD55 (hCD55) on TKO/hCD55 pig PBMCs.

Figure 2

The expression of Sda in WT or GTKO pigs. PBMCs from WT, GTKO, or GTKO/β4GalNT2KO pigs were stained for the expression of Gal (by isolectin BSI-B4) and Sda (with Dolichos biflorus agglutinin, DBA). The results are reported as geometric mean fluorescence (Gmean). (A) Comparison of the expression of Sda on WT (n=12) and GTKO (n=17) pig PBMCs. Data are presented as means ± SD (*P<0.05); (B) The expression of Gal and Sda on PBMCs from two wild-type pigs and three GTKO pigs with different levels of Sda expression. White histograms outlined in black represent negative controls that were unstained cells for lectin experiments. The appearance of a single histogram in GTKO or GTKO/β4GalNT2KO samples indicates an overlap with the negative control.

Figure 3

Natural anti-pig antibody levels in the serum of rhesus monkeys. Five of 20 rhesus monkeys that had not been immunologically screened were randomly selected. Both anti-pig IgM and IgG levels in the serum (diluted at 1:10) of each monkey were measured by flow cytometry using PMBCs from WT (Sda^hi and Sda^lo), GTKO (Sda^hi, Sda^mo, and Sda^lo) or GTKO/β4GalNT2KO pigs as the source of the target cells. The results are reported as Gmean. Data are presented as means ± SD.

Figure 4

The role of Sda expression in the binding of monkey serum antibody to GTKO pig PBMCs and in CDC against these cells. The IgM/IgG antibody binding and CDC were measured by flow cytometry after the co-culture of the pooled monkey sera with PMBCs from wildtype (Sda^hi and Sda^lo), GTKO (Sda^hi, Sda^mo, and Sda^lo), or GTKO/β4GalNT2KO pigs. (A) Flow cytometric histograms showing representative binding of rhesus monkey IgM/IgG antibody (top/middle row, serum diluted 1:10, Gmean values were shown) to pig PBMCs and CDC (bottom row, serum diluted 1:6, percentages were shown) against the same PBMCs. (B) Statistical analysis of pooled rhesus monkey serum antibody binding (IgM and IgG) to pig PBMCs and CDC against the same PBMCs. (C) Comparison of mean serum CDC (at serum concentrations of 50% to 1.57%) of pooled rhesus monkey against WT, GTKO, or GTKO/β4GalNT2KO pig PBMCs. Each sample was measured three times. All data are presented as means ± SD (*P<0.05, **P< 0.01, ***P< 0.001; ns, non-significant). The P value comes from three independent experiments.

Figure 5

The role of Sda and Neu5Gc expression in the binding of human vs. monkey serum antibody to GTKO pig PBMCs and in CDC against these cells. IgM/IgG antibody binding and CDC were measured by flow cytometry after co-culture of pooled human or monkey sera with PMBCs from WT, GTKO, GTKO/β4GalNT2KO, GTKO/CMAHKO, or TKO/hCD55 pigs. (A, B, D, E) Pooled human or rhesus monkey serum IgM/IgG binding (at a serum concentration of 10%) to pig PBMCs. (C, F) Pooled human or rhesus monkey serum CDC (at serum concentrations of 50% to 1.57%) against pig PBMCs. Each sample was measured three times. All data are presented as means ± SD (*P<0.05, **P< 0.01, ***P< 0.001; ns, non-significant). The P value comes from three independent experiments.

Figure 6

The role of induced antibodies against Sda in the development of acute humoral xenograft rejection in GTKO/hCD55 pig-to-rhesus monkey kidney xenotransplantation. Renal xenotransplantation was previously performed in four rhesus monkeys using kidneys from GTKO/hCD55 pigs. Two of the four recipient monkeys (R01 and R02) generated high levels of circulating antibodies against non-Gal antigens and developed acute humoral xenograft rejection on day 19. Serum samples collected before transplantation (day -6) and at the time of rejection (day 19) were used to measure IgM/IgG antibody binding and CDC against GTKO (Sda^mo) or GTKO/β4GalNT2KO pig PBMCs. (A) The FACS results of serum IgM and IgG binding to pig PBMCs (at a serum concentration of 10%). Cells were incubated with medium served as negative controls (gray lines). The number represents the geometric mean fluorescence intensity (Gmean). (B) Comparison of CDC (at serum concentrations of 50% to 1.57%) of rhesus monkey (R01 and R02) pre-transplant (day -6) and post-transplant (day 19) serum against GTKO and GTKO/β4GalNT2KO pig PBMCs.