*Purpose: Humans lack α-1,3-Gal (α-gal) epitopes, and naturally occurring α-gal antibodies (Abs) cause hyperacute rejection of porcine xenografts. We conducted 2 successful xenotransplants in brain-dead decedents using kidneys from α1,3Galactosyltranserase knockout (GTKO) pigs. However, human sera may contain Abs reactive to other non-Gal epitopes with unknown in-vivo significance. We report the first in-vivo evidence in humans of the histologic phenotype of non-Gal anti-pig IgM Abs.

*Methods: We conducted 2 successful GTKO xenotransplants in decedents registered as research donors. Sera collected before transplant were tested for the presence of anti-pig IgM Abs by flow cytometry (FC) on pig PBMCs. To establish a correlation between FC and complement-dependent cytotoxicity crossmatch (CDCXM), a serially diluted positive control (PPP) was tested for the presence of anti-pig IgM Abs by FC on pig PBMC and by CDCXM on pig endothelial cells. The pre-transplant sera of both decedents were tested for the presence of anti-pig IgM Abs by FC, and the results compared to the observations from PPP. Biopsies of both porcine kidneys were assessed for C4d deposition by immunofluorescence (IF).

*Results: Testing on PPP indicate that anti-pig IgM Abs with FC median channel shift (MCS) <700 at 1:16 dilution correspond to a negative CDCXM on endothelial cells (Fig.1). Decedent’s sera were tested against the PBMCs of their pig donor at the same dilution. Recipient-1 MCS was +628, comparable to PPP and predictive of a negative CDCXM (Fig.1B). However, recipient-2 had a +3958 MCS, a 6.3-fold increase in IgM titer and predictive of a positive CDCXM (fig.1B). The 48 hrs C4d staining was significantly stronger in porcine kidney transplanted to recipient-2 (Fig.1D) compared to kidney transplanted to recipient-1 (Fig.1C). However, in both recipients there was no histologic evidence of acute AMR (ptc-itis, vasculitis, or glomerulitis), a phenotype that has been seen in human ABO-incompatible transplants.

*Conclusions: Our study demonstrates that strong non-Gal anti-pig IgM Abs in human recipients predict C4d deposition on kidney xenografts. However, C4d deposition does not correlate with histologic findings of acute AMR.

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