Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, Cooper DKC, Bianchi J. Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs. Xenotransplantation 2012; 19: 305–310. © 2012 John Wiley & Sons A/S. Abstract  Serum anti‐galactose‐α1,3‐galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti‐Gal IgM was present soon after birth, and rose to a peak at 4–6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti‐Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti‐Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.

Summary α1,3‐Galactosyltransferase gene‐knockout pigs transgenic for porcine cytotoxic T‐lymphocyte antigen 4 immunoglobulin (pCTLA4‐Ig) have been produced to reduce T‐cell‐mediated rejection following xenotransplantation. The level of soluble pCTLA4‐Ig in their blood was greatly in excess of the therapeutic level in patients, rendering the pigs immune‐incompetent. Soluble pCTLA4‐Ig produced by these transgenic pigs was evaluated for binding to porcine and human (h) B7 molecules, and for its inhibitory effect on allogeneic and xenogeneic human T‐cell responses. Porcine CTLA4‐Ig‐expressing peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (AECs) were evaluated for their direct inhibitory effect on hCD4+ T‐cell responses. Soluble pCTLA4‐Ig and purified hCTLA4‐Ig showed similar binding to pB7 molecules, but pCTLA4‐Ig showed significantly less binding to hB7 molecules. The pCTLA4‐Ig and hCTLA4‐Ig inhibited the response of hCD4+ T cells to pAECs equally, but pCTLA4‐Ig was less successful in inhibiting the human allogeneic response. The hCD4+ T‐cell response to PBMCs from pCTLA4‐Ig pigs was significantly lower than that of non‐pCTLA4‐Ig pigs. Although pCTLA4‐Ig was detected in the cytoplasm of pCTLA4‐Ig‐expressing pAECs, only a minimal level of soluble pCTLA4‐Ig was detected in the supernatant during culture, and pCTLA4‐Ig‐expressing pAECs did not inhibit the xenogeneic direct human T‐cell response. High‐level tissue‐specific production of pCTLA4‐Ig may be required for sufficient immunosuppression for organ or cell (e.g. islets) transplantation.

Background An initial observation suggested high levels of anti-pig antibodies in healthy humans who had spent their childhood in the Middle-East. We tested larger cohorts to determine whether anti-pig antibody levels correlated with the geographic location in which the subject spent his/her childhood, as this might have implications for clinical trials of xenotransplantation. Methods Anti-pig IgM and IgG levels (by flow cytometry using PBMC from wild-type [WT] and α1, 3-galactosyltransferase gene-knockout [GTKO] pigs), and anti-Gal IgM and IgG levels (by ELISA) were measured in 75 volunteers. Comparisons of antibody levels were also made on the basis of subject age, gender, ABO blood group, diet, and history of vaccination. Results Antibody binding to GTKO pig cells was less than to WT cells. There was a reduction in anti-pig IgM and anti-Gal IgM, but a slight increase in anti-non Gal IgG, with age. Women had higher levels of anti-Gal IgM than men. Blood group A subjects had higher levels of anti-pig IgM and IgG than those of group AB. Diet had no influence on antibody levels. Typhoid or MMR vaccination was associated with lower anti-nonGal IgG or anti-Gal IgG, respectively, whereas influenza vaccination was associated with higher anti-nonGal IgG. There were some significant variations in antibody levels associated with location during childhood, with subjects from the middle-east demonstrating higher anti-nonGal IgG and anti-Gal IgG. Conclusion Clinical trials of xenotransplantation may be influenced by various factors, including the geographic location of the recipient during childhood, possibly associated with exposure to different microorganisms.

Background Bortezomib, a proteosome inhibitor used to treat multiple myeloma, has been administered (+/- plasma exchange +/- intravenous immunoglobulin [IVIg]) in attempts to reduce antibodies against human leukocyte antigens (HLA) in sensitized patients undergoing organ transplantation. To our knowledge, bortezomib has not been investigated for its effect on natural anti-pig antibodies. If bortezomib could reduce the production of anti-pig antibodies, this would likely be beneficial to the outcome of pig organ grafts in primates. Methods Nine patients received bortezomib either to reduce anti-HLA antibody levels before organ allotransplantation or to treat antibody-mediated rejection. Patients at the Mayo Clinic (Group 1; n=4) received bortezomib alone, whereas at the UPMC (Group 2; n=5) this was combined with plasmaphereses +/- IVIg in some cases. Anti-pig IgM and IgG levels against wild-type (WT) and α1,3-galactosyltransferase gene-knockout (GTKO) pig aortic endothelial cells (flow cytometry – relative MFI) and anti-Gal IgM and IgG (ELISA – OD480nm) were measured pre- and post-bortezomib therapy. Results Mean anti-pig IgM levels were 11.2 (WT) and 1.9 (GTKO) pre-bortezomib and 9.4 (WT: P=0.02) and 1.7 (GTKO: P=0.33) post-bortezomib. Mean anti-pig IgG levels were 4.3 (WT) and 1.5 (GTKO) pre-bortezomib and 3.6 (WT: P=0.21) and 1.4 (GTKO: P=0.20) post-bortezomib. Mean anti-Gal IgM and IgG levels were 0.7 and 1.1, respectively, pre-treatment, and 0.6 (P=0.03) and 1.1 (NS), respectively, post-treatment. When the data were analyzed in Groups 1 and 2 separately, there were no significant differences between the pre- and post-bortezomib levels of anti-pig, anti-nonGal, or anti-Gal IgM or IgG. Conclusion From this limited study, we conclude that bortezomib might reduce anti-Gal IgM levels in primates, but, in this respect alone, is unlikely to have any significant effect on the outcome of GTKO pig organ transplantation.