Preformed IgM Antibodies Research Articles

Allosensitization Does Not Increase the Risk of Xenoreactivity to ??1,3-Galactosyltransferase Gene-Knockout Miniature Swine in Patients on Transplantation Waiting Lists

The recent availability of alpha1,3-galactosyltransferase knockout (GalT-KO) miniature swine has eliminated anti-Gal antibodies as the major barrier to xenotransplantation, potentially bringing this modality closer to clinical application. Highly-allosensitized patients, who have poor prospects of receiving a suitable cross-match negative human organ, might be the first patients to benefit from xenotransplantation of porcine organs. However, concerns exist regarding cross-reactivity of alloreactive anti-human leukocyte antigen (HLA) antibodies against xenogeneic swine leukocyte antigen (SLA) antigens. We have investigated this question using sera from such patients on GalT-KO target cells. Using flow cytometry and complement-dependent cytotoxicity (CDC) assays, we have tested a panel of 88 human serum samples from patients awaiting cadaveric renal allotransplantation for reactivity against: 1) human; 2) standard miniature swine; and 3) GalT-KO peripheral blood lymphocytes (PBL) and cultured endothelial cells. Anti-swine IgM and IgG antibody binding, as well as CDC, were significantly attenuated on GalT-KO versus standard swine. No correlation was found between the degree of anti-human panel reactive antibodies (PRA) and xenoreactivity against either standard or GalT-KO miniature swine. Treatment of sera with dithiothreitol (DTT) showed that the majority of remaining lymphocytotoxicity against GalT-KO swine was mediated by preformed IgM antibodies. Patients with high alloreactivity but low anti-GalT-KO xenoreactivity were readily identified. Highly allosensitized patients awaiting renal transplants appear to be at no increased risk of xenosensitization over their non-sensitized cohorts, and could therefore be candidates for xenotransplantation using GalT-KO swine donors.

Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts.

The elimination of circulating anti-porcine preformed antibodies is crucial for avoiding hyperacute vascular rejection (HAVR) of primarily vascularized xenograft in discordant pig to baboon model. Previously described methods used for eliminating natural antibodies, however, constantly removed both anti-porcine IgM and IgG antibodies, as well as often complement proteins. To study specifically the role of preformed anti-porcine IgM antibodies, a specific anti-IgM monoclonal antibody (mAb) has been designed and evaluated in vivo. Iterative injections of anti-IgM mAb (LO-BM2) at high dose (20 mg/kg) depleted to undetectable level the circulating IgM and therefore anti-porcine IgM antibodies but did not change the concentration of anti-pig IgG antibodies. The serum concentration of IgM and IgG antibodies was assessed by ELISA and the level of anti-pig natural IgM and IgG antibodies by flow cytometry (FC). Anti-rat sensitization was assessed by specific ELISA as well as the serum concentration of LO-BM2. Iterative injections of LO-BM2 allowed to specifically eliminate the anti-porcine IgM antibodies to undetectable levels at ELISA. Despite a normal serum level of anti-porcine IgG and complement proteins, HAVR was avoided. Without immunosuppression, the specific elimination of preformed anti-porcine IgM prolonged the survival of a renal xenograft in baboon up to 6 days, whereas without IgM antibody elimination, the renal xenografts were hyperacutely rejected within hours. The lost of activity of LO-BM2 after 10 days was concomitant to an IgM and IgG antibody rebound, which caused an acute vascular rejection of the xenograft. Specific elimination of natural anti-porcine IgM antibodies allows to avoid HAVR of a pig to baboon renal xenograft, whereas anti-porcine IgG antibodies and complement proteins were present in the serum. This result confirms previous in vitro reports and demonstrates for the first time in vivo that preformed IgM antibodies alone are responsible for HAVR, while preformed anti-porcine IgG antibodies are unable alone to cause HAVR. Anti-IgM therapy appears as an important tool to transiently but completely eliminates xeno-IgM antibodies in vivo.