Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes.
BACKGROUND
The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose alpha (1,3) galactose (alphaGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine alphaGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies.


RESULTS
Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations.


CONCLUSIONS
The small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.
Zahorsky-Reeves
Joanne L
JL
Cardiothoracic Surgery Research, The Saban Research Institute of Childrens Hospital, The Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA. jreeves@mednet.ucla.edu
Gregory
Clare R
CR
Cramer
Donald V
DV
Patanwala
Insiyyah Y
IY
Kyles
Andrew E
AE
Borie
Dominic C
DC
Kearns-Jonker
Mary K
MK
D000595:Amino Acid Sequence; D000818:Animals; D000910:Antibodies, Heterophile / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry; D005690:Galactose / Q000737:chemistry / Q000276:immunology; D015870:Gene Expression; D050438:Genes, Immunoglobulin Heavy Chain; D022781:Hepatocytes / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016031:Liver Transplantation / Q000276:immunology*; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013552:Swine / Q000276:immunology*
D000910:Antibodies, Heterophile; D007135:Immunoglobulin Variable Region; D004247:DNA; D005690:Galactose