Identification of an anti‐idiotypic antibody that defines a B‐cell subset(s) producing xenoantibodies in primates

Abstract

Summary Synthetic anti‐idiotypic antibodies represent a potentially valuable tool for the isolation and characterization of B cells that produce xenoantibodies. An anti‐idiotypic antibody that binds to a subset of B cells producing antibodies encoded by the variable‐region heavy chain 3 (VH3) germline genes DP35 [immunoglobulin variable‐region heavy chain 3‐11 (IGHV3‐11)], DP‐53 and DP‐54 plus a small number of VH4 gene‐encoded antibodies in humans has recently been identified. These germline progenitors also encode xenoantibodies in humans. We tested whether the small, clearly defined group of B cells identified with this anti‐idiotypic antibody produce xenoantibodies in non‐human primates mounting active immune responses to porcine xenografts. Peripheral blood B cells were sorted by flow cytometry on the basis of phenotype, and cDNA libraries were prepared from each of these sorted groups of cells. Immunoglobulin VH gene libraries were prepared from the sorted cells, and the VH genes expressed in each of the sorted groups were identified by nucleic acid sequencing. Our results indicate that xenoantibody‐producing peripheral blood B cells, defined on the basis of binding to fluorescein isothiocyanate (FITC)‐conjugated galactose α(1,3) galactose–bovine serum albumin (Gal‐BSA) and the anti‐idiotypic antibody 2G10, used the IGHV3‐11 germline gene to encode xenoantibodies and were phenotypically CD11b+ (Mac‐1+) and CD5–. This novel reagent may be used in numerous applications including definition of xenoantibody‐producing B‐cell subsets in humans and non‐human primates and immunosuppression by depletion of B cells producing anti‐Gal xenoantibodies.

The Anti-Non-Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

“…The similarity in the structure of the xenoantibodies that initiate rejection of porcine xenografts provides new opportunities for the design of novel approaches to limit xenoantibody-mediated rejection. Structure-based drug design or the elimination of B cells producing xenoantibodies may be useful as adjunct therapeutics to prolong xenograft survival (45). Specific elimination of anti-gal B cells using alpha-gal linked toxins in mice has been performed without toxicity to the mice and without affecting production of antibodies with other specificities (46).…”

Xenotransplantation: immunological hurdles and progress toward tolerance

“…A study performed by Fischer‐Lougheed et al . in rhesus macaques demonstrated that anti‐Gal secreting cells were CD5 − CD11b + , similar to the B‐1b cells in GalTKO mice . Unfortunately, the same approach, which relies on using the anti‐Gal IgM receptor on antibody‐secreting cells to define xenoantibody‐producing cells, has little applicability in the current era, when the relevant xenoantibodies under study are directed to poorly defined non‐Gal antigens.…”

Xenotransplantation: Current Challenges and Emerging Solutions

“…Indeed, depletion of the anti-Gal antibody leads to more favorable outcomes, further implicating B-cells in the rejection of xenotransplants 71 – 73 . The phenotypic characteristics of anti-Gal antibody-producing subpopulations of B-cells in humans are not clearly identified 72 . One study has shown that splenic B-cells produce anti-Gal antibodies, whereas peritoneal B-cells do not, although they do express anti-Gal receptors 73 .…”