Mac-1-Negative B-1b Phenotype of Natural Antibody-Producing Cells, Including Those Responding to Galα1,3Gal Epitopes in α1,3-Galactosyltransferase-Deficient Mice

Abstract

Human natural Abs against Galα1-3Galβ1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21^−/low IgM^high B220^low CD5⁻ Mac-1⁻ 493⁻ cells) in the spleens of α1,3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of α1,3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21^−/low IgM^high CD5⁻ CD43⁺ Mac-1⁺). However, these became Mac-1⁻ and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1⁺ B-1b cells and Mac-1⁻ B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1⁻ splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.