Development and characterization of anti-Gal B cell receptor transgenic Gal-/- mice.
BACKGROUND
The successful clinical application of pig-to-primate xenotransplantation is currently limited by the development of an acute vascular rejection, which is thought to involve an induced humoral immune response to the galactose alpha1,3 galactose (alpha-Gal) antigen. Successful xenotransplantation may require the development of novel methods for removal or neutralization of anti-Gal antibodies and anti-Gal-producing B cells. The large diversity of the B-cell repertoire makes it difficult, however, to isolate and study anti-Gal B-cell development.


METHODS
We have established a transgenic mouse model for investigating anti-Gal B cells by introducing a transgene encoding both heavy and light chains for an anti-Gal IgM antibody into an alpha-galactosyltransferase-deficient (Gal-/-) background. We have characterized the frequency, phenotype, and function of transgenic anti-Gal B cells by multiparameter flow cytometric analysis and ELISA.


RESULTS
ELISA analysis of serum from animals with the transgene in an alpha-galactosyltransferase-deficient background (Tg Gal-/-), from transgenic animals with a heterozygous alpha-galactosyltransferase background (Tg Gal-/+), and from nontransgenic alpha-galactosyltransferase-deficient littermates (Gal-/-) demonstrated elevated expression of anti-Gal antibodies in Tg Gal-/- mice compared with nontransgenic Gal-/- animals and a lack of transgene expression in the Tg Gal-/+ mice. Anti-Gal antibody expression in Tg Gal-/- mice could be increased by immunization with an ovalbumin-Gal glycoconjugate in vivo and through stimulation with lipopolysaccharide in vitro. Multiparameter flow cytometric analysis indicates that 50% to 80% of splenic and peritoneal B cells expressed the transgene and excluded endogenous immunoglobulin gene rearrangements. The majority of these B cells expressed anti-Gal receptors on the surface, as identified by staining with a fluorescein isothiocyanate-bovine serum albumin-Gal glycoconjugate. FACS analysis of the Tg Gal-/- B cells identified them as a population of CD21highCD23lowIgMhigh marginal zone B cells in the spleen and CD5-CD23low B1 cells in the peritoneal cavity.


CONCLUSIONS
These observations suggest that this model can be used to study the regulation of anti-Gal B cells and can establish a reliable source of functional anti-Gal B cells, which could be used to test the effectiveness of alpha-Gal-specific immunosuppressive reagents.
Xu
Hui
H
Nextran Inc., Princeton, New Jersey 08540, USA.
Sharma
Ajay
A
Lei
Ying
Y
Okabe
Jeannine
J
Wan
Hua
H
Chong
Anita S-F
AS
Logan
John S
JS
Byrne
Guerard W
GW
D000818:Animals; D015703:Antigens, CD; D001402:B-Lymphocytes / Q000276:immunology*; D001854:Bone Marrow Cells / Q000276:immunology; D004187:Disaccharides / Q000172:deficiency* / Q000235:genetics* / Q000276:immunology; D007072:Immunoglobulin D / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D051917:Leukosialin; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D023421:Models, Animal; D011947:Receptors, Antigen, B-Cell / Q000235:genetics* / Q000276:immunology*; D012795:Sialoglycoproteins / Q000276:immunology; D013154:Spleen / Q000276:immunology; D014183:Transplantation, Heterologous
D015703:Antigens, CD; D004187:Disaccharides; D007072:Immunoglobulin D; D007075:Immunoglobulin M; D051917:Leukosialin; D011947:Receptors, Antigen, B-Cell; D012795:Sialoglycoproteins; C497863:Spn protein, mouse; C055075:galactosyl-(1-3)galactose