The structure of anti-Gal immunoglobulin genes in naïve and stimulated Gal knockout mice.
BACKGROUND
Naturally occurring antibodies (Nabs) that bind to terminal galactose alpha1,3-galactose carbohydrate structures (Gal) are present in humans and Old World monkeys but are negatively regulated in other mammalian species because they express Gal epitopes on their cell surfaces. A Gal knockout mouse (Gal-/-) model, generated by homologous disruption of alpha1,3-galactosyltransferase gene, is capable of producing natural anti-Gal Abs.


METHODS
To study the genetic control of the anti-Gal response, we have generated anti-Gal hybridomas from Gal-/- mice and analyzed VH genes of anti-Gal Abs from naïve animals and from mice stimulated by rat heterotopic heart transplantation.


RESULTS
Six immunoglobulin (Ig)M anti-Gal hybridomas derived from naïve Gal-/- mice exhibited anti-Gal binding activity with some cross-reactivity to related carbohydrate structures. These naïve anti-Gal Abs used five different VH genes in a germline configuration. Anti-Gal IgM hybridomas isolated after a rat heterotopic heart xenograft (4 days) utilized germline VH gene segments from the VH7183 family and exhibited less cross-reactivity. In contrast to mice 4 days after xenograft, we have predominantly isolated IgG anti-Gal hybridomas from mice 21 days after rat heterotopic heart xenografts, indicating an isotype switch. Nine of the IgG anti-Gal hybridomas secreted IgG3 subclass and one produced IgG1. Sequence analysis of the VH gene usage from the induced anti-Gal IgG antibodies demonstrated a restricted gene utilization (VHJ606-V14A).


CONCLUSIONS
Our results demonstrate that the anti-Gal response in naïve Gal-/- mice is encoded by multiple germline progenitors. In response to a xenograft, the induced anti-Gal Abs exhibited a restricted gene usage and somatic mutations, indicating a positive selection.
Xu
H
H
Nextran Inc., Princeton, NJ 08540, USA.
Sharma
A
A
Chen
L
L
Harrison
C
C
Wei
Y
Y
Chong
A S
AS
Logan
J S
JS
Byrne
G W
GW
Shama
A
A
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology; D001483:Base Sequence / Q000235:genetics; D003429:Cross Reactions; D004187:Disaccharides / Q000235:genetics / Q000276:immunology*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D016027:Heart Transplantation / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats; D012016:Reference Values; D014183:Transplantation, Heterologous / Q000276:immunology*
D000906:Antibodies; D004187:Disaccharides; D010446:Peptide Fragments; C055075:galactosyl-(1-3)galactose