A High-Affinity Natural Autoantibody from Human Cord Blood Defines a Physiologically Relevant Epitope on the FcεRIα

Abstract

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the α domain of the high-affinity IgE receptor (FcεRIα) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcεRIα Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcεRIα had the same sequence. This single IgM Ab, named CBMα8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcεRIα recognized by both CBMα8 and the previously isolated anti-FcεRIα Abs from autoimmune and healthy donors. This common epitope on FcεRIα coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMα8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcεRIα Abs are rendered less likely to compete with IgE for binding to FcεRIα.

The splenic autoimmune response to ADAMTS13 in thrombotic thrombocytopenic purpura contains recurrent antigen-binding CDR3 motifs

“…32 The anti-ADAMTS13 mAbs showed somatic hypermutation typical for an antigen-driven, affinity matured immune response, which is a process dependent on CD4 1 T-cell help. [40][41][42]48 The average nucleic acid mutation rate was 12% for the V H and the V L chain regions, with the highest mutation rate in mAbs using the V H 1-3 germ-line gene (average, 15%; range, 13.5-18.4%) and the lowest in V H 1-69 mAbs (average, 4.8%; range, 2.9-5.7%).…”

“…24,[40][41][42] Briefly, enrichment of anti-ADAMTS13-bearing surface Fabs of both IgG 4 -k/l-phage libraries and an IgM Fab-k/l-phage library generated from cord blood, representing the naïve immune repertoire, 42 was achieved by 5 selection rounds on full-length recombinant ADAMTS13 (rADAMTS13), expressed in human HEK293 cells (.99% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gift from Fritz Scheiflinger, Baxter), and coated at a concentration of 1 mg/mL onto a microtiter plate. Phagemid DNA was prepared from the last panning round by removing the gene III fragment by restriction digestion with NheI and SpeI, followed by religation, and were subsequently used to transform XL1-blue cells to produce single clones secreting soluble anti-ADAMTS13 Fab fragments.…”

“…To investigate the presence of anti-ADADMTS13 antibodies in the natural antibody repertoire, IgM-k and -l phage libraries, generated from umbilical cord blood lymphocytes of 7 donors, 42 BLOOD were selected by panning on rADAMTS13. ADAMTS13-specific IgM (n 5 9) were composed of k L-chains in combination with V H genes from the IgV H 1 superfamily (3 clones), IgV H 3 superfamily (3 clones), and IgV H 6 (3 clones).…”

DARPins against a functional IgE epitope

“…The experiment was performed according to previous described protocols [47][48][49] with minor modifications. Briefly, 1 × 10 5 cells were seeded in 96-well tissue-adherent cell culture plates in 100 l antibiotic-free RPMI-1640 medium (Biochrom AG, Berlin, Germany) containing 10% fetal calf serum (FCS).…”

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

“…Particularly, we are interested in the umbilical cord blood libraries containing B lymphocytes that presumably have not been exposed to exogenous antigens and have been used as a source of naturally occurring unmutated or minimally mutated pre-immune antibodies (Casali and Schettino 1996;Ridings et al 1997). These germlineencoded natural antibodies could possibly play a vital role in the development and physiology of the human B cell repertoire as well as have the ability to bind a variety of exogenous antigens (Bobrzynski et al 2005;Cooke et al 1993;Messmer et al 1999;Rodman et al 2001). Furthermore, the human neonatal IgM antibody repertoire was found to be invariant and directed toward a limited set of self-antigens that could represent the targets for unmutated antibody specificities (Mouthon et al 1995).…”