Ruud De Wildt

Comparable heavy and light chain pairings in normal and systemic lupus erythematosus IgG(+) B cells.

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the presence of high immunoglobulin serum titers, but the mechanism by which these arise remains unclear. It has been suggested that the disease is associated with specific antibody features, including variable gene use, the presence of charged complementarity‐determining region residues and / or an aberrant process of secondary light chain rearrangement. To study this in more detail, we compared variable, diversity and joining gene segment use, somatic mutation, and heavy and light chain pairings in single peripheral IgG+ B cells between one normal (209 B cells) and two SLE (156 B cells) donors. In contrast to others, we found no systematic differences, indicating that the memory B cell repertoires in normal and SLE donors are shaped in a similar way.

Isolation of receptor–ligand pairs by capture of long-lived multivalent interaction complexes

We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab–antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab–antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor–ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.

Isolation and Characterization of Single Anti‐U1A‐specific B Cells from Autoimmune Patients

Patients with systemic lupus erythematosus (SLE) or SLE-overlap syndromes often produce autoantibodies directed to UlRNA-binding proteins.] Previously, we isolated and characterized autoantigen-binding Ab fragments directed to the U1 RNAassociated A protein (UlA) from several variable (V) gene combinatorial phage libraries.2 Although these libraries have proven to be very successful, heavy (VH) and light (V,) chains are randomly combined during construction of such libraries. Because we were interested in the utilization of the original VHNL pairings of the encoding autoantibodies, we developed a single cell culture system for B cells using mouse thymoma EL-4 B5 cells.3 In combination with a preselection via recombinant antigen and single cell sorting using fluorescence-activated cell sorting (FACS), we were able to enrich for autoantigen-specific B cells. VH and VL genes originating from single U1A-specific B cells were cloned in a phage display vector for expression of single-chain variable fragments (scFv). Human mononuclear cells were isolated from SLE patients and selected against U 1 A-coated plates or biotinylated U1 A coated to streptavidin-coupled superparamagnetic microbeads (MACS). Adhering cells were collected from the plates via trypsin treatment or were directly used in the case of U1A-bound MACS (FIGURE 1). The selected lymphocytes were plated as single CD 19/CDZO-positive cells using FACS with an automatic cell deposit unit. Single selected B cells were seeded on 96-well plates containing 20,000 irradiated EL4 B5 cells and 10% supernatant of phorbol myristate acetate (PMA)and phytohemagglutinin (PHA)-activated human T cells (FIGURE 2). Cultures were grown for 16-11 days and tested in ELISA for antigen (Ag)-specific antibodies and total Ig production. Cell cultures that were positive in the Ag-specific ELISA screening were used for RNA isolation. An RT reaction was performed with an oligo-dT primer and the cDNA was used in a first PCR with VH family-specific primers or a mixture of V-kappa or V-lambda primers (in the case of the light chains) as in Marks et aL4 These first PCR products were used in a second