8 The Recovery of Immunoglobulin Sequences from Single Human B Cells by Clonal Expansion

The development of phage-display technology and the construction of huge libraries of antibody (Ab ) fragments have provided an unlimited source of binders to virtually any antigen (Ag ) (1 ). However, it is unlikely that the heavy (VH ) and light (VL ) chains of the Abs obtained from these libraries resemble original in vivo pairings. In certain autoimmune diseases and immunological processes, such as B-cell tolerance, these V_H and V_L combinations can be of crucial importance. To be able to determine the original V_H and V_L combinations of Abs, we have set up a single B-cell culture system. This comprises the sorting of individual lymphocytes into culture wells using flow cytometry, a culture system to expand these cells (2 ) and polymerase chain reaction (PCR) amplification of their variable-region genes, thereby immortalizing the V_H and V_L regions from individual human B cells. The method relies on the clonal expansion of single B cells in which cell-cell interactions (CD40-CD40L), as well as soluble factors, have been shown to be essential. One advantage beyond conventional hybridoma technology is that this method circumvents laborious plating and screening; the advantage compared to phage-display technology is that original V_H and V_L pairings can be isolated. This system has been used to analyze V_H and V_L pairings of human immunoglobulin G⁺ (IgG+) B cells of unknown specificity (3 ) and, combined with a selection on the Ag U1A, a frequent autoantigenic protein target in patients with systemic lupus erythematosus, to analyze pairings in Ag-specific B cells (4 ). The efficiency of the system makes it possible to analyze large numbers of B cells and should therefore allow rare B-cell activities to be studied.