A final extension of 5 min at 72C was performed. The principles of allelic exclusion state that each B cell expresses a single light and heavy chain pair. Here, we show that B cells with both kappa and lambda light chains (Ig and Ig) are enriched Haloperidol Decanoate in some patients with the systemic autoimmune disease systemic lupus erythematosus (SLE), but not in the systemic autoimmune disease control granulomatosis with polyangiitis. Detection of dual Ig and Ig expression by flow cytometry could not be abolished by acid washing or by DNAse treatment to remove any bound polyclonal antibody or complexes, and was retained after two days in culture. Both surface and intracytoplasmic dual light chain expression was evident by flow cytometry and confocal microscopy. We observed reduced frequency of rearrangements of the kappa\deleting element (KDE) in SLE and an inverse correlation between the frequency of KDE rearrangement and the frequency of dual light chain expressing B cells. We propose that dual expression of Ig and Ig by a single B cell may occur in some patients with SLE when this may be a consequence of reduced activity of the KDE. = 26) and SLE patients (= 56) were analysed for cell surface expression of Ig and Ig by flow cytometry. (A and B) Representative contour plots of isotype controls for cell surface expression of Ig and Ig by CD19+ cells from a healthy donor and a patient with SLE, respectively. (C) Representative contour plot of cell surface expression of Ig and Ig by CD19+ cells in healthy donors showing clear separation of B cells expressing Ig and Ig. (D) Four representative contour plots of highly variable Haloperidol Decanoate dual Ig and Ig expression by CD19+ B cells in patients with SLE. (E) Frequencies of viable CD19+ B cells expressing both Ig and Ig light chains in healthy donors (= 26) SLE patients (= 56) and patients with GPA (= 23). Each dot shows an individual patient, bars represent averages. (F) Examples of PCR amplification of Ig gene rearrangements from six consecutive single B cells expressing Ig and failure to amplify Ig gene rearrangements from six consecutive single B cells expressing Ig?from healthy donors.?? In contrast, samples of eight cells pooled and five single cells from patient 43 sorted for dual Ig and Ig light IFNGR1 chain expression gave products for both Ig and Ig. These were not originally consecutive wells; the PCR products were re run in adjacent lanes to compare band sizes. Representative gels of three independent experiments are shown. We confirmed that the bands from these five single cells were indeed Ig and Ig?gene rearrangments by sequencing. V and J pairings for these rearrangments are illustrated in G. Statistical analyses were performed by MannCWhitney tests assuming nonparametric distributions * 0.05; ** 0.005; *** 0.0001; **** = 0.00001. Expression of light chains by single cells was investigated by PCR. B cells from healthy individuals were sorted for expression of either Ig or Ig, and cDNA from each cell was tested in PCR reactions for amplification of Ig or Ig rearrangements. The PCR efficiency for amplification of light chain genes that agreed with surface light chain expression was approximately 41%. No examples of wells with both Ig+ and Ig+ PCR Haloperidol Decanoate amplicons, or amplification of the wrong light chain were obtained from 288 single Ig+ or Ig+ B cells from healthy donors. An example of amplicons from seven consecutive wells following amplification of Ig from Ig or Ig expressing B cells from a healthy donor is illustrated in Fig. ?Fig.1F.1F. SLE B cells expressing both Ig+ and Ig+ were placed at 1 cell per well in 384 wells of 96\well PCR plates. Of these, 14 wells gave PCR amplicons of both Ig and Haloperidol Decanoate Ig. (Fig. ?(Fig.1F),1F), 43 gave amplicons for Ig only and 61 gave amplicons for Ig only. The PCR amplicons of Ig and Ig from each of five single cells illustrated in Fig. ?Fig.1F1F were sequenced and were confirmed to be productively rearranged Ig and Ig rearrangements (Fig. ?(Fig.1G).1G). Experimentally, these were not adjacent reactions. PCR products were run alongside positive controls with eight dual positive cells per well to allow direct comparison of band sizes. Although the PCR efficiency for dual Ig+ or Ig+ B cells was low, the frequency of detection of amplicons for both Ig and Ig from a single cell was significantly greater in dual Ig and Ig+ positive cells from SLE patients than in the single Ig+ or Ig+ positive cells in healthy donors by 2 analysis (= 0.001). The reason for PCR inefficiency for dual Ig+, Ig+ positive cells is not clear. If the presence.