Expression of both Igκ and Igλ light chain by CD19⁺ B cells in SLE. CD19⁺ cells from healthy donors (n = 26) and SLE patients (n = 56) were analysed for cell surface expression of Igκ and Igλ by flow cytometry. (A and B) Representative contour plots of isotype controls for cell surface expression of Igκ and Igλ by CD19⁺ cells from a healthy donor and a patient with SLE, respectively. (C) Representative contour plot of cell surface expression of Igκ and Igλ by CD19⁺ cells in healthy donors showing clear separation of B cells expressing Igκ and Igλ. (D) Four representative contour plots of highly variable dual Igκ and Igλ expression by CD19⁺ B cells in patients with SLE. (E) Frequencies of viable CD19⁺ B cells expressing both Igκ and Igλ light chains in healthy donors (n = 26) SLE patients (n = 56) and patients with GPA (n = 23). Each dot shows an individual patient, bars represent averages. (F) Examples of PCR amplification of Igκ gene rearrangements from six consecutive single B cells expressing Igκ and failure to amplify Igκ gene rearrangements from six consecutive single B cells expressing Igλ from healthy donors.   In contrast, samples of eight cells pooled and five single cells from patient 43 sorted for dual Igκ and Igλ light chain expression gave products for both Igκ and Igλ. 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 Igκ and Igλ gene rearrangments by sequencing. V and J pairings for these rearrangments are illustrated in G. Statistical analyses were performed by Mann–Whitney tests assuming nonparametric distributions *p < 0.05; **p < 0.005; ***p < 0.0001; **** = p < 0.00001.

(A) PBMCs from SLE patient 119 were stained for CD19, IgD, IgG, Igκ, and Igλ and CD19⁺ B cells expressing single Igκ ( quadrant 1), single Igλ (quadrant 3), or both Igκ and Igλ ( quadrant 2) were identified by flow cytometry. (B) Cells in quadrants 1, 2, and 3 from A were then regated to identify cells with surface IgD and IgG. (A, B) One representative plot out of two independent experiments is shown. (C) Quantification of flow cytometric analysis of the frequency of Igκ and Igλ light chain expression in different B‐cell subsets in SLE; transitional (TS) B cells (CD19⁺ IgD⁺ CD27⁻ CD10^hi), mature naive (MN) B cells (CD19⁺ IgD⁺ CD27⁻ CD10^lo), memory (Mem) B cells (CD19⁺ IgD⁻ CD27⁺), and plasmablasts (PB) (CD19⁺ CD38^hi CD27^hi). Each dot shows an individual patient, bars show averages.

Cell surface expression of Igκ and Igλ light chains following DNAse treatment, acid washes, and cell culture. (A) Flow cytometry analysis of light chain expression by CD19⁺ SLE B cells following incubation with DNase, or two acid wash steps using two different acidic wash buffers; 0.2 M glycine (pH3.0) and RPMI‐1640 media wash (pH2.5) prior to immunostaining and flow cytometric analysis. Representative contour plots from three independent experiments with three different donors are shown. (B) Immunoglobulin light chain cell surface expression on sorted Igκ⁺, Igλ⁺ CD19⁺ B cells from an SLE patient was determined by flow cytometry on day 0 or following coculture with allogeneic CD3⁺CD4⁺ T cells with PMA for 2 days. Isotype controls and two replicates (Rep1 and Rep2) out of two independent experiments with two different donors are shown.

Dectection of surface and intracellular Igκ and Igλ light chains by CD19⁺ B cells in SLE. (A) B cells from a patient with SLE gated to identify B cells with surface Igκ and Igλ, remain Igκ⁺ and Igλ⁺ when reanalysed for cytoplasmic Igκ and Igλ. Fluorescence minus one (FMO) controls ae illustrated. One representative dot plot out of ten independent experiments is shown. (B) This was quantified and found to be consisent in ten patients previously seen to show dual light chain expression. Intra‐DP refers to the percentage of B cells that are double positive for surface Igκ and Igλ, that are then shown to be Igκ and Igλ following permeabilisation and use of antibodies to Igκ and Igλ conjugated to different fluorochromes. Bars show the percentage of intracellular double‐positive cells out of the total surface double‐positive B cells for ten patients analysed. (C) B cells from healthy controls and SLE patients were stained for Igκ and Igλ expression and analysed by confocal microscopy. Representative images from two independent experiments with four donors are shown. Original magnification ×60.

Relationship between frequency of KDE rearrangement to the iRSS and dual light chain expression. (A) Sorting strategy to avoid biases resulting from gating. Cells expressing Igλ were sorted, irrespective of whether they also expressed Igκ prior to isolation of genomic DNA. One representative dot plot out of 34 independent experiments is shown. (B) Representation of qRT‐PCR amplification of rearranged KDE/iRSS junctions. (C) Relative copy numbers (RCN) of iRSS‐KDE rearrangments in gDNA isolated from FACS sorted CD19⁺ Igλ ⁺ B cells from healthy controls (n = 11) and SLE patients (n = 23) were determined by qRT‐PCR as shown in (B). RNaseP was used as a reference gene and data was adjusted in each assay so that cell line NALM6 had a value of 2. Each dot shows an individual patient, bars show means. (D) Representation of qRT‐PCR amplification of intronic sequence between the iRSS and IgKJ. (E) Relative quantification by qRT‐PCR of an intronic region of the Igκ light chain, located 5’ to the iRSS site, in gDNA isolated from CD19⁺ Igλ ⁺ B cells from healthy controls (n = 9) and SLE patients (n = 14) standardised to RNaseP. Each dot shows an individual patient, bars show means. (F) RCN of iRSS‐KDE rearrangements standardised to RNaseP is inversely correlated with the frequency of dual light chain expressing cells. Each dot shows an individual patient, and regression line is illustrated. Statistical analysis was performed by Mann–Whitney tests assuming nonequal variance (C, E) or Spearman's test for correlation (F) where **p < 0.01.

Comparison of iRSS‐KDE junctions and sequences flanking the junctions in B cells from healthy donors and patients with SLE. (A) KDE rearrangements to the iRSS were PCR amplified from gDNA isolated from CD19⁺Igλ⁺ B cells from healthy controls (n = 2) and SLE patients (n = 2). PCR products were cloned, sequenced, and aligned to germline sequences. (B) Bars show the frequency of sequences that terminate at specific breakpoints after iRSS‐KDE rearrangement. (C) Frequency of N‐nucleotide additions to the iRSS‐KDE junction in healthy donors (n = 2) and SLE patients (n = 2) was determined as shown in (A). Bars show percentage of sequences with each number of nucleotide additions to the junction.