1. Volume 11, Issue 3, Pages 317-327 (September 1999)
Frequencies of Multiple IgL Chain Gene Rearrangements in Single Normal or κL Chain– Deficient B Lineage Cells 
Tamotsu Yamagami, Edwin ten Boekel, Jan Andersson, Antonius Rolink, Fritz Melchers 
Immunity 
Volume 11, Issue 3, Pages (September 1999)
DOI: /S (00)

2. Figure 1
Single Cell PCR Analyses of κL and λL Chain Gene Rearrangements in Different B Cell Subpopulations of B6/JCκ− F1 Mice
The rearrangement status of single small pre-BII cells, immature B cells of bone marrow (or spleen), and mature B cells of spleen are shown. Positions that are boxed indicate the presence of a rearrangement in the cell. The open circles indicate a nonproductive and the closed circles a productive VκJκ rearrangement. The single cell PCR analyses are subject to false negative results due to lack of detection of a given rearrangement. False positives are also seen. They are probably due to very low levels of contamination especially visible in the germline configuration (∼2%). Examples of such false positives are small pre-BII cell numbers 58, 120, and 183 of B6/JCκ− F1 mice. These cells show germline configuration together with Vκ to Jκ1 rearrangement that should not be detected from a single κL chain gene allele (see primer designs in Yamagami et al. 1999).
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3. Figure 4
Frequencies of All VκJκ Rearrangemnts on One Allele in Single B Cells
Frequencies of VκJκ rearrangements in (A) single pre-BII cells (open squares), bone marrow immature B cells (open circles), spleen immature B cells (closed circles), and spleen mature B cells (closed triangles) of B6/JCκ− F1 mice; (B) single λL+sIg+ bone marrow (open circles) and spleen (closed circles) immature B cells, spleen mature B cells (closed triangles), and κL+sIg+ bone marrow immature B cells (circles with cross) of B6/JCκ− F1 mice; (C) single bone marrow (open circles) and spleen (closed circles) immature B cells and spleen mature B cells (closed triangles) of Cκ−/JCκ− F1 mice; and (D) single small pre-BII cells of B6/JCκ− (open squares) and Cκ−/JCκ− (closed squares) F1 mice. Frequency zero (0) indicates a cell that has only the germline configuration on the κL chain gene allele. Frequency 1, 2, 3, and 4 indicates a cell that shows one, two, three, and four VκJκ rearrangement(s), respectively. The sum of cells of each compartment does not make 100% in total because some cells did not yield any PCR product (Figures 1, 2, 3). (E) Frequencies of productive rearrangements in the different VκJκ joints found in κL chain alleles of small pre-BII and mature B cells of B6/JCκ− and Cκ−/JCκ− F1 mice. Open squares, B6/JCκ− small pre-BII cells; closed squares, B6/JCκ− mature B cells; open circles, Cκ−/JCκ− small pre-BII cells; and closed circles, Cκ−/JCκ− mature B cells.Figure 1Yamagami et al. 1999Figure 1
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4. Figure 4
Frequencies of All VκJκ Rearrangemnts on One Allele in Single B Cells
Frequencies of VκJκ rearrangements in (A) single pre-BII cells (open squares), bone marrow immature B cells (open circles), spleen immature B cells (closed circles), and spleen mature B cells (closed triangles) of B6/JCκ− F1 mice; (B) single λL+sIg+ bone marrow (open circles) and spleen (closed circles) immature B cells, spleen mature B cells (closed triangles), and κL+sIg+ bone marrow immature B cells (circles with cross) of B6/JCκ− F1 mice; (C) single bone marrow (open circles) and spleen (closed circles) immature B cells and spleen mature B cells (closed triangles) of Cκ−/JCκ− F1 mice; and (D) single small pre-BII cells of B6/JCκ− (open squares) and Cκ−/JCκ− (closed squares) F1 mice. Frequency zero (0) indicates a cell that has only the germline configuration on the κL chain gene allele. Frequency 1, 2, 3, and 4 indicates a cell that shows one, two, three, and four VκJκ rearrangement(s), respectively. The sum of cells of each compartment does not make 100% in total because some cells did not yield any PCR product (Figures 1, 2, 3). (E) Frequencies of productive rearrangements in the different VκJκ joints found in κL chain alleles of small pre-BII and mature B cells of B6/JCκ− and Cκ−/JCκ− F1 mice. Open squares, B6/JCκ− small pre-BII cells; closed squares, B6/JCκ− mature B cells; open circles, Cκ−/JCκ− small pre-BII cells; and closed circles, Cκ−/JCκ− mature B cells.Figure 1Yamagami et al. 1999Figure 1
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5. Figure 4
Frequencies of All VκJκ Rearrangemnts on One Allele in Single B Cells
Frequencies of VκJκ rearrangements in (A) single pre-BII cells (open squares), bone marrow immature B cells (open circles), spleen immature B cells (closed circles), and spleen mature B cells (closed triangles) of B6/JCκ− F1 mice; (B) single λL+sIg+ bone marrow (open circles) and spleen (closed circles) immature B cells, spleen mature B cells (closed triangles), and κL+sIg+ bone marrow immature B cells (circles with cross) of B6/JCκ− F1 mice; (C) single bone marrow (open circles) and spleen (closed circles) immature B cells and spleen mature B cells (closed triangles) of Cκ−/JCκ− F1 mice; and (D) single small pre-BII cells of B6/JCκ− (open squares) and Cκ−/JCκ− (closed squares) F1 mice. Frequency zero (0) indicates a cell that has only the germline configuration on the κL chain gene allele. Frequency 1, 2, 3, and 4 indicates a cell that shows one, two, three, and four VκJκ rearrangement(s), respectively. The sum of cells of each compartment does not make 100% in total because some cells did not yield any PCR product (Figures 1, 2, 3). (E) Frequencies of productive rearrangements in the different VκJκ joints found in κL chain alleles of small pre-BII and mature B cells of B6/JCκ− and Cκ−/JCκ− F1 mice. Open squares, B6/JCκ− small pre-BII cells; closed squares, B6/JCκ− mature B cells; open circles, Cκ−/JCκ− small pre-BII cells; and closed circles, Cκ−/JCκ− mature B cells.Figure 1Yamagami et al. 1999Figure 1
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6. Figure 5
Primary and Secondary IgL Chain Gene Rearrangements in the Small Pre-BII Cell Compartment during B Cell Development in Mouse Bone Marrow
DHJH-rearranged pre-BI cells expressing surrogate L chain (inside pre-BII cells) initiate VH to DHJH rearrangements at the transition to pre-BII cells. When these rearrangements result in a productively rearranged IgH chain locus that can make a μH chain (green rod) capable of pairing with surrogate L chain, a pre-B cell receptor (green rod on the surface of large pre-BII cells) can be deposited on the surface, and the corresponding large pre-BII cells expand by proliferation. When large pre-BII cells have used up surrogate L chain protein, which is no longer synthesized in these cells, they come to rest as small pre-BII cells. At the transition of large to small pre-BII cells, VL to JL rearrangements are initiated at the κL and λL chain loci. Primary rearrangements can be productive, and the produced IgL (κL, closed circles; λL, open triangles) chain capable of pairing with the preexisting μH chain to form IgM on the cell surface, the BCR (shown for κL and λL chains as the top three cells in transition to the immature B cell pool). If the BCR recognizes an autoantigen in bone marrow, the cells are stopped from further development, and surface IgM is downregulated (small pre-BII cell number 3 from top). The cells, now expressing μH chains and κL chains only in the cytoplasm, i.e., with a pre-B cell phenotype, begin to “edit” their BCR. Other primary rearrangements can be productive but may yield IgL chains that cannot pair with the preexisting μH chain (small pre-BII cell number 4 from top), expressing μH chain and IgL chain only in the cytoplasm. Still other primary rearrangements can be out-of-frame, in that case the cell remains IgL chain negative (small pre-BII cell number 5 from top). These three types of small pre-BII cells (number 3, 4, and 5 from top) engage in secondary and subsequent rearrangements (horizontal sinusoid arrow) as long as they have not managed to generate a productively VLJL-rearranged locus producing a pairing IgL chain that yields a BCR that is not autoreactive. It is assumed that secondary and subsequent rearrangements first occur at the first κL chain allele in the order in which the Jκ and RS elements are positioned and then at the second allele (see series of small pre-BII cells at the bottom of that compartment). Not shown in the figure are the VL to JL rearrangements at the λL chain locus that occur independently of those at the κL chain locus (see Yamagami et al. 1999) at 5–10 times lower frequencies, again in- and out-of-frame, yielding pairing and nonpairing λL chains, and the corresponding BCR expressing nonautoreactive or autoreactive immature B cells. Productive VLJL rearrangements yielding pairing IgL chains and hence, lead these cells to enter the sIgM+ immature B cell compartment are indicated (arrow pointing upward). From the nonautoreactive immature B cell pool, first in the bone marrow and then in spleen, mature B cells are selected into the peripheral pools (Rolink et al. 1998).
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