BCR Ligation Induces Receptor Editing in IgM+IgD− Bone Marrow B Cells In Vitro  Marc Hertz, David Nemazee  Immunity  Volume 6, Issue 4, Pages 429-436 (April 1997) DOI: 10.1016/S1074-7613(00)80286-1

Figure 1 BCR Ligation Does Not Induce Death of Bone Marrow B Cells (A and B) Bone marrow cells from 3-83μ/δ (A) or 3-83μ/δ/Bcl-2 (B) mice were cultured either alone or with 10 μg/ml of anti-Id MAb 54.1, anti-κ MAb 331812, or control MAb 5Ci. The absolute number of B220+ cells was determined at various times by counting viable cell numbers and determining the percentage of B220+ cells in flow cytometry assays. (C and D) Bone marrow B cells from B10.D2 (C) or B10.D2/Bcl-2 (D) mice were cultured as in (A) and (B). Data points represent the means ± standard deviation of 2–12 independent experiments. Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)

Figure 2 BCR Ligation Increases RAG-2 mRNA Expression in Bone Marrow B Cells (A) Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis of RAG-2 mRNA expression levels in response to BCR ligation. Cultures were harvested after 24 hr of treatment. All antibodies were used at 10 μg/ml final concentration as in Figure 1. Control transcript Gas was amplified as a ubiquitously expressed gene and used to standardize loading. (B) Summary of RT–PCR analysis of RAG-2 mRNA expression levels. Relative intensity is calculated as RAG-2:Gas ratio of radioactive signal. Bars represent means ± standard deviation of six independent experiments. Statistical significance determined by JMP 3.15 statistical software (SAS Institute, Cary, NC). Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)

Figure 3 Induction of Vκ-Jκ1 Joining after BCR Ligation in Bone Marrow Cultures (A) Autoradiograph of samples analyzed by PCR and Southern blot for endogenous Vκ-Jκ1 rearrangements (top panel) and control, α-actin gene (bottom panel). Cell lysates were made after 48 hr treatment in culture and used as template in PCR assays for rearrangement products. Lanes 1–6 show results for control lysates made with mixtures of various proportions of VκJκ1-positive and VκJκ1-negative hybridoma cell lines. Lanes 8 and 10 show PCR results from untreated cultures of 3-83μ/δ and 3-83μ/δ/Bcl-2 bone marrow, respectively. Lanes 9 and 11 show results from the anti-Id MAb 54.1 (10 μg/ml) treated cultures of 3-83μ/δ and 3-83μ/δ/Bcl-2 bone marrow, respectively. (B) Standard curve derived from (A) relating the band intensity to fraction of cells bearing a Vκ-Jκ1 rearrangement. (C) Estimated fraction of control and anti-Id-treated B cells that bear Vκ-Jκ1 rearrangement after 48 hr of treatment. Data shown are the means of six independent experiments ± the standard deviation. Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)

Figure 4 BCR Ligation Increases the Proportion of Immature B Cells Expressing (Endogenous) λ-Light Chain (A–D) Flow cytometric analysis of bone marrow cultures 72 hr after no treatment (A) or treatment with either 10 μg/ml control MAb 5Ci (B), anti-Id MAb 54.1 (C), or anti-κ 331812 (D). Bone marrow cells (2 × 106) were stained with anti-B220-PE and anti-λ-FITC and analyzed on a FacsCalibur flow cytometer. (E) The effect of BCR ligation on the percentage of B cells that express endogenous λ-light chains after 72 hr in culture. Data represent the means ± standard deviation of ten independent experiments. Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)

Figure 5 BCR Ligation Enhances Receptor Editing in Immature B Cells from Normal, Nontransgenic Mice (A–E) Flow cytometric analysis of κ- and λ-light chain surface expression in B220+ gated bone marrow cells from B10.D2 mice after 48 hr of treatment in culture. Cells were stimulated with no added antibody (A), 100 μg/ml control MAb 54.1 (B; this anti-3-83-Id MAb does not bind to normal, non-Tg B cells), 1 μg/ml anti-κ MAb (C), 10 μg/ml anti-κ MAb (D), or 100 μg/ml anti-κ MAb (E). The extreme down-modulation of κ-light chain expression in (D) and (E) is not associated with cell death, but is the result of receptor down-modulation and epitope blocking by the treatment antibody. (F) Percentages of B cells expressing λ-light chain in B10.D2 bone marrow cultures after 48 hr. Percentages represent the means ± standard deviation of six to nine independent experiments. Asterisk indicates a statistical significance of p < 0.05 determined by JMP 3.15 statistical software. Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)

Figure 6 B Cell Maturation Stage and CFSE Analysis of Cell Division of λ+ Cells in Treated and Control Cultures (A) Absolute number of λ+ B cells in immature IgD− and mature IgD+ B cell subpopulations in Ig-Tg bone marrow cultures. λ+ cell counts were determined from IgD+B220+ or IgD− B220+ cells from Ig-Tg bone marrow cultures after 48 hr in untreated culture, or cultures treated with 10 μg/ml control MAb or anti-Id MAb. (B) Absolute number of λ+ B cells in immature CD23− and mature CD23+ B cell subpopulations in normal bone marrow cultures. λ+ cell counts were determined from CD23+B220+ or CD23−B220+ cells from normal B10.D2 bone marrow cultures after 48 hr in untreated culture, or cultures treated with 10 μg/ml control MAb or anti-κ MAb. (C) Histogram of total CFSE-labeled B220+ cells. Peaks of CFSE fluorescence intensity represent successive cell division; with each division the cells lose approximately half of the FL1 mean fluorescence. Broken vertical lines mark 0, 1, and 2 cell divisions. (D) Histogram of CFSE labeling in gated λ+ cells from Ig-Tg bone marrow cultures 48 hr after no treatment or treatment with 10 μg/ml anti-Id MAb. Histograms are overlaid and show that few λ+ cells have undergone cell divisions. (E) Histogram of CFSE labeling in gated λ+ cells from normal bone marrow. Cells were gated on λ+ lymphocytes as in (D) and show overlaid histograms of cultures either untreated or treated with 10 μg/ml control MAb or 10 μg/ml anti-κ MAb. Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)

Figure 7 Two Ways That BCR Ligation Could Promote Receptor Editing This schematic depicts a model of immature B cell development. The cell depicted at the left is the immediate descendant of a pre-B-cell that actively rearranged light chain genes and was able to form a functional light chain. This cell may transiently continue to express the recombination machinery and “test” its antigen receptor on the cell surface. In the absence of tolerogen, further maturation terminates light chain recombination (represented by the cell in the center marked “recombination−“) and allows further maturation, ultimately allowing export to the periphery (represented by the cell marked “mature B” at right). Antigen may cause an immature B cell that has stopped expressing the recombination machinery to reexpress it and to undergo secondary light chain rearrangements (pathway 1). Alternatively, antigen may block the developmental progression of immature B cells that express the recombination machinery (pathway 2). In either case, antigen would cause immature B cells to express the recombination machinery and to initiate secondary light chain gene rearragements. Immature B cells that fail to make a new light chain that lacks self-reactivity eventually undergo cell death. Immunity 1997 6, 429-436DOI: (10.1016/S1074-7613(00)80286-1)