Transient IL-7/IL-7R Signaling Provides a Mechanism for Feedback Inhibition of Immunoglobulin Heavy Chain Gene Rearrangements  Dipanjan Chowdhury, Ranjan Sen  Immunity  Volume 18, Issue 2, Pages 229-241 (February 2003) DOI: 10.1016/S1074-7613(03)00030-X

Figure 1 IL-7-Dependent Accessibility of VHJ558 Genes (A) The top line is a schematic representation of the murine VH gene locus showing the approximate location of gene segments analyzed in this paper. The lower line shows an expanded view of the VHA.1 and JH3 regions with the location of the Sau3AI restriction enzyme sites used in the ligation mediated-PCR (LM-PCR) accessibility assays. First strand synthesis utilized primer 1, PCR amplification was carried out with primer 2, and radioactive labeling with primer 3. (B) CD19+ pro-B bone marrow cells from RAG2−/− mice were cultured for 4 days with (right panel) or without IL-7 (20 ng/ml, left panel). These cells were stained simultaneously either with FITC-anti-CD43 and PE-anti-CD19 (upper panel) or with FITC-anti-CD25 and PE-anti-HSA (lower panel) and then analyzed on a Becton Dickinson FACS Calibur. (C) Nuclei from these cells were treated with 0 U (lanes 1 and 4), 2 U (lanes 2 and 5) or 10 U (lanes 3 and 6) of Sau3AI. The extent of cleavage was estimated by LM-PCR using primers specific for the VHA.1 gene segment (left panel) and JH3 (right panel). To control for quality and quantity of linker ligated DNA, a fragment within the Cμ region was amplified in the same PCR reaction. Cμ amplification was independently tested to be in the linear range. Representative data from one of three experiments using three preparations of bone marrow cells are shown. (D) The results in (C) were quantified by phosphorimager analysis, and the intensity of the VHA.1 (IVHA.1) and JH3 (IJH3) bands were normalized to their respective Cμ (ICμ) signal. The y axis of the graph represents the relative intensity of VHA.1 and JH3 signals compared to the corresponding Cμ signal (IVHA.1/ICμ or IJH3/ICμ ) expressed as a percentage. Immunity 2003 18, 229-241DOI: (10.1016/S1074-7613(03)00030-X)

Figure 2 VH Activation and Accessibility in Bone Marrow Pro-B and Pre-B Cells (A) Hardy classification of early B cell development listing some of the cell surface markers used to distinguish the different cell populations (Hardy et al., 1991). In the IgH locus, DJH recombination is seen in fraction B cells, and VDJ recombination is seen in fraction C cells. Light chain recombination occurs in D fraction cells. (B) B/C fraction pro-B cells and D fraction pre-B cells were purified from bone marrow of 6- to 8-week BALB/c mice by magnetic depletion of erythroid, myeloid, T cells, and IgM+ B cells, followed by sorting for the B220+CD43+HSA+ cells for the pro-B and B220+CD43−HSA+ cells for the pre-B fraction, respectively. The middle panel shows the composition of the cell preparation after magnetic depletion and indicates the gates used to purify pro- and pre-B cells. The left and right panels show the purified populations used for chromatin immunoprecipitation and accessibility studies. (C) Chromatin immunoprecipitation assays were carried out using purified pro-B (blank bars), pre-B cells (stippled bars), and thymocytes (black bars). Formaldehyde crosslinked chromatin prepared from cells as described in the Experimental Procedures was incubated with anti-acetylated histone H3 antibody (α-AcH3). Antibody-bound DNA was collected by adsorption to protein A-agarose, decrosslinked, and purified. The input DNA, which is DNA purified from chromatin prior to immunoprecipitation, and immunoprecipitated DNA were quantitated using picogreen fluorescence, and real-time PCR using SYBR Green Universal Mix was carried out with 0.5 ng of each DNA sample. Real-time PCR gives a value corresponding to the amount of target sequence in each DNA sample and allowed us to calculate (as described in the Experimental Procedures) the relative increase in target sequence in the immunoprecipitated DNA over the input DNA. The y axis indicates the relative enrichment of target genes as fold difference between the input fraction and the immunoprecipitated fraction, and the x axis indicates the different gene segments analyzed. The locations of these VH gene segments are shown in Figure 1A. The μ enhancer region (μE) was used as a positive control and the β-globin gene as a negative control. The results shown are an average of two experiments done in duplicate from two independently sorted cell populations with error bars representing the standard deviation. (D and E) Nuclei from the pro-B cells and pre-B cells were treated with 0 U (lanes 2 and 4) or 10 U (lanes 1 and 3) of Sau3AI, and the extent of cleavage was estimated by LM-PCR using the primers shown in Figure 1A. Phosphorimager quantitation was done as described in the legend of Figure 1D. Genomic DNA made from pro-B cells and pre-B cells were quantitated using picogreen fluorescence. Radioactive PCR was done using 0 (lane 1), 0.04 ng (lane 2), 0.4 ng (lane 3), and 4 ng (lane 4) of DNA, and the products were visualized after fractionation through 6% polyacrylamide gel (lowest panel in [D]). Representative data from one of two experiments using two independently sorted bone marrow cell preparations are shown. (F and G) Nuclei from thymocytes were treated with 0 U (lane 1), 0.4 U (lane 2), 2 U (lane 3), and 10 U (lane 4) of Sau3AI, and the extent of cleavage was estimated by LM-PCR using the primers shown in Figure 1A. Representative data (F) from one of two experiments using two independently prepared thymocyte populations is shown. Phosphorimager quantitation was done as described in the legend of Figure 1D and represents the average of two independent experiments (G). Immunity 2003 18, 229-241DOI: (10.1016/S1074-7613(03)00030-X)

Figure 3 IL-7R-Mediated Signaling in Bone Marrow Pro-B and Pre-B Cells (A) Whole-cell lysates from 0.5 × 106 pro-B cells (lane 1) and pre-B cells (lane 2) purified as described in Figure 2 legend were fractionated through a 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and probed with anti-phospho Stat5 antibody (top panel). The blot was then stripped and reprobed with anti-Stat5 antibody to determine total Stat5 levels (lower panel). 32D cells starved of IL-3 for 12 hr served as a negative control (lane 3), and these cells restimulated with IL-3 for 30 min were used as a positive control (lane 4). Representative data from one of three experiments using three independently sorted bone marrow cells is shown. (B) The phospho-Stat5 and Stat-5 bands were quantitated by scanning multiple exposures of the Western blot on X-ray film using Epson Expression 1600 Scanner. Relative intensity (indicated on the y axis) is the ratio of the intensity of phospho-Stat5 band over the intensity of the corresponding total Stat5 band. The results shown are an average of three independent experiments using independently sorted cell populations with the error bars representing the standard deviation. Immunity 2003 18, 229-241DOI: (10.1016/S1074-7613(03)00030-X)

Figure 4 Deacetylation of VH J558 Genes (A) Schematic representation of the murine VH locus showing the approximate location of three representative gene segments from the VHJ558 gene locus analyzed in this study. (B) The temperature-sensitive Abelson virus-transformed pre-B cell line, 103-(4) (Chen et al., 1994) was incubated at 34°C (permissive, P) or at 39°C (restrictive, R) for 12 or 24 hr, respectively, in the presence or absence of IL-7 (0.2 ng/ml) as indicated. Chromatin immunoprecipitation and real-time PCR analysis were done as described in the legend to Figure 2, using VH primers shown in (A). The μ enhancer region (μE) served as a positive control and β-globin gene as a negative control. The results shown are the average of three independent temperature shift experiments. (C and D) Nuclei from the 103-(4) cells incubated at permissive or restricted temperature for 24 hr in the presence or absence of IL-7 as indicated, were treated with 0 (lane 1), 2 U (lane 2), and 10 U (lane 3) of Sau3AI, and the extent of cleavage was estimated by LM-PCR using the VHA.1 primers shown in Figure 1A. The JH3 gene could not be analyzed because it has been deleted by recombination. Representative data, after normalization to Cμ sequences, from one of three independent experiments is shown (D). Phosphorimager quantitation was done as described in the legend to Figure 1D and is the average of three independent experiments. Immunity 2003 18, 229-241DOI: (10.1016/S1074-7613(03)00030-X)

Figure 5 Reactivation of the Allelically Excluded VH Gene Segments (A) Splenic B cells from anti-HEL transgenic mice (stippled and blank bars) and wild-type littermates (hatched and filled bars) were cultured for 3 days with anti-CD40 antibody (10 μg/ml) in the presence or absence of IL-7 (20 ng/ml) and IL-4 (800 U/ml) (Schrader et al., 1999). Chromatin immunoprecipitation and real-time PCR analysis were done as described in the Figure 2 legend, using the same VH primers. The μ enhancer region (μE) served as a positive control and β-globin gene as a negative control (data not shown). VH10 and VH81X gene sequences could not be amplified from B cell DNA from wild-type littermates presumably reflecting their deletion by 5′ VH gene rearrangements. The results shown are an average of three independent experiments done in duplicate with the error bars representing the standard deviation. (B and C) Nuclei from splenic B cells from anti-HEL transgenic mice (B) or normal littermates (C) were treated with 0 (lanes 1 and 4), 3 U (lanes 2 and 5), or 12 U (lanes 3 and 6) of Sau3AI, and the extent of cleavage was estimated by LM-PCR using the primers shown in Figure 1A. Phosphorimager quantitation was done as described in the legend to Figure 1D. Representative data, after normalization to Cμ sequences, from one of three experiments using three preparations of splenic cells is shown. Immunity 2003 18, 229-241DOI: (10.1016/S1074-7613(03)00030-X)