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IMMUNOBIOLOGY
From the Departments of Immunology, Pediatrics, and
Cell Differentiation (Institute of Molecular Embryology and Genetics),
Kumamoto University School of Medicine; the Biomedical Research Center,
Osaka University Medical School, Japan; and the Food and Drug
Administration, Center for Biologic Evaluation and Research, Division
of Cellular and Gene Therapies, Bethesda, MD.
Secondary rearrangements of immunoglobulin gene segments that
generate a new antibody repertoire in peripheral B cells have been
described as receptor revision and occur by as yet unknown mechanisms.
To determine the importance of recombination activating gene (RAG)
expression in receptor revision, heterozygous rag1/green fluorescent protein (gfp) knockin mice were used to examine the location of RAG1 expression in the germinal centers (GCs) of lymphoid follicles after immunization with a variety of T-cell-dependent antigens. Immunization of rag1/gfp heterozygous mice or
rag1 homozygous knockout mice reconstituted with
rag1/gfp heterozygous spleen cells caused the
down-regulation of RAG1/GFP signal in GCs. Although some
RAG1/GFP+ cells appeared in regions surrounding the peanut
agglutinin (PNA)+GL-7+ GC area,
RAG1/GFP+ cells did not accumulate in the central region.
In addition, the stimulation of spleen B cells with anti-µ antibody
plus interleukin-4 (IL-4) or with anti-CD40 monoclonal antibody
plus IL-7 did not induce GFP signals at detectable levels in vitro.
These results clearly demonstrate that RAG1 re-expression either does
not occur or is at extremely low levels in antigen-driven B cells in
GCs of secondary lymphoid follicles, suggesting that other mechanisms may mediate the gene rearrangements observed in receptor revision.
(Blood. 2001;97:2680-2687) B cells are generated in the fetal liver or the
adult bone marrow (BM) after immunoglobulin gene rearrangements that
create a primary repertoire of random antibody (Ab)
specificity.1-3 B cells that react to various
self-antigens (Ags) at immature stages are thought to experience anergy
after encountering Ags or to be eliminated by apoptotic mechanisms in
the BM microenvironment.4-6 It has been suggested that a
substantial proportion of B cells reactive to self-Ags undergoes a
process known as receptor editing in which secondary immunoglobulin
gene rearrangements replace variable (V) gene segments, generating a
new Ab specificity as a part of the primary Ab
repertoire.7-11 B cells recruited to the circulation are
stimulated by Ags and helper-T (Th) cells in the T cell area of
secondary lymphoid organs.12-14 Such Ag-driven B cells
subsequently undergo rapid proliferation to become centroblasts located
in the dark zone of germinal centers (GCs) and then arrest cell cycling
and become small centrocytes in the light zone.15-20
Recent studies proposed an interesting model in which mature B cells
also undergo a similar process, termed receptor editing, in GCs of
secondary lymphoid follicles after stimulation by T-dependent (TD)
Ags.21-25 This notion has been supported by the detection of rag1/rag2 transcripts and by the evidence of
immunoglobulin gene rearrangements in situ, suggesting that receptor
editing occurs in the GC light zone.21 In addition,
stimulation with anti-CD40 monoclonal antibody (mAb) plus interleukin-4
(IL-4), or lipopolysaccharide (LPS) plus IL-4, induced the
up-regulation of rag1/rag2 transcripts and immunoglobulin
gene rearrangements in mature surface immunoglobulin M
(sIgM)+ B cells in vitro.22,24-26 These
results suggested that mature B cells frequently undergo receptor
editing in GCs after stimulation with Ags. The receptor editing
mechanism might provide a more flexible and diverse Ab specificity
response than the one produced solely by the somatic mutation
mechanism. The receptor editing in GCs could be a requisite molecular
event for an immediate and effective response producing
high-affinity Abs against various antigenic stimuli. Alternatively,
receptor editing could salvage B cells carrying anti-self specificities.
The reactivation of rag gene expression has been suggested
as one mechanism for the immunoglobulin gene rearrangements that occur
in receptor revision.27,28 To demonstrate direct in vivo evidence of the induction of RAG molecules in GCs and to measure the
frequency of cells undergoing receptor editing (or receptor revision),
we examined lymphoid organs for RAG1/green fluorescent protein (GFP)
signal using immunized or unimmunized rag1/gfp knockin mice.
Unimmunized heterozygous rag1/gfp knockin mice express GFP that can be detected at high sensitivity by microscopic observation of
splenic sections. Surprisingly, when we examined GCs of immunized mice,
we observed that the RAG1/GFP signal was not significantly induced in
GCs after stimulation with TD-Ags. This observation suggests that the
RAG proteins do not play a major role in receptor revision during the
clonal expansion of antigen-reactive B cells at the GC area after
stimulation by TD-Ags.
Cells and cell culture
Mice
Immunization with TD-Ags Heterozygous rag1/gfp knockin mice were immunized with 200 µL prewashed sheep red blood cell (SRBC) (Nippon Bio-Test Laboratories, Tokyo, Japan) in phosphate-buffered saline, 25 µg dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) in complete Freund's adjuvant (CFA) emulsion, or 100 µg nitrophenyl-chicken gamma globulin (NP-CGG) (provided by Dr Hitoshi Ohmori, Okayama University, Japan) in CFA emulsion. We introduced Ags by intraperitoneal injection for analysis of spleens or by injection into each hind footpad with 20 µg NP-CGG in CFA for analysis of lymph nodes.Cell surface staining, flow cytometric analysis, and cell sorting Cell surface staining was performed as described previously31 with various mAbs. Phycoerythrin (PE)-anti-B220 mAb (RA3-6B2; Pharmingen, San Diego, CA), biotinylated-anti-IgM mAb (R6-60.2; Pharmingen), biotinylated-anti-Fas mAb (Jo2; Pharmingen), and PE-anti-IgD mAb (11-26; Southern Biotechnology Associates, Birmingham, AL) were purchased. Anti-CD43 mAb (S7) and anti-heat-stable antigen (HSA) mAb (M1/69) were purified from the culture supernatants of hybridoma cells by protein-G Sepharose column chromatography and were biotinylated as described previously.31 For 5-parameter analyses of individual cells, streptavidin-R670 (Gibco-BRL, Rockville, MD) was used as the secondary reagent for mAbs. Cells were analyzed by flow cytometry (Facscan; Becton Dickinson, San Jose, CA) with lymphoid gating using side and forward scatters.Detection of rag messenger RNA Total RNAs from FACS-sorted GFP+ cells of the rag1+/gfp+ spleen cells were extracted using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacture's protocol. The possible contamination of genomic DNA in polymerase chain reaction (PCR) assay was avoided by DNase I (Boehringer Mannheim, Mannheim, Germany) treatment as described.32 The complementary DNAs (cDNAs), synthesized from RNAs with MuLV reverse transcriptase (PerkinElmer, Foster City, CA), were used for PCR assay using Ampli Taq Gold (PerkinElmer) with gene-specific primers for rag1 messenger RNAs (mRNAs) as described previously.33,34 The amplification was carried out as follows: 10 minutes at 95°C; 35 cycles of 40 seconds at 94°C, 40 seconds at 60°C, 60 seconds at 72°C; and 10 minutes at 72°C. PCR products were resolved on 2% agarose gels and stained with ethidium bromide.32 As the control for RNA preparation, hypoxanthine-guanine phosphoribosyltransferase (HPRT) amplification was compared. PCR primers were as follows: 5'-TGCAGACATTCTAGCACTCTGG-3' and 5'-ACATCTGCCTTCACGTCGAT-3' for RAG1, 5'-CCATCCTGGTCGAGCTGGAC-3' and 5'-GCTCAGGTAGTGGTTGTCGG-3' for GFP, 5'-GCTCAGGTAGTGGTTGTCGG-3' and 5'-GCTGGTGAAAAGGACCTC-3' for HPRT, and 5'-CCTAAGGCCAACCGTGAAAAG-3' and 5'-TCTTCATGGTGCTAGGAGCCA-3' for -actin.
Reconstitution of spleen cells into rag1 knockout mice Spleen cells (1 × 107 cells) from heterozygous rag1/gfp-knockin mice (9 weeks after birth) were resuspended in 0.3 mL RPMI 1640 medium and injected into rag1-knockout mice intravenously. The mice, 14 days after reconstitution, were used for immunization with various TD-Ags, or the spleen cells were obtained for in vitro culture with various stimuli.Detection of anti-DNP Abs Ninety-six-well plates coated with DNP-chicken egg ovalbumin were incubated with serially diluted serum samples, and bound Abs were detected with biotinylated-goat-antimouse immunoglobulin Abs (isotype-specific for IgM and IgG1, respectively, obtained from Southern Biotechnology Associates), followed by streptavidin-alkaline phosphatase using a substrate of p-nitrophenyl phosphate disodium tablet (Sigma). After each incubation step of 3 hours at room temperature, the plates were washed 3 times with washing buffer Tris-buffered saline containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2% bovine serum albumin, and 0.05% Tween-20. Enzyme reaction was carried out as described elsewhere and was measured using the Bio-Rad enzyme-linked immunosorbent assay (ELISA) reader at 410 nm. The average of triplicate wells is shown.In vitro stimulation B cells were recovered from spleens of rag1 knockout mice that had been reconstituted with spleen cells (GFP-SPL) from rag1/gfp knockin mice. These recovered B cells were cultured with RPMI-1640 complete medium with various combinations of the following stimulatory reagents: 10 µg/mL affinity-purified F(ab')2 goat antimouse µ-chain-specific Ab (ICN, Costa Mesa, CA), 20 µg/mL LPS (Sigma Chemical, St Louis, MO), 1 µg/mL rat antimouse CD40 mAb (LB429),29 500 U/mL recombinant mouse IL-4 (provided by Dr K. Nakanishi, Hyogo College of Medicine, Nishinomiya, Japan), or 100 U/mL recombinant mouse IL-7 (provided by Dr F. Melchers, Basel Institute for Immunology, Switzerland) for various periods. After stimulation in vitro, cells were recovered and GFP signal was detected by flow cytometry. For control in experiments in which we attempted RAG1/GFP induction, we prepared in vitro cell lines from the BM of heterozygous rag1/gfp knockin mice using a BM culture method.35 After culturing for 3 weeks, B220+GFP+ cells, B220+GFPlow cells, and B220+GFP cells could be observed. These cells
were used for comparison of the GFP signal. Cellular responses to
various stimulatory reagents were examined by the incorporation of
[3H]-thymidine (TdR; Amersham, Buckinghamshire, United
Kingdom). Cells (1 × 105 cells per well) were cultured
in 96-well microtiter plates in culture medium with various stimulatory
reagents for 48 hours and were pulsed with [3H]-TdR
during the last 16 hours. After pulse labeling, cells were harvested
using a cell harvester, and [3H]-TdR incorporation was
measured by scintillation counter. The average of triplicate wells
is shown.
Preparation of tissue sections, immunohistochemistry, and confocal laser microscopic analysis Lymphoid organs from rag1/gfp knockin mice or littermates embedded in OCT compound (Tissue-Tek, Sakura Finetechnical, Tokyo, Japan) were rapidly frozen in liquid nitrogen. Tissues were prepared for histochemical analysis after they were sectioned at 10 µm, mounted on glass slides, and subsequently treated with 2% 3-amino-propyltriethoxysilane (Sigma) in acetone. Sections were processed immediately for confocal laser scan microscopy (Olympus, Tokyo, Japan) to detect the fluorescent signal emitted from GFP. Frozen specimens were prepared in a rectangular shape by cutting to the rim of the organs using a cryostat, and then serial sections were mounted onto glass slides. Immediately after drying the fixed sections, the fluorescence pictures were captured under confocal laser microscopy. Multiple marks were made at the edge of the each glass slide to identify a starting point. The turns of the microscope knobs from this starting point in both vertical and horizontal directions were counted. Using these counts from the initial start point of each slide, the microscopic focal area was oriented precisely onto the same GC without observing the view. By this method, the same GC on adjacent sections was easily identified. Using this method, the GFP signals from 2 contiguous slides could be compared and then identified with the corresponding GC by hematoxylin-eosin (H&E) or PNA staining. For marking of GCs, the mounted section was fixed with pure acetone and stained with biotinylated PNA (Molecular Probes, Eugene, OR). Biotinylated PNA was developed with diaminobenzidine (DAB) staining using streptavidin-horseradish peroxidase (HRP; Kierkegaard & Perry Laboratories, Gaithersburg, MD). For staining of GL-7, nonconjugated rat mAb GL-7 (Ly-77; Pharmingen) was used in combination with biotinylated-mouse antirat chain-specific mAb (MRK-1; Pharmingen)
and developed with DAB staining as described above. H&E staining was
conducted as described elsewhere.
RAG1/GFP+ B cells in secondary lymphoid organs In previous reports, expression of RAG1 and RAG2 was demonstrated in the whole area of PNA+ GCs, especially at the region of centrocytes, after immunization with various TD-Ags.21,22,24,25 Typically, a priming injection of NP-CGG with CFA at a footpad induced RAG1 expression in GCs at days 5, 12, and 21.To examine the pattern of RAG1 expression in greater detail,
rag1/gfp knockin mice were used to localize the expression
of RAG1 at the single-cell level in vivo after immunization with TD-Ags. When peripheral B cells were examined, the GFP signal was
observed in B220+ populations from spleen, Peyer patch, and
lymph nodes (Figure 1A). Because most
splenic B cells do not normally express RAG proteins or transcripts,
our observation of RAG1/GFP signal could potentially be explained by
the persistence of GFP protein that had been expressed in the
pre-B-cell stage of development under the control of the
rag1 gene transcriptional elements. Indeed, RAG1/GFP signal
generated from pro-B/pre-B cells of heterozygous rag1/gfp
knockin mice was previously shown to be clearly
detectable.30 The spleen contained nearly 20%
GFP+B220+ cells, most of which were probably
freshly recruited from the BM and could express low levels of RAGs in
normal conditions.36,37 To investigate further,
B220+ cells were separated into 3 populations based on GFP
signal. Expression of rag1 mRNA was detected in the
population with highest GFP expression by PCR analysis (Figure 1A,
right panel), though it was not detected in the cells with weak or no
GFP expression. The rag mRNA+ fraction with the
highest GFP expression contained 5% of the B220+ cells and
was presumably made up of freshly recruited cells. Similar
GFPhigh+ cells were much rarer in the Peyer patch, axillary
lymph nodes, and mesenteric lymph nodes. Recent reports used similar
approaches to look for RAG molecules using mice expressing RAG1/RAG2
transgenes in a bacterial artificial chromosome vector (RAG1/RAG2
transgenic mice)36,37 or expressing RAG2/GFP fusion
protein (RAG2/GFP knockin mice).38 Percentages of the
GFP+ cells were between 5% to 20%, similar to the results
of RAG1 transgenic (TG) mice, whereas RAG2/GFP knockin mice showed
fewer GFP+ cells. Percentages of GFP+ spleen
cells in heterozygous rag1/gfp knockin mice were comparable to previous results from RAG1 TG mice using similar gating (Figure 1B).
To determine the maturation stage of the GFP-expressing splenic B
cells, we looked at several markers that correlated with B-cell
maturation. The maturation of splenic B cells was accompanied by
increased expression of sIgM Localization of RAG1/GFP+ cells in peripheral lymphoid organs after immunization After a single injection of SRBC, GCs were clearly visible by PNA staining on days 5, 10, and 15 (Figure 2A); however, there was no up-regulation of GFP signal in the central region of GCs until day 15, in comparison to the other follicular regions. Other TD-Ags, DNP-KLH, and NP-CGG showed similar results (data not shown). The linear-shape accumulation of GFP signal was observed in the spleen sections shown in Figures 1C and 2A (on day 0). Because no similar GFP fluorescence was detected on the sections of control wild-type (WT) mice, we believe that the signal was generated by the RAG1/GFP knockin in B-lineage cells. These cells are presumably recruited from the BM under nonimmunized conditions.
The observation of no RAG1/GFP signal in the centrocyte region of the GCs was surprising because others had reported RAG re-expression after immunization.21-25 Loss of GFP expression in GCs could be due to differences of Ags or the immunization protocol. Therefore, we immunized mice with the same batch of NP-CGG and by the same immunization protocol described previously.24 Figure 2B shows the results in the draining lymph nodes after the administration of NP-CGG with CFA in footpads. On days 8 and 16 after immunization, no RAG1/GFP signal appeared in the central area of GCs, which are identified by H&E staining and positive staining with PNA and GL-7.18,43-45 These results demonstrated the down-regulation of RAG1 expression in most of Ag-driven B cells at the central area of GCs. Although RAG1/GFP signal was obviously down-regulated in the whole GC
area, we saw slightly positive GFP signals on the periphery of GCs that
were clearly demarcated by H&E, PNA, and GL-7 (Figure 2B). The
GFP+ cells seen at the margins of GCs might receive signals
for further maturation and activation in the process of Ag immunization
in vivo. GFP+ cells, though they are not present in the
centrocyte area, seem to be present in the region surrounding the GC
architecture. RAG1/GFP+ cells may appear at the extra-GC
area of lymphoid organs after Ag immunization in vivo. However, these
results did not demonstrate the re-expression of RAG1 in the centrocyte
area of GCs. The GFP signal was compared between
Fas+B220+ GC B-cell fractions before and after
immunization with TD-Ags (Figure 2C).46 There is no
up-regulation of GFP signal in the Fas+ GC B cells after Ag
(SRBC) immunization, whereas the Fas GFP signal in RAG1/GFP+ spleen cells adoptively transferred into RAG1-deficient mice We observed GFP+ cells in the follicular area under nonimmunized conditions, which made it difficult to determine whether the RAG1/GFP signal originated from B cells newly arriving from the BM or from Ag-driven B cells that re-express RAG1. To further investigate this question, we reconstituted rag1 knockout mice with spleen cells from heterozygous rag1/gfp mice (GFP-SPL mice). Because rag1 knockout mice generate neither B nor T cells in the periphery,47,48 any mature lymphocytes detected in reconstituted mice would originate from the RAG1/GFP cells. The spleens of these reconstituted GFP-SPL mice contained only a small number of GFP+ mature lymphoid cells 7 days after transplantation. We immunized the GFP-SPL mice with DNP-KLH and observed no up-regulation of RAG1/GFP signal in the GC-like region (Figure 3A). On FACS analysis, no obvious increase of GFP signal appeared in sIgM+ mature B cells after immunization (Figure 3B), whereas the immunization induced Ab production (Figure 3C). Only a limited number of B cells expressed faint GFP signals. Control spleens of unmanipulated heterozygous mice (RAG1/GFP) demonstrated GFP expression, presumably from newly arrived B cells from the BM (G3), as shown in Figure 1A. These results are in accord with the results of similar transfer experiments reported previously by Nussenzweig and colleagues.36
Absence of increased expression of RAG1/GFP signal in mature spleen B cells stimulated in vitro Previous reports demonstrated the re-expression of RAG1 and RAG2 in spleen B cells by various B-cell activators, including LPS plus IL-4 and anti-CD40 mAb plus IL-7 in vitro.22,24-26 To study whether B-cell antigen receptor (BCR) cross-linking or any of these other combinations induces RAG1/GFP signal, spleen cells obtained from GFP-SPL mice were stimulated by similar methods. BCR cross-linking with anti-µ Ab plus IL-4 did not obviously up-regulate the expression of RAG1/GFP+ cells, suggesting that RAG1 expression is hardly detectable after stimulation with Ags. LPS plus IL-4 or stimulation with anti-CD40 mAb plus IL-7 could not induce an apparent increase of RAG1/GFP+ cells (Figure 4). To demonstrate that these stimulatory reagents were effective on the purified B cells in vitro, proliferative responses to the same stimulatory reagents were examined in vitro. As the control for RAG1/GFP signal, pre-B cells maintained by the Whitelock and Witte35 method were used. Line 5 shows intermediate GFP signal, and line 7 shows a mixture of bright positive cells and cells with various levels of GFP signal during culture in the presence of IL-7. We also examined whether the same stimuli, reported previously by others,22,24-26 can induce the expression of endogenous rag1 mRNA in vitro. Stimulation with any combinations did not induce rag1 transcripts in spleen cells obtained from GFP-SPL mice (Figure 4). As the comparison, freshly obtained spleen cells from the WT mice were also stimulated in vitro. Fresh spleen cells do express the rag1 mRNA detected by the RT-PCR method, as described previously,36,38 but there is no further induction by the stimulation in vitro. The stimulation with anti-µ Ab + IL-4 or anti-µ Ab + IL-7 down-regulated the rag1 mRNA induction in culture (data not shown). Our results demonstrated that peripheral B-lineage cells seldom induce the expression of RAG1 by stimulation.
Many reports from several investigators have shown that BCR cross-linking or stimulation with LPS could induce receptor editing, as evidenced by the up-regulation of rag1/rag2 transcripts and immunoglobulin gene rearrangements in spleen B cells.22,24-26 These in vitro experiments could not determine the localization of B cells undergoing receptor editing. Although the expression of PNA should have been a good marker for GC B cells, it would have been difficult to decide whether the rag1/rag2 mRNA was induced in mature sIgM+ B cells by stimulation with BCR cross-linking or simply that the in vitro stimulation maintained the survival of rag1+ B lineage cells that have been freshly recruited from the BM. Mice stimulated with multiple injections of TD-Ags would still receive a supply of virgin B cells from the BM into regional lymph nodes through the hematogenous route. B cells with high-affinity receptors will participate in effective Ab production, but most of the B cells would re-enter the next round of maturation acquiring further hyper-mutation at V gene segments of immunoglobulin genes. Vigorous consumption of B cells in the periphery might accelerate the generation and recruitment of fresh B cells from the BM. The results demonstrated that BCR cross-linking does not induce the up-regulation of RAG1/GFP signal in the centrocyte area of GCs. Although the data cannot rule out the possibility that a very low number (below 0.1%) of total cells would participate in receptor editing in Ag-driven B cells in the spleen, the peripheral BCR+ B cells will seldom undergo secondary immunoglobulin gene rearrangements under normal conditions. Two independent analyses using RAG1/RAG2 TG mice and RAG2/GFP knockin mice studied the expression of RAGs in the secondary lymphoid tissues on immunization with TD-Ags.36,38 These studies did not unequivocally determine whether re-expression of RAG molecules occurs in Ag-stimulated B cells. The results of RAG1/RAG2 TG mice showed a nonresponsiveness of RAG induction after stimulation both in vivo and in vitro.36 In RAG2/GFP knockin mice, 2% to 20% of splenic B cells expressed GFP after immunization, whereas less than 1% of these cells expressed GFP in unimmunized mice.38 Most GFP+ cells in the spleen were phenotypically identical to pre-B and immature B cells.38 Our experiments studied the expression of RAG1/GFP in GCs of the Ag-immunized spleen. GFP expression was not detected in the central region of GCs, nor was it down-regulated. Regardless of whether GFP+B220+ cells were in the central GC area, the GFPLow+ cells surrounding the GC area (Figure 2) might have been reported previously as RAG+ cells.38 Similar RAG+ B cells were also observed in a human
tonsillar population.49 Human GC B cells are classified by
surface markers of the CD38+sIgD The evidence in our model mouse provides critical information regarding receptor editing in the secondary lymphoid follicles. Our results show an absence of RAG1 expression at the centrocyte stage. Centrocytes, generated from the Ag-driven centroblasts with arrested cell cycling, will be selected to enrich the B cells with high-affinity BCR through the follicular dendritic cells network. Because this selection process could be a critical filter to eliminate the self-reactive B-cell clones, it seems reasonable to suppress the re-expression of RAGs, which could cause receptor editing events at the centrocyte stage of the GCs. This may also be beneficial to maintain the allelic exclusion of immunoglobulin genes in the mature B cells of the peripheral lymphoid organs, as suggested by Nussenzweig and colleagues.36
We thank Dr Ohmori, Department of Biotechnology, Faculty of Engineering, Okayama University, for providing us NP-CGG and the immunization protocol routinely performed in his laboratory. We thank Drs Michelle Nussenzweig, and Garnett Kelsoe for the critical discussion and the kind help to prepare the manuscript. We thank Drs Marjorie Shapiro and Ejaz Shamim for critical reading of the manuscript. We thank Drs Seiji Inui and Kazuhiko Kuwahara in our laboratory for the help on the ELISA assay and Ms Y. Mukohmatsu (Department of Cell Differentiation) for cell sorting.
Submitted August 18, 2000; accepted December 1, 2000.
Supported by grants from The Ministry of Education, Culture, Sports, Science, and Technology. Supported in part by a grant from Novartis Foundation for the Promotion of Science.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nobuo Sakaguchi, Department of Immunology, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811, Japan; e-mail: nobusaka{at}kaiju.medic.kumamoto-u.ac.jp.
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© 2001 by The American Society of Hematology.
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  J. H. Wang, F. W. Alt, M. Gostissa, A. Datta, M. Murphy, M. B. Alimzhanov, K. M. Coakley, K. Rajewsky, J. P. Manis, and C. T. Yan Oncogenic transformation in the absence of Xrcc4 targets peripheral B cells that have undergone editing and switching J. Exp. Med., December 22, 2008; 205(13): 3079 - 3090. [Abstract] [Full Text] [PDF]
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Z. K. Mirnics, E. Caudell, Y. Gao, K. Kuwahara, N. Sakaguchi, T. Kurosaki, J. Burnside, K. Mirnics, and S. J. Corey Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center J. Immunol., April 1, 2004; 172(7): 4133 - 4141. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
as shown in G1, G2, G3
-like and VpreB, a pre-BCR component. It was mentioned that some
CD38