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IMMUNOBIOLOGY
From the Departments of Medicine, Pediatrics, and
Microbiology, Division of Developmental and Clinical Immunology,
University of Alabama at Birmingham and the Howard Hughes Medical
Institute, Birmingham, AL; Deutsches Rheuma-Forschungszentrum, Berlin,
Germany; and the Department of Immune Regulation, Tokyo Medical and
Dental University, Japan.
Surrogate light chain expression during B lineage differentiation
was examined by using indicator fluorochrome-filled liposomes in an
enhanced immunofluorescence assay. Pro-B cells bearing surrogate light
chain components were found in mice, but not in humans. A limited
subpopulation of relatively large pre-B cells in both species expressed
pre-B cell receptors. These cells had reduced expression of the
recombinase activating genes, RAG-1 and RAG-2. Their receptor-negative pre-B cell progeny were relatively small, expressed RAG-1 and RAG-2, and exhibited
selective down-regulation of VpreB and Since the discovery that B cells belong to a
discrete lymphoid lineage,1 much has been learned about
this pathway of cellular differentiation (reviewed in
2-5). B lymphopoiesis occurs in hemopoietic tissues,
primarily embryonic liver and bone marrow in mammals. In these sites,
lymphoid progenitors lacking immunoglobulin (Ig) expression (pro-B
cells) give rise to large B lymphocyte precursors (pre-B cells)
containing µ heavy chains (HCs).6-9 The replicating
pre-B cells later exit the cell cycle to generate small pre-B cells
that in turn become IgM-bearing B cells.10-14 The Ig V(D)J
gene rearrangement events underlying this progression follow an orderly
sequence in which D-JH rearrangement is followed by
V-DJH rearrangement in pro-B cells.15-17 Pre-B
cells generated through a productive VDJH rearrangement
undergo several rounds of cell division before exiting the cell cycle.
V-JL rearrangement in the kappa or lambda light chain (LC)
loci preferentially occurs in these small resting pre-B
cells.18-20 A productive VJL rearrangement allows the assembly of B-cell receptors (BCRs), composed of µHC, LC,
and the Ig Differential expression of a large cohort of genes is required for the
progression of B lineage differentiation,5,17,23 and the
nonrearranging VpreB and SLCs, the unique components of pre-BCR, are easily detected on pre-B
cell lines of mouse and human origin46-49 and on murine pro-B cell lines,48 but their cell surface expression by
primary B lineage cells has been remarkably difficult to
demonstrate.49-57 This difficulty has clouded the issue of
exactly when and where SLCs exert their function during B lineage
differentiation. Even in studies in which pre-BCR expression has been
demonstrated, only a few pre-B cells could be identified by cell
surface staining with anti-SLC antibodies.49,50,54,56,58
The most easily identifiable SLC-bearing cells in mice have in fact
been pro-B cells,53 whereas in human B lineage cells the
appearance of cell surface SLCs has not been detectable before the
onset of µHC expression in most studies.49,56,59 With
rare exceptions,59,60 human pro-B cell lines have also
been reported to lack cell surface SLCs.49,55,56,58
Species differences may thus exist regarding when cell surface SLC
expression begins. Given the very low levels at which SLC-containing
receptors are expressed on primary B lineage cells, technical
differences and variability in the antibody reagents used for
SLC detection could also account for some of the discordant results.
The paradox posed by our understanding of the importance of pre-BCR in
B lineage differentiation versus the uncertainty about when pro-BCR and
pre-BCR are expressed led us to use an amplified immunofluorescence
assay that can detect as few as 50 to 100 molecules per
cell61 and a panel of monoclonal anti-VpreB/ Cells
Antibodies
Immunofluorescence analysis and cell sorting The enhanced indirect immunofluorescence system61 used fluorochrome-filled liposomes conjugated to Fab fragments of sheep antidigoxigenin antibodies as a second-step reagent. Viable cells pre-incubated with 200 µg/mL IgG and 20 µg/mL anti-Fc IIR
antibody in phosphate-buffered saline containing 0.5% bovine serum
albumin for 10 minutes at 4°C were incubated with a
digoxigenin-conjugated anti-VpreB, anti- 5, or anti-pre-BCR
antibodies for 15 minutes before washing and incubation with
antidigoxigenin conjugated fluorescent liposomes for 1 hour on ice with
agitation, followed by washing and analysis by flow cytometry. Staining
specificity was assessed by pre-incubation with a 100- to 1000-fold
excess of the unlabeled primary antibody. B lineage cells enriched by magnetic cell sorting (Miltenyi Biotec) of bone marrow using anti-human CD19 or anti-mouse B220 antibodies were sorted using FACStar (Becton Dickinson) or MoFlow (Cytomation, Fort Collins, CO) instruments. Cells
fixed by incubation in 0.05% paraformaldehyde solution at 4°C for 1 hour were permeabilized by treatment with 0.2% Tween 20 or 0.1%
saponin in phosphate-buffered saline at room temperature for 20 minutes
and were blocked with serum for 10 minutes before antibodies were added
for intracellular staining.
Cell cycle analysis Cells purified by immunofluorescence cell sorting were fixed in 95% ice-cold ethanol for more than 30 minutes before treatment with RNase A (50 µg/mL) for 30 minutes at 37°C, followed by staining with FITC-conjugated anti-Ki-67 or control antibodies (BD PharMingen) for 30 minutes on ice. The cells were then washed before incubation, with 40 µg/mL propidium iodide for 15 minutes at room temperature, and flow immunocytometric analysis.Reverse transcription-polymerase chain reaction assays Subpopulations of pro-B and pre-B cells purified by 2 sequential fluorescence-activated cell sorting (FACS) sorts were lysed in TRIzol reagent (Gibco, Grand Island, NY) before preparation of total RNA, as recommended by the manufacturer (Gibco). First-strand cDNA synthesis was performed using the SuperScript preamplification system (Life Technologies) in parallel with a control synthesis reaction without reverse transcriptase (RT) to test for genomic DNA contamination. Protocols for polymerase chain reaction (PCR) of human gene products involved denaturing at 94°C for 3 minutes, amplification by 36 cycles of 94°C for 1 minute, 30 seconds for annealing at 55°C for RAG-1 and RAG-2, 60°C for TdT and B29, or 65°C for -actin and VDJ-Cµ, 72°C for 30 seconds,
and extension at 72°C for 5 minutes. Primers for PCR
amplification were as follows: TdT,
5'-ACACGAATGCAGAAAGCAGGA-3', 5'-AGGCAACCTGAGCTTTTCAAA-3'; RAG-1, 5'-ATGACAGCAGATGACCTCCTA-3',
5'-TACCTCCAGAAGTTTATGAAT-3'; RAG-2,
5'-TTCTTGGCATACCAGGAGACA-3', 5'-CTATTTGCTTCTGCACTGAAA-3'; 5/14.1, 5'-ACTGTCGGATCCTCGCAGAGCAGG-3',
5'-CAGTCAAGCTTCTATGAACATTCT-3'; VpreB,
5'-GTAGAGGCATGCCAGCCGGTGCTG-3', 5'-CTTGAAGCTTTCGAGGGACACGTGT-3'; B29, 5'-GAATCTCTCGCCACCCTCACC-3',
5'-CCTTGCTGTCATCCTTGTCCA-3'; VDJ-Cµ,
5'-GGGTCGACACGGCCGTGTATTACTGT-3', 5'-TGGTGGCAGCAAGTAGACATC-3'; and
-actin, 5'-GCGGGAAATCGTGCGTGACAT-3',
5'-GTGGACTTGGGAGAGGACTGG-3'. Primers used for V-J PCR
amplification have been previously described.63 PCR
protocols for mouse gene products involved 36 cycles of amplification
with annealing at 58°C for -actin, VDJ-Cµ, B29,
and 5; 60°C for RAG-2; and 65°C for
TdT, RAG-1, and VpreB for 1 minute followed by extension at 72°C for 1 minute and
94°C denaturation for 1 minute. Primers for mouse PCR
amplification were as follows: TdT,
5'-GAAGATGGGAACAACTCGAAGAG-3', 5'-CAGGTGCTGGAACATTCTGGGAG-3'; RAG-1, 5'-TGAAAAGGCACCCGAAGAAGC-3',
5'-GGTGCCACTCCACGGTCACTT-3'; RAG-2,
5'-CACATCCACAAGCAGGAAGTACAC-3', 5'-GGTTCAGGGACATCTCCTACTAAG-3'; 5, 5'-GTTGGGTCTAGTGGATGGTGT-3',
5'-TTGGTCTGTTTGGAGGGTTGG-3'; VpreB,
5'-GCCACCATCCGCCTCTCCTGT-3', 5'-CCCCACGGCACAGTAATACAG-3'; B29,
5'-TCAGAAGAGGGACGCATTGTG-3', 5'-TTCAAGCCCTCATAGGTGTGA-3'; VDJ-Cµ, 5'-CGCGCGGCCGCTGCAGCAGCCTGGGGC TGAG-3',
5'-GGAATGGGCACATGCAGATCTC-3'; Vk-Ck,
5'-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3',
5'-CTCATTCCTGTTGAAGCTCTTGACAATGGG-3'; and -actin,
5'-CGCAGCTCAGTAACAGTC-3', 5'-TACGAGGGCTATGCTCTC-3'. Cellular cDNA
was serially diluted for template use in semiquantitative RT-PCR assays
of VpreB and 5 transcripts.
Surrogate light chain expression on B lineage cell lines of human and mouse origin Pro-B, pre-B, and B cell lines were examined with conventional and enhanced indirect immunofluorescence assays in a preliminary survey of SLC expression during human and mouse B lineage differentiation. In the enhanced immunofluorescence assay, cells were incubated first with anti-SLC antibodies conjugated with digoxigenin and then with fluorochrome-loaded liposomes bearing antidigoxigenin antibodies. This method yielded approximately a 2-log enhancement of the mean fluorescence intensity for anti-VpreB and anti-5 staining of human
and mouse pre-B cells over that observed by conventional indirect
immunofluorescence (Figure 1).
Specificity was verified by the inhibition of staining with
unconjugated anti-VpreB or anti-5 antibodies. SLC components were
also detected on mouse pro-B cell lines, with similar enhancement seen
with the fluorochrome-loaded liposomes. In contrast, SLC components
could not be detected on human pro-B cell lines (Figure 1) despite
their production of intracellular VpreB and 5 SLC
proteins.56,58 This species distinction held for all mouse
(SCID7, 38B9, 40E1, 63-12, D1F9, Raw8.1) and human (Nalm16, RS4;11,
JEA2, and REH) pro-B cell lines included in the analysis. Cell surface
expression of SLC on mouse pro-B cell lines versus its absence on human
pro-B cell lines was confirmed using the entire panel of monoclonal
antibodies against VpreB (HSL96, 4G7, VP245) and 5/14.1 epitopes
(HSL11, LM34). When mouse and human pre-B cell lines were examined for cell surface reactivity with anti-µHC antibodies, they were
universally found to express this pre-BCR component, whereas none of
the pro-B cell lines produced µHC (data not shown).
Analysis of surrogate light chain expression by primary B lineage cells Normal B lineage cells identifiable by their expression of the human CD19 or mouse B220 markers were examined for SLC expression using conventional and enhanced immunofluorescence staining methods. Although the usual difficulty was encountered in detecting SLC expression on bone marrow cells by conventional immunofluorescence, SLC-bearing B lineage cells were easily detected by the enhanced immunofluorescence method in human and mouse bone marrow samples (Figure 2). Specificity of this immunofluorescence staining was validated by the results of blocking experiments with unlabeled antibody. The remarkable contrast between the staining results achieved with both methods was emphasized by the fact that we were unable to unambiguously detect SLC expression on mouse bone marrow cells by conventional indirect immunofluorescence (Figure 2B, lower panels). The frequency of SLC+ B lineage cells among bone marrow mononuclear cells identified by the enhanced immunofluorescence method was relatively low (range, 0.6%-4.8%).
Enhanced immunofluorescence was used for the detection of the VpreB and
In bone marrow samples from 6 juvenile mice, the SLC+
subset comprised 6.3 ± 1.5% of the B220+ B lineage
cells. As in humans, all SLC+ cells in mice were
CD19+, and none expressed Mouse, but not human, pro-B cells express cell surface surrogate light chain To test the implied species variability in SLC expression, SLC-bearing cells in human bone marrow were purified by cell sorting on the basis of cell surface CD19 and VpreB expression and the absence of or LC. When permeabilized before
immunofluorescence analysis, the
CD19+VpreB+LC cells were found to
contain µHC (Figure 4A, top
panel), thereby indicating that all SLC-bearing cells in
human bone marrow samples are pre-B cells, whereas pro-B cells lack
cell surface SLC.
Most of the B220+VpreB+ LC Pre-BCR expression is limited to a subpopulation of pre-B cells Previous studies using conventional indirect immunofluorescence have identified pre-BCR components on few, if any, of the µHC+ pre-B cells.49-51,53,55-57 It seemed possible that this reflected the relative insensitivity of the methods used for detecting pre-BCR expression. To our surprise, even with the fluorochrome-filled liposomes, we were unable to detect SLC components on most pre-B cells in human and murine bone marrow samples. When the cell surface SLC subpopulation of
CD19+CD34 /LC cells was
isolated from human bone marrow samples and examined for intracellular
µHC, they were found to contain µHC as a clear indication of their
pre-B cell status (Figure 4A, lower panel). Correspondingly, when the
subpopulation of mouse B220+LC bone marrow
cells lacking cell surface VpreB was isolated, µHC expression was
found in most, but not all, of these cells (Figure 4B, lower panel).
This subpopulation of
B220+VpreBLCµHC
cells represents pro-B and possibly non-B lineage cells that lack cell
surface SLC. These composite results indicate that most of the pre-B
cells in mice and humans (60%-80%) lack pre-BCR.
Characterization of the pre-BCR+ and
pre-BCR /LC and
VpreBCD34CD19+/LC,
were purified by 2 rounds of cell sorting. Analysis of these subpopulations indicated that most of the pre-BCR+
subpopulation (ie, VpreB+) were relatively large cells,
whereas the pre-BCR subpopulation of pre-B cells was
composed primarily of relatively small cells (Figure
5). In accordance with the cell size
difference, the analysis of Ki-67 expression and DNA content indicated
that a greater proportion of the pre-BCR+ pre-B cells were
in the G1/S/G2/M stages of the cell cycle. The same trends were observed for the pre-BCR+ and
pre-BCR subpopulations of mouse pre-B cells, though the
strategy for identifying these subpopulations in mouse bone marrow
samples was necessarily different. The mouse pre-B cell population of B220+ cells was identified by expression of the BP-1
antigen in the absence of /LC expression. This strategy was used
because all BP-1+/LC cells were shown to
express intracellular µHC.64 Thirty percent of the
latter subpopulation expressed cell surface IgM after overnight culture, thereby confirming that these cells are immediate B cell precursors. Pre-BCR expression is therefore restricted to a
subpopulation of relatively large cycling pre-B cells, whereas
receptor-negative pre-B cells are predominantly small, resting
pre-B cells.
Gene profile analysis of the pro-B and pre-B cell subpopulations Pro-B cells in human bone marrow were identified in this analysis as CD19+CD34+SLC cells, and the 2 pre-B cell subpopulations were isolated as in the previous experiments.
The mouse pro-B cell subpopulations, SLC+ and
SLC, were isolated from RAG-2/
mice to ensure the absence of more mature B lineage cells. Mouse pre-B
cells were isolated from wild-type bone marrow as
CD19+BP-1+/LC cells, and
the VpreB+ and Vpre-B subpopulations were
then separated. Two rounds of cell sorting were conducted to ensure
purity (more than 99.5%) of the subpopulations.
Several notable changes in gene expression were evident in this
analysis (Figure 6). As anticipated,
full-length µHC transcripts were either absent or were present only
in trace levels in pro-B cells, and Tdt expression was
detected exclusively in the pro-B subpopulations. RAG-1 and
RAG-2 transcripts were down-regulated in the
pre-BCR+ subpopulation, though RAG-2 transcripts
could still be demonstrated, whereas both RAG-1 and
RAG-2 expression were clearly evident in the
VpreB
Since discovery of the SLC components, VpreB and SLC components could not be detected on human pro-B cells with
monoclonal antibodies recognizing the VpreB and SLC expression for mouse pro-B cells was reaffirmed in these studies,
though a physiological role of this type of pro-BCR remains enigmatic.
Cell surface expression of SLC is restricted to a subpopulation of the
murine pro-B cells, and the SLC+ phenotype is faithfully
reproduced by pro-B cell lines in which the SLC may be associated with
surrogate HC proteins.48,51 One of the surrogate HC
proteins has been identified as BILL-cadherin,66 a
transmembrane glycoprotein that apparently lacks signal transducing capability. Ig The pattern of pre-BCR expression conserved in mice and humans was
characterized by the restriction of receptor expression to a limited
subpopulation of the pre-B cells. Although the entire pre-B population
exhibited the hallmark features of intracellular µHC presence and LC
absence, the pre-BCR+ and pre-BCR Bone marrow DNA labeling studies initially revealed that large dividing
pre-B cells give rise to small postmitotic pre-B cells that become cell
surface IgM+ B cells after a 1- to 2-day
interval.10,13,14 V-JL rearrangements in the
This analysis of SLC expression supports a B-cell differentiation
scheme that differs for mice and humans primarily in the earlier onset
of cell surface SLC expression in mice (Figure
7). The extinction of pre-BCR expression
by selective down-regulation of SLC production appears to have a
profound impact on the pre-B cell differentiation process. The most
well-documented function of pre-BCR is the promotion of pre-B cell
growth and survival in conjunction with costimulatory signals provided
by stromal cells or interleukin-7.73 Although evidence for
pre-BCR promotion of HC allelic exclusion and LC gene rearrangement has
also been suggested,74-76 these events can proceed in the
absence of pre-BCR.38,68 The implication that pre-BCR
expression is not directly involved in these differentiation events is
reinforced by our observation that the loss of pre-BCR expression and
withdrawal from the cell cycle coincide with the reinstatement of
RAG-1 and RAG-2 expression and VJL
rearrangement. The selective SLC down-regulation to extinguish pre-BCR
expression is thus an important prerequisite to B-cell differentiation.
This sequence of events also ensures that SLC and conventional
The model of primary B-cell differentiation supported by this analysis
of SLC expression begs the question of how the VpreB and
The current observations are also relevant to the RAG and SLC
expression observed for B lineage cells in secondary lymphoid tissues.56,81-86 Initially considered to reflect a
reactivation of the pre-B cell gene program in germinal center B cells,
further analysis of this phenomenon instead suggests an efflux of bone marrow pre-B cells to the periphery in response to inflammatory stimuli.86 However, a subpopulation of peripheral B cells
with a restricted V gene repertoire has been reported to express SLC and
We thank Dr Larry Gartland for help with flow cytometry; Drs Peter Burrows, Flavius Martin, and John Kearney for helpful discussions; and Marsha Flurry and Ann Brookshire for help in preparing the manuscript. We thank Dr Alan Fantel (University of Washington, Seattle) for providing fetal bone marrow tissue samples.
Submitted August 13, 2001; accepted November 8, 2001.
Supported by National Institutes of Health grant AI39816 (M.D.C.). M.D.C. is a Howard Hughes Medical Institute Investigator.
Y.-H.W. and R.P.S. contributed equally to this manuscript.
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: Max D. Cooper, Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, WTI 378, 1824 6th Ave S, Birmingham, AL 35294-3300; e-mail: max.cooper{at}ccc.uab.edu.
1. Cooper MD, Peterson RDA, Good RA. Delineation of the thymic and bursal lymphoid system in the chicken. Nature. 1965;205:143-146[CrossRef][Medline] [Order article via Infotrieve]. 2. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983;302:575-581[CrossRef][Medline] [Order article via Infotrieve]. 3. Rajewsky K. Clonal selection and learning in the antibody system. Nature. 1996;381:751-758[CrossRef][Medline] |
Top
Abstract
Introduction
/
, µ heavy chains;
, Ig
, VpreB/
, BILL-cadherin;
, calnexin; Tdt, terminal
deoxyribonucleotidyl transferase. Pro-B cells depicted in this scheme
express cell surface CD19.