Increased Frequency of Pre-germinal Center B Cells and
Plasma Cell Precursors in the Blood of Children with Systemic
Edsel Arce,*†Deborah G. Jackson,*†Michelle A. Gill,*†Lynda B. Bennett,*†
Jacques Banchereau,* and Virginia Pascual2*†
We have analyzed the blood B cell subpopulations of children with systemic lupus erythematosus (SLE) and healthy controls.
We found that the normal recirculating mature B cell pool is composed of four subsets: conventional naive and memory B
cells, a novel B cell subset with pregerminal center phenotype (IgD?CD38?centerin?), and a plasma cell precursor subset
(CD20?CD19?/lowCD27?/??CD38??). In SLE patients, naive and memory B cells (CD20?CD38?) are ?90% reduced,
whereas oligoclonal plasma cell precursors are 3-fold expanded, independently of disease activity and modality of therapy. Pre-
germinal center cells in SLE are decreased to a lesser extent than conventional B cells, and therefore represent the predominant
blood B cell subset in a number of patients. Thus, SLE is associated with major blood B cell subset alterations. The Journal of
Immunology, 2001, 167: 2361–2369.
laboratory abnormality in this disease (3). Despite the low circu-
lating lymphocyte levels, B cells play a major role in the patho-
genesis of SLE in both humans and murine SLE models, as they
are responsible for the hypergammaglobulinemia and autoantibody
production that characterize this disease (4, 5). Most studies on
lupus B cells have been performed on mice with lupus-like syn-
dromes (6–9) rather than human SLE (10–14). Interestingly,
MRL/lpr mice expressing surface Ig but lacking secreted Ig de-
velop nephritis, suggesting that B cells may play a role in the
pathogenesis of SLE nephritis that is independent from serum au-
toantibodies (15). With regard to humans, SLE B cells exhibit,
upon signaling through the Ag receptor, increased Ca2?flux and
early protein tyrosine phosphorylation (12). SLE B cells express
high levels of costimulatory molecules CD80 and CD86 (13) as
well as CD40 ligand (CD40L)/CD154 (14). High levels of soluble
CD40L are also found in the serum of active SLE patients (16, 17).
In recent years our laboratory has developed methods to isolate
and characterize mature peripheral B cells. Using anti-IgD and
ymphocyte counts are known to be significantly de-
creased in systemic lupus erythematosus (SLE)3and lym-
phopenia of ?1500 cells/?l is the most prevalent initial
anti-CD38 Abs, four mutually exclusive peripheral B cell popula-
tions can be isolated (reviewed in Refs. 18 and 19). Single-positive
IgD cells correspond to follicular mantle cells (Bm1 ? Bm2),
whereas single-positive CD38 cells correspond to germinal center
(GC) cells (Bm3 ? Bm4). Double-negative B cells correspond to
the memory population (Bm5), whereas double-positive cells rep-
resent a combination of cells at a transitional stage between fol-
licular mantle and GC (Bm2?) and single-isotype IgD?GC cells
(20). More recently, CD27 has been reported as marker of memory
B cells within both the sIgD?and sIgD?peripheral B cell com-
partments (21, 22). The phenotypic summary of these populations
is depicted in Table I.
These studies and those by others (23–30) have led to the pro-
posal of a model of T cell-dependent, Ag-dependent mature B cell
differentiation: naive B cells (Bm1 and Bm2) are activated in as-
sociation with Ag-specific T cells and interdigitating cells within
the extrafollicular areas. The activated B cell blasts either undergo
terminal differentiation toward plasma cells (extrafollicular reac-
tion) or become GC founder cells (Bm2?). In GCs, Bm2? differ-
entiate into centroblasts (Bm3) that proliferate and accumulate
point mutations into the Ig variable region genes, yielding three
types of mutants: high affinity, low affinity, and autoreactive mu-
tants. These mutants will be selected while they differentiate into
centrocytes (Bm4), their survival depending on their affinity for the
Ag trapped within immune complexes bound to follicular dendritic
cells. The high affinity mutants will pick up the Ag, process it, and
present it to GC T cells, which are induced to express CD40L and
secrete cytokines (i.e., IL-4 and IL-10), key elements for survival,
proliferation, and isotype switching. These high affinity centro-
cytes differentiate into either memory B cells (Bm5) or plasma
cells. Low affinity mutants that do not bind FDC-bound Ag will die
by apoptosis, whereas autoreactive mutants are eventually deleted
because they do not receive T cell help. During secondary humoral
immune responses, recirculating memory B cells can be activated
in extrafollicular areas, giving rise to plasma cells and GC founder
Although extensive information has accumulated on the mature
B cells that populate peripheral lymphoid organs such as human
tonsils, little is known about blood B cell subsets. We have thus
*Baylor Institute for Immunology Research, Dallas, TX 75204; and†Department of
Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390
Received for publication February 15, 2001. Accepted for publication June 5, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants 1-R29-AI42862-01
and R01-AR46589-01, NIAID-DAIT-99-12, and the Lupus Foundation of America
(to V.P.) and the Robert Wood Johnson Foundation Minority Medical Faculty De-
velopment Program (to E.A.). Parts of this work have been previously presented in
abstract format at the 1999 and 2000 Annual Meetings of the American College of
Rheumatology (12–17 November 1999, Boston, MA and 28 October–2 November
2000, Philadelphia, PA).
2Address correspondence and reprint requests to Dr. Virginia Pascual, Baylor
Institute for Immunology Research, Dallas, TX 75204. E-mail address:
3Abbreviations used in this paper: SLE, systemic lupus erythematosus; CD40L,
CD40 ligand; GC, germinal center; JDM, juvenile dermatomyositis; SLEDAI, SLE
disease activity index.
Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00
analyzed the peripheral blood B cell compartment of healthy
adults, healthy children, and children suffering from rheumatic dis-
eases including juvenile dermatomyositis (JDM) and, most partic-
ularly, SLE. These studies have permitted us to identify a novel
blood B cell population expressing a partial GC phenotype and an
oligoclonal plasmablast population. Although these populations
are not restricted to SLE patients, the disproportionate depletion of
conventional naive and memory B cells in SLE make pre-GC cells
and plasmablasts predominate in SLE blood.
Materials and Methods
Samples and patient populations
Blood samples from 35 healthy children, 68 children with SLE, 10 with
JDM, and 17 healthy adults were drawn after informed consent in accor-
dance with our institutional internal review board was obtained. All pedi-
atric SLE patients included in this study fulfil the established American
College of Rheumatology criteria for SLE (31). The patients’ clinical and
serological data were gathered during clinic visits, and the corresponding
SLE disease activity index (SLEDAI) was recorded in the chart (32). The
average ? SD age and the sex ratio for each of the groups were: 1) healthy
children group, 12.15 ? 3.15 years, 3:1 female/male; 2) pediatric SLE
group with SLEDAI ?10 (n ? 36), 14 ? 2.67 years, 5:1 female/male; 3)
pediatric SLE group with SLEDAI ?10 (n ? 32), 13 ? 3.15 years, 6:1
female/male; 4) JDM group, 9.2 ? 3.8 years, 4:1 female/male; and 5) adult
group, 36.8 ? 6.21 years, 3:2 female/male. SLE patients belong to different
ethnic backgrounds, including Caucasian (32.3%), African-American
(25.3%), Hispanic (23.9%), and Oriental (4.2%). The healthy children con-
trol group had a similar ethnic distribution. Therapy guidelines for child-
hood SLE are similar to those for adult SLE patients. Most of the included
patients were being treated with oral prednisone and hydroxychloroquine,
and those with type III/IV nephritis and/or major extrarenal organ involve-
ment were receiving i.v. cyclophosphamide (?20% of patients) and/or
methylprednisolone (?40% of patients). Blood samples were drawn at
least 4 wk after the last i.v. pulse of either of these medications had been
administered. Selected patients with JDM had active disease and were
treated with oral prednisone and/or i.v. methylprednisolone at doses com-
parable to those given the SLE patients (10/10).
Flow cytometric analysis of blood B cells
Two methods have been used to assess blood B cells. The first analyzes
purified B cells, whereas the second analyzes total blood and has the con-
siderable advantage of necessitating only 0.5 ml (rather than 10–20 ml) of
blood. Samples from 44 SLE patients, 22 healthy children, 10 JDM, and 17
healthy adults were analyzed using enriched B cells, whereas samples from
24 SLE patients and 13 healthy children were assessed using whole blood.
The validity of the whole blood method has been established on three
patients and yielded comparable results, therefore permitting us to pool the
results of a 30-mo-long study. Absolute numbers of cells were calculated
from the relative size of total B cells and B cell subpopulations and the
absolute leukocyte and/or PBMC counts.
Isolation of peripheral blood B cells
Mononuclear cells were isolated using gradient centrifugation over a Hys-
topaque cushion. The resulting population was enriched for B cells using
negative depletion with magnetic beads coupled to anti-CD2, CD3, CD4,
CD14, CD16, CD56, and glycophorin A (stem cell). The enriched B cells
were stained with fluorochrome-labeled Abs (FITC, PE, Tricolor, PerCP,
and allophycocyanin). The following were used: anti-human CD3-FITC,
CD7-FITC, CD14-PE, CD19-allophycocyanin, CD20-PerCP (BD Bio-
sciences, Mountain View, CA); CD10-FITC, CD40-PE, CD71-FITC,
CD79a-FITC (Immunotech Research, Quebec, Canada); CD23-PE, CD56-
FITC (Caltag, South San Francisco, CA); CD38-PE, CD5-PE, CD138-
FITC, ? and ? light chain-PE (Serotec, Oxford, U.K.); CD154-FITC
(Ancell, Bayport, MN); and anti-human IgD-FITC, IgM-PE, IgG-PE, IgE-
FITC, IgA-FITC (Southern Biotechnology Associates, Birmingham, AL).
Stained cells were analyzed using flow cytometry (FACSCalibur, BD Bio-
sciences). All experiments were analyzed after gating on live cells accord-
ing to forward side scatter/side light scatter. A minimum of 100,000 cells
was used for each staining condition, and 5,000–50,000 events were re-
corded for analysis. Selected populations of cells were sorted for immu-
nohistochemistry or molecular studies using the FACSVantage (BD Bio-
Labeling of cell surface Ags from whole blood samples
Whole blood was collected into tubes containing heparin or ACD and
stained with the following Abs: IgD-FITC,CD38-PE, CD20-PerCP, and
CD19-allophycocyanin and corresponding isotype controls. We used 50 ?l
blood and 3 ?l of each Ab per tube for each staining. After staining, the
blood was lysed with FACS Lysing Solution (BD Biosciences), rinsed with
PBS, centrifuged at 1200 rpm for 10 min, and resuspended in 1% para-
formaldehyde. Samples were then analyzed on a BD Biosciences flow cy-
Amplification of the centerin gene
Real-time PCR was performed using an ABI Prism 7700 sequence detector
(PE Biosystems, Foster City, CA). The RT-PCR conditions were 30 min at
48°C and 10 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min
at 60°C. The Taqman PCR core kit reagents (PE Biosystems), Multiscribe
reverse transcriptase (PE Biosystems), and RNase inhibitor (PE Biosys-
tems) were used according to the manufacturer’s suggested concentrations
for a multiplex reaction. The 18S ribosomal RNA and Centerin standard
curves were generated using a serial dilution of a known quantity of Raji
total RNA. Ribosomal RNA analysis was performed using the ribosomal
RNA control reagent kit (PE Biosystems). The centerin probe (6-FAM-
tcaccagaaccatggccgtcagaag-TAMRA) was used at a concentration of 250
nM, and the forward and reverse centerin primers (forward aagggaaggtt
gtagacataatcca; reverse gcttctcccacttggctttaaa) were used at a concentration
of 900 nM.
Sequencing of Ig VHgenes
Total RNA from between 1,000 and 100,000 sorted B cells was prepared
using the mini-RNEASY kit, (Qiagen, Valencia, CA) following the man-
ufacturer’s protocol. RT-PCR was performed on 10% of the total RNA
generated from the sorted cells using the Titan RT-PCR kit (Roche, Indi-
anapolis, IN). The VHregion of IgM transcripts was amplified using either
a VH4 or a VH5 leader primer in combination with a ?-constant region
reverse primer, as previously described (33, 34). The VHregion of IgG was
amplified using identical forward primers with a ?-specific constant region
reverse primer. The VHfragments were excised from a low melt agarose
gel and reamplified using heminested reverse primers and the high fidelity
PFU polymerase (Stratagene, La Jolla, CA). The PCR fragments were ei-
ther t-tailed with Taq polymerase (Promega, Madison, WI) and subse-
quently cloned into the pCRII-TOPO vector or directly cloned into the
pCR-blunt-II-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced in
both directions using an automated DNA sequencer (ABI-377; Advanced
Biotechnologies, Columbia, MD).
Table I. Surface marker expression of mature human B cell subpopulations
CD19 CD20sIgM sIgD CD23CD38CD10CD71 CD77CD27CD138
Naive (Bm1 ? Bm2)
GC Founder (Bm2?)
GC (Bm3 ? Bm4)
2362BLOOD B CELL SUBPOPULATIONS IN PEDIATRIC SLE
Analyses of DNA sequences
Sequences were edited and analyzed using the DNAstar software package
(DNAstar, Madison, WI). Cloned products were searched against the
IMGT (the international ImMunoGeneTics database, http://imgt.cines.fr:
The data obtained in this study were evaluated using a two-tailed t test and
multivariable statistical analysis, as well as the Pearson correlation ratio.
T and B cells are profoundly decreased in SLE blood
Although lymphopenia has been described in SLE (3), the extent
of T and B cell decrease remains uncharacterized. Therefore, we
measured the absolute numbers of CD3?, CD14?, and CD20?/
19?cells in the blood of 1) 68 children suffering from SLE, 2) 35
age-matched healthy controls, 3) 10 children with JDM to control
for the effect of steroid treatment, and 4) 17 healthy adults. SLE
patients were divided into two groups according to their disease
activity index (SLEDAI over or under 10) measured at the time of
blood sampling. The ages (mean and SD) of the SLE patients and
healthy controls were comparable (see Materials and Methods). As
previously reported (36, 37), when compared with adults healthy
children display significantly more blood CD3?T cells (1687 ?
1139 vs 881 ? 202 cells/?l; p ? 0.002) and CD19?B cells
(394 ? 196 vs 129 ? 67 cells/?l; p ? 0.0001; Fig. 1). Children
with JDM, treated with steroid regimens similar to those of SLE
patients, display numbers of CD19?cells comparable to those in
healthy controls (Table II and Fig. 2). The slight difference (not
statistically significant) may reflect the lower average age of the
JDM group (9.2 ? 3.8 vs 12.1 ? 3.5 years in JDM and healthy
Children with SLE showed significantly fewer circulating T
cells than healthy children (450 ? 300 vs 1700 ? 380 cells/?l;
p ? 0.0001). Although patients with the highest disease activity
(SLEDAI, ?10) had lower numbers of T cells than patients with
lower disease activity (SLEDAI, ?10; 310 ? 167 vs 510 ? 467
cells/?l), this difference was not statistically significant. SLE pa-
tients had fewer circulating monocytes than healthy children
(144 ? 149 vs 313 ? 326 cells/?l), but this difference did not
reach statistical significance (p ? 0.06; Fig. 1).
Blood CD19?B cells in SLE patients were reduced by 81%
compared with those in age-matched healthy controls (82.6 ? 77.5
vs 394 ? 196 cells/?l; p ? 0.0001). There was no difference in the
number of circulating B cells between the two patient groups (Ta-
ble II), suggesting that B cell lymphopenia in SLE is independent
of disease activity. Although most of our patients had been treated
for weeks to years with steroids at the time of study, the T and B
cell lymphopenia is not a consequence of this therapy, as newly
diagnosed patients (3 of 68) were also found to have similarly
decreased numbers of T and B cells before they had entered into
Mean and SD of blood B cell, T cell, and monocyte numbers from healthy adults, healthy children, and children with SLE.
Table II. Mean and SD numbers of cells per microliter in each of the studied populations of healthy donors and patientsa
Mean ? SD
Mean ? SD
Mean ? SD
Mean ? SD
Healthy adults (17)
Healthy children (35)
Total SLE (68)
SLEDAI ?10 (36)
SLEDAI ?10 (32)
129.6 ? 67.5b
394.0 ? 196.1c
470.7 ? 298.4c
82.6 ? 77.5
75.2 ? 81.1
90.5 ? 74.0
97.7 ? 49.7c
270.9 ? 157.9c
428.4 ? 294.6c
28.0 ? 40.3
28.6 ? 40.2
27.1 ? 41.6
18.1 ? 18.7d
57.8 ? 59.3c
37.4 ? 31.2d
19.9 ? 24.5
21.4 ? 27.7
18.2 ? 20.6
1.4 ? 1.7c
6.3 ? 9.2e
4.2 ? 5.5c
18.7 ? 22.2
19.5 ? 12.8
18.0 ? 19.9
aSuperscript letters indicate the statistical significance between control and SLE groups.
bp ? 0.006.
cp ? 0.001.
ep ? 0.001.
2363The Journal of Immunology
any therapy (64.2 ? 72.1 B cells/?l; n ? 3). Additionally, nine of
the patients treated with i.v. solumedrol and cyclophosphamide
who were included in this study have been followed after discon-
tinuation of these drugs for periods between 6 mo and 2 years
without finding statistically significant differences in the number of
B cells (data not shown).
Circulating naive and memory B cells are considerably reduced
Our earlier studies on tonsillar B cells showed that CD38
expression permits us to distinguish plasmablasts/plasma cells
and GC B cells from naive and memory B cells (reviewed in
Refs. 18 and 19). Thus, CD19?CD20?CD38?blood cells
include both naive and memory B cells. As shown in Table II,
healthy children displayed significantly more conventional
mature (CD19?CD20?CD38?) B cells than adults (270 ? 157
vs 97 ? 49 cells/?l; p ? 0.0001). In contrast, SLE patients showed
a marked reduction (?90%) in these cells compared with age-
matched controls (28.0 ? 40.3 cells/?l; p ? 0.0001). This reduc-
tion does not appear to be related to disease activity (27.1 ? 41.6
cells/?l for SLEDAI ?10; 28.6 ? 40.2 cells/?l for SLEDAI ?10;
The blood memory B cell population is best identified as
CD20?CD27?cells. We calculated the ratio of memory/naive B
cells in healthy children and children with SLE and found no dif-
ference between the two groups (0.46 ? 0.30 and 0.49 ? 0.35 in
healthy and SLE children, respectively).
B cells with pre-GC phenotype recirculate in blood of healthy
and SLE children
Our initial studies on SLE total blood and enriched blood B
cells revealed a strikingly high percentage of circulating
CD20?IgD?CD38?cells. A closer analysis of samples from non-
SLE patients revealed that cells with similar phenotype were also
present in the blood of healthy children, adults, and children with
autoimmune diseases other than SLE, prompting us to report their
characterization (Fig. 3). In absolute numbers healthy children
have the highest numbers of IgD?CD38?cells (57.6 ? 53.3 cells/
?l), followed by patients with JDM (37.4 ? 31.2 cells/?l). The
number of IgD?CD38?cells in SLE patients (21.4 ? 27.7 cells/?l
SLEDAI ?10, 18.2 ? 20.6 cells/?l SLEDAI ?10) is comparable
to that in adults (18.1 ? 18.7 cells/?l; Table II). Due to the more
drastic reduction in conventional CD20?CD38?cells in SLE pa-
tients, this population overall represents 29 ? 17.7% of SLE blood
B cells (range, 6–77%), whereas it represents 13.2 ? 8 and 18.5 ?
14.9% of the total blood B cells in healthy adults and children,
respectively (Fig. 4).
In both patients and controls these cells express high CD20, a
characteristic of GC B cells (data not shown). When sorted and
analyzed with Giemsa staining, IgD?CD38?cells appear very
Blood B cell and plasma cell precursor (CD19?) numbers in SLE patients and controls. F, Median values.
adult (a), healthy child (b), and a child with SLE (c)
stained with anti-CD38-PE and anti-IgD-FITC Abs.
Double-positive cells display a pre-GC phenotype.
Enriched blood B cells from a healthy
2364 BLOOD B CELL SUBPOPULATIONS IN PEDIATRIC SLE
similar to the tonsilar Bm2 (IgD?CD38?CD23?) cell subset: they
are larger than naive B cells and display a full cytoplasmic rim
(Fig. 5, a and b). Using real-time PCR, these cells were found to
transcribe centerin (Fig. 6), a GC-specific serpin not expressed in
conventional naive and memory blood/tonsil B cells (36). Yet, the
blood IgD?CD38?cells seem less committed toward GC differ-
entiation than the GC founder cells (Bm2?) that were previously
identified within tonsils (37), as they mostly lack expression of
CD10 and CD77, and only about one-fifth of these cells (21.5 ?
16.7% of 17 samples analyzed) express CD71.
One of the characteristics of tonsilar IgD?CD38?cells is the ini-
tiation of somatic mutation within Ig VHgenes (38). Therefore, blood
IgD?CD38?cells were sorted from eight different SLE patients, and
their VHIg RNA was amplified using primers specific to the small
VH4 and VH5 family leader peptide and ? constant region. Fifty-six
independent clones were sequenced and aligned to their closest germ-
line counterparts, revealing the presence of low grade somatic muta-
tion within 66% of the transcripts (1–7 bp substitutions/mutated VH
region; Table III). The same population in healthy adults showed a
higher rate of mutation (80% transcripts), with a range of 1–13 bp
(CD19?CD20?CD38?), pre-GC B cells (CD20?
CD38?), and plasma cell precursors (CD20?CD19low
CD27?/??CD38??) in healthy adults, healthy chil-
dren, and SLE patients.
Percentage of conventional B cells
cytoplasm next to three larger cells with more abundant cytoplasm corresponding to IgD?CD38?B cells. b, Sorted blood IgD?CD38?B cells. c and d,
Sorted blood CD19?/lowCD20?CD27?CD38??plasmablasts at ?40 and ?100 magnifications, respectively.
a, Wright-Giemsa staining of cytospun, magnetic bead-purified blood B cells; arrows show two resting naive B lymphocytes with scant
2365 The Journal of Immunology
substitutions/mutated VHregion (data not shown). Thus, blood
IgD?CD38?cells have initiated the process of somatic mutation.
Taken together, our data indicate the presence in blood of a subset of
B cells that may represent the link between naive and GC cells.
Increase in SLE blood of CD20?CD19?CD38??clonally
expanded plasma cell precursors that can be further subdivided
into CD27?and CD27??
Most SLE patients display a distinct population of CD20?
CD19?/lowCD38??blood cells (Fig. 7, A and B). Upon staining
with CD27, these cells can be further subdivided into a CD27?and
a CD27??population. Although the ratio of CD27?/CD27??
varies, the predominant population expresses CD27 with intensity
comparable to that of memory (CD27?) B cells (Fig. 7B). After
sorting and Wright Giemsa staining, the majority of these cells do
not look like mature plasma cells but like plasmablasts/early
plasma cells (39, 40), as they have larger, less peripheral nuclei
and less abundant cytoplasms (Fig. 5, c and d). The majority of
these cells express both surface and intracytoplasmic Ig, with a ??
ratio close to 1 (43.5 ? 17.9% ?), whereas only a small percentage
(15.5 ? 8.8%) of them expresses the mature plasma cell marker
CD138 or syndecan.
As shown in Table II, SLE patients have a 3-fold expansion
of this population compared with healthy controls. This expan-
sion does not correlate with disease activity as measured by the
SLEDAI (18.0 ? 19.9 cells/?l for SLEDAI ?10; 24.1 ? 33.1
cells/?l for SLEDAI ?10).
We sorted these cells and analyzed 38 IgG VHgene transcripts
from four different SLE patients. All but two transcripts showed a
high frequency of somatic mutations (mean, 16 ? 8.5 mutations/
mutated transcript). However, a striking finding was the identifi-
cation in three of four patients of clonally related transcripts. An
example of the VHsequences corresponding to an expanded clone
(seven related transcripts), with unique and shared mutations, is
displayed in Fig. 8. The pattern of nucleotide mutation within this
clone strongly suggests that it is the product of an Ag-driven re-
sponse, as there is a high ratio of replacement vs silent substitution,
especially concentrated within the second hypervariable region
and the third framework. Clonally related, somatically mutated
transcripts were also found in the blood plasma cell precursors
isolated from two healthy adults (data not shown), suggesting that
these cells in health and disease are the product of oligoclonal
SLE serum does not alter the survival of normal blood B cells
To determine whether the consistently low numbers of blood B
cells and/or the activated B cell phenotype that we observed in our
SLE patients were due to soluble serum factors, we purified naive
blood and tonsilar B cells from healthy donors and cultured them
in the presence of autologous sera, sera from four lymphopenic
SLE patients with different SLEDAI, and sera from two patients
with JDM. The percentage and absolute numbers of viable cells
were calculated at 24, 48, 72, and 96 h using a hemocytometer
after trypan blue staining. Apoptotic cells were also analyzed by
flow cytometry using forward side scatter/side light scatter and
annexin V binding/propidium iodine staining. No consistent dif-
ferences were observed (data not shown), thus suggesting that a
soluble factor(s) is not responsible for mature B cell death and
subsequent lymphopenia in all SLE patients.
B cell subsets in the blood of healthy children
Our study shows that blood B cells in all age groups include at
least four subsets: 1) naive (CD19?CD20?IgD?CD38?CD27?) B
cells, 2) pre-GC (CD19?CD20?IgD?CD38?CD27?) B cells, 3)
memory (CD19?CD20?CD38?CD27?) B cells, and 4) plasma
cell precursors (CD19?CD20?CD27?/??CD38??). When com-
paring children to adults, naive and memory B cells are 2.4-fold
more abundant, whereas pre-GC B cells and plasma cell precursors
are 3- and 4-fold expanded, respectively, in children.
A puzzling observation is the detection in blood of sIgM?
sIgD?B cells bearing a phenotype similar to that of tonsil GC B
cell founders. As GC B cells, these cells express CD38 and cen-
terin, but, unlike GC founders (Bm2?) and centroblasts (Bm3),
they lack the expression of CD10 and CD77. Furthermore, they are
smaller than centroblasts, hence their denomination as pre-GC
cells. Importantly, these cells have initiated the process of somatic
mutation, which is another hallmark of GC reactions; sequencing
the VHIg transcripts from sorted sIgD?sIgM?CD38?blood B
cells from healthy adults revealed a mutation frequency similar to
that described for tonsilar GC B cell founders (1–12 bp muta-
tions/VHregion in 80% transcripts) and higher than that of naive
B cells (1–2 bp mutations/VHregion in 50% transcripts) (33, 39).
Thus, IgM?IgD?CD38?blood B cells may represent the link be-
tween naive (Bm1 and Bm2) and GC founders (Bm2?). It remains
to be established whether these cells result from 1) activation in
lymphoid sites and recirculation in the blood, or 2) activation in
nonlymphoid sites followed by recirculation in the blood and later
homing to peripheral lymphoid organs.
Table III. Mutation analysis of VHtranscripts from SLE B cell subpopulations
B Cell Subpopulation
1.4 ? 1.6
15.6 ? 8.6
and Daudi, IgD?CD38?pre-GC B cells, IgD?and IgD?CD38?(naive
and memory) B cells, and the Jurkat T cell line. Quantification of centerin
message was performed using real-time RT-PCR as described in Materials
and Methods. Bars represent the relative expression of centerin in each of
the tested samples using Raji RNA as standard. Values were normalized
according to the ribosomal RNA expression in each of the samples.
Expression of centerin message in the Burkitt’s lines Raji
2366BLOOD B CELL SUBPOPULATIONS IN PEDIATRIC SLE
Plasma cell precursors constitute another underestimated circu-
lating cell population. We show herein that they represent ?1.4%
of the total B cell compartment in healthy adults and ?3.3% in
healthy children. In the context of certain infections and malig-
nancies, higher numbers of plasmablasts have been described in
the blood (reactive plasmacytosis) (40, 41). These cells have been
reported to characteristically lack the plasma cell marker CD138,
but they acquire it in vitro upon exposure to IL-6 (41). Addition-
ally, these cells express variable levels of CD27 (Refs. 42 and 43,
and our own observation), suggesting caution when using CD27 to
enumerate memory cells, especially in clinical situations where
plasmacytosis may be expected.
Blood B cell subsets in children with SLE
Our studies reveal that children with SLE suffer profound B cell
lymphopenia due to a dramatic reduction in all mature B cell
subsets. SLE B cell lymphopenia does not correlate with any
modality of therapy, SLEDAI, or anti-dsDNA or complement
titers. SLE B cell lymphopenia could be due to 1) a reduction in
the number of bone marrow B cell precursors, 2) shortened mature
B cell life span, or 3) accelerated activation/differentiation of
naive cells into downstream phenotypes including GC, memory,
or plasma cells that would subsequently home into lymphoid
clonally related VH5/? transcripts isolated from
sorted plasmablasts from the blood of a SLE pa-
tient. The transcripts display unique and common
mutations while sharing the same V-D and D-J
junctions (only the V-D junction is shown). There is
a high ratio of R/S nucleotide substitutions espe-
cially within CDR2 (R/S ? 2 and 5 in transcripts
SLE 7 and SLE 3, the least and most mutated VH
regions from this clone, respectively) and FW3
(R/S ? 3 and 8 in SLE 7 and SLE 3, respectively).
The nucleotide sequences corresponding to these tran-
scripts have been submitted to GenBank under acces-
sion numbers 384526, 384527, 384534, 384543,
384551, and 384565.
Amino acid translation of seven
from two SLE patients (A and B). Squares depict the
CD19lowCD38??plasma cell precursor population. Pa-
tient A was recently diagnosed and untreated at the time
the sample was obtained, whereas the sample from patient
B was obtained 1 mo after cytoxan and solumedrol pulses.
B, Enriched B cells from a SLE patient stained with
anti-CD20-PerCP, anti-CD19-allophycocyanin, anti-
CD38-PE, and anti CD27-FITC. CD19lowCD38??cells
gated in B are represented within a rectangle in D and
divided by a dotted line according to the intensity of CD27
staining. The same CD19lowCD38??population is en-
closed by a dotted circle (A) and a dotted rectangle (C).
This experiment is representative of 12 individual
A, Enriched (?95% pure) blood B cells
2367The Journal of Immunology
Killing of B cells by soluble factors (i.e., anti-lymphocytic Abs)
has been implicated as a cause of SLE lymphopenia (44, 45). Al-
though this mechanism may operate in some SLE patients, our
results suggest that it is unlikely to explain the universal lym-
phopenia observed in this disease, as incubation of blood naive B
cells from healthy donors with serum from active SLE patients
failed to disclose any significant reduction in the number of viable
cells. Additionally, the B and T lymphocyte propensity to undergo
spontaneous and induced apoptosis has been recently described to
be grossly intact in SLE (46).
The lymphopenia that we describe cannot be explained by bone
marrow aplasia, as the neutrophil and platelet counts were within
normal limits in the population that we studied. Furthermore, bone
marrow aspirates from SLE patients, usually obtained in the con-
text of severe blood cytopenias, have rarely revealed aplasias (47–
49). Therefore, only a selective lymphoid cell precursor defect
could explain the reduced numbers of T and B cells that we ob-
served in the blood of our SLE patients. The increased proportion
of CD38?B cells in SLE blood may provide us with some clues
regarding the lymphopenia and perhaps some ethiopathogenic fac-
tors in this disease. In trying to induce naive B cells to become GC
B cells in vitro, we identified IFN-? as one of the most efficient
signals to up-regulate CD38 expression on naive B cells (50). In-
terestingly, high levels of IFN-? have been described in the serum
of SLE patients (51), and the PBMCs of patients without circulat-
ing IFN-? display high levels of oligoadenylate synthetase and Mx
protein, a signature of exposure to IFN-? (52, 53). The potential
role of this cytokine in SLE development is further suggested by
the large proportion of patients receiving IFN-? therapy who de-
velop autoimmune, including SLE-like, syndromes (reviewed in
Ref. 54). Finally, and perhaps best explaining the generalized lym-
phopenia of SLE patients, administration of IFN-? to newborn
mice inhibits T and B cell development in the bone marrow, thy-
mus, and spleen by 80% (55). Therefore, all these findings make it
tempting to speculate that SLE may be associated with a deregu-
lation of IFN-? production. Consistent with this hypothesis, the
blood pre-GC (IgD?CD38?) B cell subpopulation is reduced to a
lesser extent in SLE patients compared with controls and repre-
sents the predominant blood B cell population in many SLE
In contrast to the reduction in all mature B cell subsets, children
with SLE present a 3-fold expansion of blood plasma cell precur-
sors that make up to 8.7% of their blood B cell compartment.
Plasma cells expressing CD138 and high levels of CD27 have been
recently reported in the blood of 13 adult SLE patients (43). In our
study only a small proportion of the CD20?CD19lowCD38??
cells in the 68 patients analyzed display this more mature pheno-
type, whereas the majority lack CD138, express two levels of
CD27 (comparable and higher than memory B cells), and upon
sorting and Giemsa staining do not show a mature plasma cell
Blood plasma cell precursors are post-GC cells, as they express
highly mutated and isotype-switched Ig transcripts. Additionally,
there is a high degree of clonal relatedness within this subset, as
numerous transcripts share the same VDJ rearrangement while dis-
playing common and unique nucleotide substitutions. This sug-
gests that they are the products of a recent clonal expansion that
probably occurred in a GC, given the presence of unique muta-
tions. This expansion may be explained by increased IL-10, a ma-
jor plasma cell differentiation factor (56). Indeed, high levels of
IL-10 are found in the serum of SLE patients, and treatment of
these patients with anti-IL-10 Abs has shown beneficial effects
(57–59). Alternatively, the recently identified B lymphocyte stim-
ulator (BLyS/BAFF/TALL-1), a TNF family cytokine (60–63),
may contribute to the disease, as it seems to prominently enhance
humoral responses. BLyS transgenic mice show hypergamma-
globulinemia and an autoimmune lupus-like disease (61). Further-
more, the survival of lupus-prone mice is increased by treatment
with a BLyS antagonist (63). Although altered expression of BLyS
and/or its receptors may play a role in human SLE, significant
differences between the B cell phenotype found in BLyS trans-
genic mice and human SLE exist, as these transgenic mice display
B cell expansion in the blood rather than the profound B cell lym-
phopenia that we describe in our patients. SLE may thus be best
explained by the combined ectopic expression of cytokines such as
?-IFN, IL-10, and BLyS. The etiology of this disease may be
explained at the level of cells that produce these cytokines, which
include APC such as dendritic cells.
We thank Dr. Karolina Palucka for very helpful discussions. Steven Scholl
and Elizabeth Kraus provided excellent technical assistance.
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