Defined Blocks in Terminal Plasma Cell
Differentiation of Common Variable
Immunodeficiency Patients
This information is current as
of June 18, 2017.
Nadine Taubenheim, Marcus von Hornung, Anne Durandy,
Klaus Warnatz, Lynn Corcoran, Hans-Hartmut Peter and
Hermann Eibel
J Immunol 2005; 175:5498-5503; ;
doi: 10.4049/jimmunol.175.8.5498
http://www.jimmunol.org/content/175/8/5498
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References
The Journal of Immunology
Defined Blocks in Terminal Plasma Cell Differentiation of
Common Variable Immunodeficiency Patients1
Nadine Taubenheim,* Marcus von Hornung,* Anne Durandy,† Klaus Warnatz,‡
Lynn Corcoran,§ Hans-Hartmut Peter,‡ and Hermann Eibel2*
C
ommon variable immunodeficiency (CVID)3 is the second most frequent primary immunodeficiency in humans
with a prevalence of ⬃1 in 30,000 (1). The major hallmark of CVID is a significant reduction or absence of Abs in the
serum of the patients leading to recurrent bacterial infections predominantly of the respiratory and gastrointestinal tracts. Furthermore, an increased incidence of granulomatous inflammation, autoimmune disorders, and gastrointestinal malignancies has been
described (2). The syndrome can occur in both sporadic and familial forms and covers a heterogeneous group of disorders of
which the underlying molecular bases are largely unknown. Recently, a homozygous deletion in the ICOS gene has been identified in a small group of CVID patients (3). However, in the majority of the patients, the characterization of genetic defects
remains elusive. Several functional defects have been described
affecting proliferation, activation, and cytokine production in T
cells (4, 5). In B cells, early and late differentiation stages may be
affected resulting in defective up-regulation of surface molecules
such as CD86 and CD70 (6, 7), impaired signaling (8), and somatic
*Clinical Research Unit for Rheumatology, University Hospital of Freiburg, Freiburg,
Germany; †Institut National de la Santé et de la Recherche Médicale (INSERM) Unité
429, Hôpital Necker-Enfants Malades, Paris, France; ‡Division of Rheumatology and
Clinical Immunology, Department of Medicine, University Hospital of Freiburg,
Freiburg, Germany; §The Walter and Eliza Hall Institute of Medical Research,
Parkville, Victoria, Australia
Received for publication April 28, 2005. Accepted for publication July 22, 2005.
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.
1
This work was supported by the European Community through Grants QLCT-200101536 and LSHM-CT-2004-005264 to H.E. and the Grants EURO-POLICY-PID PL
006411, INSERM, the French Rare Disease Program and the Assistance-Publique,
Hopitaux de Paris (to A.D.).
2
Address correspondence and reprint requests to Dr. Hermann Eibel, Clinical Research Unit for Rheumatology, University Hospital Freiburg, Zentrum Klinische
Forschung, Breisacher Strasse 66, 79106 Freiburg, Germany. E-mail address:
[email protected]
3
Abbreviations used in this paper: CVID, common variable immunodeficiency; CSR,
class switch recombination; CT, circle transcript; AICDA, activation-induced cytidine
deaminase; EGFP, enhanced GFP; GC, germinal center; GLT, germline transcript;
HD, healthy donor; LN, lymph node; MFI, mean fluorescence intensity; SHM, somatic hypermutation.
Copyright © 2005 by The American Association of Immunologists, Inc.
hypermutation (SHM) (9), as well as defective formation of memory B cells (10 –13).
Class switch recombination (CSR) as well as SHM of the V
regions take place in germinal centers (GC) of secondary lymphoid
organs and lead to the generation of Ab-secreting plasma cells and
Ag-specific memory cells. The GC reaction is initiated by the activation of B cells, which up-regulate BCL-6 and migrate into B
cell follicles where they undergo a phase of strong proliferation. At
the same time, the specificity of the BCR is modified by SHM,
allowing for affinity maturation due to positive selection of B cells
for BCRs with the highest affinity.
BCL-6 is a zinc finger protein that acts as a transcriptional repressor. BCL-6 mRNA can be found in a variety of tissues (14),
but on the protein level, the expression is mainly restricted to lymphocytes (14 –16), with the highest expression in GC centroblasts
and centrocytes. PRDM1, encoding the transcriptional repressor
Blimp-1, represents a central target gene of BCL-6 (17). Blimp-1
was proposed to be a key regulator of terminal plasma cell differentiation because ectopic expression of the protein was sufficient to
drive the differentiation of mature B cells to plasma cells (18).
The defective expression of proteins essential for the differentiation of B cells to GC B cells or plasma cells, such as BCL-6 or
Blimp-1, could be responsible for the phenotypic features observed
in CVID. In this report, we describe for the first time defects in the
GC reaction and plasma cell development in lymph nodes (LN)
from CVID patients. We revealed three different stages by which
plasma cell differentiation takes place in the control LN and could
show distinct blocks in this process in the LN from three CVID
patients.
Materials and Methods
Patients
In this study patients were only included with established diagnosis of
CVID (ESID criteria, 具www.esid.org典). The patients were regularly followed in our outpatient clinic and received monthly i.v. or weekly s.c.
replacement therapy, respectively. LN biopsies were from patients who
developed during their follow-up regional mediastinal and/or LN swellings
to exclude malignant lymphoma. None of the biopsies of the three CVID
patients described here showed malignant lymphomas. Control tissues
were obtained from patients undergoing LN dissection within the scope of
lung cancer treatment.
0022-1767/05/$02.00
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Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by defective Ab production and recurrent
bacterial infections. The largely unknown causes are likely to comprise a diverse set of genetic or acquired defects. In this study,
we investigated terminal B cell differentiation in lymph nodes from CVID patients. Up to the germinal center B cell stage, B cell
differentiation was normal but terminal plasma cell development was found to be impaired. Using differential Blimp-1 and
Syndecan-1 expression in controls, we defined three different plasma cell subsets that correspond to progressive developmental
stages locating to different sites in the lymph node. In the CVID patients, we could only detect one or two of these subsets indicating
a defective differentiation. Thus, terminal plasma cell differentiation was found to be impaired despite normal expression of
Blimp-1. B cells reaching only the first stage of plasma cell differentiation were further unable to undergo isotype switching and
to up-regulate activation markers on B cells stimulated in vitro. The Journal of Immunology, 2005, 175: 5498 –5503.
The Journal of Immunology
5499
Table I summarizes the clinical and immunological features of the patients. Informed consent was obtained from all patients and controls using
biopsy material and peripheral blood for scientific purposes according to a
local ethics committee approved research protocol (no. 239/1999).
endogenous peroxidases. Immunohistochemical stainings were then performed according to the procedures specified in Elite Vectastain kits, using
the Nova Red, Vector SG, and Vector AP substrates (Vector Laboratories).
Reagents
Flow cytometry
Primary Abs used for immunohistochemistry: anti-IgD-AP (Southern Biotechnology Associates), anti-Syndecan-1 (Serotec), anti-Ki67 (BD Pharmingen), anti-BCL 6 and anti-CD23 (DakoCytomation), anti-BOB-1 and antiOCT-2 (Santa Cruz Biotechnology) and anti-Blimp-1 (19). Abs and
reagents used for flow cytometry: anti-CD19 allophycocyanin and antiCD38 FITC (Caltag Laboratories), anti-IgM-bio (Sigma-Aldrich), antiCD19 PC5 (IO-Test), SA-allophycocyanin, anti-CD27 PE, anti-CD69
FITC and anti-CD86 PE (BD Pharmingen).
PBMC and single cell suspensions of the LN were stained with fluorescence-conjugated Abs. Cells were acquired using a FACSCalibur analyzer,
and data were analyzed using CellQuest software (BD Biosciences). Dead
cells were excluded by gating on propidium iodide-negative cells and on
viable lymphocytes according to their forward and side scatters.
Results
Defective B cell differentiation in GC from CVID patients
Cell preparation and stimulations
Detection of SHM in the IgH V regions
Frequency and characteristics of SHMs in the variable region of the IgM H
chain (VH region) was studied in purified CD19⫹CD27⫹ B cells as described previously (9). PBMCs were labeled with anti-CD19 mAb and
anti-CD27 mAb (Immunotech) and then purified by FACS sorting. The
purity of sorted CD19⫹CD27⫹ B cells was ⬎99%. SHMs were identified
in the VH3-23 (GenBank accession no. AB019439) region after PCR amplification using VH3-23 and C␮ primers and cloning as previously described (9).
Engraftment of immunodeficient mice with purified T and B cells
Human T and B cells from PBMC were isolated with CD4-positive selection kit and anti-CD19 PanB magnetic beads, respectively (Dynal).
Purified cells were transferred into Rag-2/common-␥-chain-deficient
mice (originally described by Colucci et al. (22)). Within 24 h of their
isolation, 1–3 ⫻ 107 cells in a 50-␮l suspension were directly injected into
the spleens of the mice (6:1 T/B cell ratio). Blood samples were taken from
the tail vein and human IgM and IgG concentrations were measured in the
serum by ELISA. Commercial coating Abs, alkaline phosphatase-conjugated detection Abs and standards for IgM and IgG (Jackson ImmunoResearch Laboratories) were used. The assays were developed with AP substrate p-nitrophenyl-phosphate (Sigma-Aldrich). After 3 wk, mice were
sacrificed and spleens were taken for immunohistochemical analyses.
Immunohistochemistry
Methanol/acetone (1:1) fixed 10-␮m cryosections of LN were treated with
0.1% phenylhydrazine in PBS for 30 min at room temperature to inactivate
In immunohistochemical stainings of LN from CVID patients, we
analyzed the expression of different GC and plasma cell markers to
detect possible defects during late B cell differentiation.
Large granulomas were detected in the lymph node of one patient whereas in another patient a lymphoma had developed. In the
lymph nodes of three patients, we found the GCs to be highly
enlarged compared with those found in control tissues. Analysis of
Ki67 illustrated that these GC consisted of a high number of proliferating cells, correlating well with high expression levels of the
transcriptional repressor BCL-6. Therefore, B cell differentiation
in the LN of these CVID patients seems not to be disturbed until
the centrocyte stage. At later stages of B cell differentiation,
Blimp-1 and Syndecan-1 are expressed in plasma cells and/or in a
subset of GC B cells, which are committed to plasma cell differentiation (23). The expression of Blimp-1 and Syndecan-1 in control LN revealed three different subsets of plasma cells that could
be further distinguished by their localization in the LN (Fig. 1):
Blimp-1⫹Syndecan-1⫺ cells represented a relatively small number
of late GC B cells and were found only in the light zone, whereas
Blimp-1⫹Syndecan-1⫹ were found both in and around the GC
(arrows in Fig. 1). Finally, Syndecan-1⫹ cells with low expression
of Blimp-1 (Blimp-1lowSyndecan-1high) were situated in large
numbers only outside of GC. Because plasma cell differentiation
requires exit from the cell cycle, all of these cells expressed only
very low levels of Ki67.
The LN from all patients contained cells expressing Blimp-1
and low levels of Ki67. Expression of Syndecan-1 could be detected only in the LN from patient P3. All Blimp-1-positive cells
in the tissues from the patients P1 and P2 therefore were Blimp1⫹Syndecan-1⫺, whereas the LN from patient P3 additionally contained Blimp-1⫹Syndecan-1⫹ cells. Thus, Syndecan-1⫹ cells expressing low Blimp-1 levels (Blimp-1lowSyndecan-1high), which
were present in the control sections, could not be found in the LN
from any of these three CVID patients (Fig. 1A).
Table I. Characteristics of CVID patients
Classification
Patient
Age/Sex
Bryant
et al.
(Ref. 35)
Warnatz
et al.
(Ref. 10)
Serum IgM
(g/L)
(0.4–2.3)
Serum IgG
(g/L)
(7–16)a
% CD19
(6–19%)
SHM In Vivo
% Mutations/bp
(2.6–6.3)
P1
38/F
B
Ia
⬍0.2
3.2
4.2
NA
P2
45/F
B
Ia
0.4
2.1
7.0
0.4
P3
29/F
B
Ia
0.3
4.0
13.7
2.9
a
b
Prior to monthly i.v. IgG substitution.
NA, not available.
b
Clinic
Infections
Splenomegaly, enlarged Chronic bronchitis,
mediastinal LN
chronic sinusitis
Splenomegaly, enlarged Chronic sinusitis
mediastinal LN,
sarcoid-like lesions
Splenomegaly, enlarged Chronic bronchitis,
mediastinal and
chronic sinusitis
abdominal LN,
bronchiectasia
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PBMC were isolated from EDTA-blood using Ficoll-Hypaque density gradient centrifugation (PAN Biotech) and cultivated in IMDM (Invitrogen
Life Technologies) supplemented with 10% FCS (Linaris). Cells were
stimulated with CD40L using irradiated NIH 3T3 fibroblasts expressing
human CD40L under the control of the human EF1␣ promoter. For BCR
stimulation, 6 ⫻ 105 PBMC were cultivated in 48-well plates in the presence of 1.5 ␮g/ml anti-IgM-F(ab)2 for 24 h. For CSR, 500 U/ml IL-4 (R&D
Systems) was added and supernatants were analyzed after 13 days of cultivation by ELISA as described elsewhere (20). Expression of activationinduced cytidine deaminase (AICDA), IgE germline transcript (GLT), and
circle transcript (CT) was examined as described in the report of Imai et al.
(21).
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PLASMA CELL DIFFERENTIATION IN CVID PATIENTS
Quantitative analysis of cell numbers within each Blimp-1⫹
subset was performed by counting cells expressing Blimp-1 and
Syndecan-1 in multiple areas of equal size inside and around GCs.
The mean number of Blimp-1⫹Syndecan-1⫺ cells was increased in
the GC of CVID patients (P1, 15.5 ⫾ 3.6; P2, 9.0 ⫾ 0.9; P3,
10.3 ⫾ 4.2; control, 5.7 ⫾ 0.6; Fig. 1B), whereas the number of
Blimp-1⫹Syndecan-1⫹ cells was lower in LN from P3 and absent
in P1 and P2 (P3, 6.3 ⫾ 1.2; control, 15.2 ⫾ 5.1). Blimp-1lowSyndecan-1high cells were only found in control LN (14.7 ⫾ 3.2) at
about equal numbers as Blimp-1⫹Syndecan-1⫹ cells.
Defective formation of memory B cells in CVID patients
Flow cytometric characterization of the LN from the patients showed
a significant reduction in the percentage of CD19⫹CD27⫹ memory B
cells (28.4 ⫾ 4.1% of LN cells compared with a mean of 59.7 ⫾
10.9% of LN cells from control tissues; Fig. 1, C and D; p ⬍ 0.001).
Thus, besides a defective generation of Blimp-1⫹Syndecan-1⫹ and/or
Blimp-1lowSyndecan-1high cells, impaired generation of memory B
cells in the LN from all three CVID patients was found.
The defective development of memory B cells was also detectable in the peripheral blood of the patients (Fig. 1, E and F). Analysis of the IgM and CD27 expression on CD19⫹ B cells of the
patients revealed a strong reduction in the percentage of
IgM⫹CD27⫹ B cells (mean value 9.0 ⫾ 2.7% of the CD19⫹ cells
(CVID) vs 18.2 ⫾ 5.7% (healthy donor; HD); p ⫽ 0.004; Fig. 1E).
The percentage of isotype-switched memory B cells (IgM⫺CD27⫹)
was even more drastically reduced with a mean value of 1.0 ⫾
0.4% of the CD19⫹ B cells (CVID) vs 17.8 ⫾ 5.6% (HD) ( p ⬍
0.001; Fig. 1E).
Altered responses of patients’ B cells to in vitro stimulation
We could previously show for a subgroup of CVID patients that
BCR stimulation leads to an impaired up-regulation of CD86, but
not of CD69 (7). To examine defects in the response to Ag encounter and to correlate those data with the immunohistochemical
results, we stimulated PBMC from CVID patients P2 and P3 for
24 h with CD40L ⫹ anti-IgM and analyzed the surface expression
of CD86 and CD69.
The B cells from patient P2 exhibited a lower CD86 expression
after stimulation (Fig. 2A, mean fluorescence intensity (MFI) 94.1)
whereas on B cells from patient P3 the expression was within the
normal range (MFI 284.7; HD range MFI 195–370). The up-regulation of CD69 was found to be much higher on B cells from
patient P3 (Fig. 2B; MFI 67) than on B cells from the controls
(MFI 17.4 –38) or from patient P2 (MFI 22). In terms of CD69
up-regulation, the cells of patient P3 therefore seemed to be more
prone to stimulation than the cells of patient P2 or the controls.
After 13 days of stimulation of PBMC via CD40 and the IL-4
receptor, only 19% of the B cells from patient P2 expressed CD38,
compared with 58% in patient P3 and 50.9 ⫾ 15.2% in HD (Fig.
2C). The analysis of other activation markers, such as CD27,
CD130, or CD180 revealed no difference between the stimulated
cells of patients and controls (data not shown).
Generation of SHM
Because the generation of SHM is closely linked to the selection of B
cells during memory and plasma B cell formation, we analyzed the
frequency and the nucleotide substitution of SHM in CD19⫹CD27⫹
B cells from patients P2 and P3. As shown in Table I, the SHM
frequency was found to be very low in B cells from patient P2, but in
normal range (with a normal pattern) in patient P3.
CSR in vitro and in transfer experiments into immunodeficient mice
As a strongly impaired formation of class-switched Abs is a
common feature of CVID, we analyzed CSR in vitro and in cell
transfer experiments to elucidate possible defects.
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FIGURE 1. Immunohistochemical and flow cytometric analysis of LN and peripheral blood cells. A,
Immunohistochemical analysis of LN sections from
patients P1, P2, and P3 and controls (C). Arrowheads
point toward different plasma cell subsets. Magnifications are ⫻100 at Ki67 stainings except for C (⫻200),
⫻200 at BCL-6 (P3), ⫻100 at C, P1 and P2, ⫻100 at
Syndecan-1 except for P3 (⫻1000) and ⫻1000 at all
Blimp-1 stainings. For the controls, four independent
samples were analyzed. B, Numbers of Blimp-1
and/or Syndecan-1 expressing cells determined in
multiple (n ⫽ 4) distinct areas of equal size in LN
from controls and the CVID patients. C, Percentages
of CD19⫹CD27⫹ B cells in LN from CVID patients
P1 (䉬), P2 (F), and P3 (Œ) and from controls in a flow
cytometric analysis. D, Dot plot of the cells from the
CVID LN and one representative control. E, Percentage of IgM⫹CD27⫹ and IgM⫺CD27⫹ cells of B cells
in the peripheral blood of patients P1, P2, and P3 and
of controls. F, IgM and CD27 expression on CD19⫹ B
cells from the peripheral blood of the CVID patients
P1, P2, and P3 and one representative of four controls.
The Journal of Immunology
We determined IgE levels in supernatants of PBMC stimulated
with CD40L ⫹ IL-4 for 13 days. The PBMC from patient P3
produced normal amounts of IgE (3744 pg/ml), whereas the stimulated cells of patient P2 secreted diminished levels of IgE (994
pg/ml; Fig. 3A).
To further characterize CSR at a molecular level in cells from
patient P2, PBMC were stimulated for 5 days with CD40L ⫹ IL-4
and expression of AICDA as well as IgE eps-GLT and IgE eps-CT
were analyzed. We could not detect IgE CTs, indicating that CSR
had not taken place. However, the first steps of CSR initiation were
not impaired because AICDA transcript and eps-GLT expression
were shown to be normal (Fig. 3B). The low but present amounts
of IgE secreted by stimulated B cells from patient P2 may result
from low numbers of memory B cells present in the peripheral
blood (Fig. 1, E and F), suggesting that the patient might have had
B cells able to undergo CSR but lost them over the time and during
the time course of the disease.
As the cells of patient P3, but not of patient P2 showed a normal
CSR after stimulation in vitro, we further examined the capacity of
the cells from these patients to undergo CSR in transfer experiments using immunodeficient mice as recipients. Mixtures of purified peripheral CD4⫹ T cells and CD19⫹ B cells (6:1) from patients P2 and P3 and from HD were transferred into the spleens of
Rag-2/common-␥-chain-deficient mice. At time points of 7, 14,
and 21 days after engraftment, serum concentration of human IgM
and IgG were determined. After 3 wk, mice were sacrificed, and
spleens were analyzed immunohistochemically for human CD4⫹
T and CD19⫹ B cells. Consistent with the in vitro results, transferred cells from patient P3 produced normal amounts of IgG (557
␮g/ml vs 503 ␮g/ml in the control), whereas cells from patient P2
failed to secrete IgG (3 ␮g/ml) (Fig. 3D). The serum levels of IgM
were ⬃10-fold higher in the mice engrafted with the cells from
patient P3 (107 ␮g/ml) than in control mice (11.2 ␮g/ml) or those
with cells from patient P2 (4.6 ␮g/ml) (Fig. 3C). In the spleens of
all mice, human CD4⫹ T cells were detected, reflecting a successful transfer in all cases and the presence of the T cells 4 wk after
engraftment (data not shown). In contrast, human CD19⫹ B cells
were detectable neither in mice engrafted with cells from HD nor
from CVID patients. This might be due to the differentiation of the
B cells into Ab-secreting CD19⫺ plasma cells, which remained
undetected, or to a shorter half-life of CD19⫹ B cells.
The transfer experiments underscore the in vitro switch experiments confirming the inability of B cells from patient P2 to undergo CSR. Conversely, the cells of patient P3 were able to switch
in both experiments.
Discussion
FIGURE 3. CSR in B cells from CVID patients. A, IgE secretion in
supernatants of PBMC stimulated for 13 days with CD40L ⫹ IL-4. B, CSR
in PBMC after 5 days of stimulation with CD40L ⫹ IL-4 (⫺, unstimulated;
⫹, CD40L ⫹ IL-4). C and D, Transfer of purified B and T cells into the
spleens of RAG-2/common-␥-chain-deficient mice. Human IgM (C) and
IgG (D) titers in the serum of mice after 7, 14, and 21 days of engraftment
with T and B cells from HD (open squares), P2 (circles), and P3 (triangles).
All experiments were reproduced at least once depending on the availability of the patient samples.
CVID is characterized by low concentrations or the lack of Abs.
We were interested in analyzing whether any of the Ab deficiencies result from blocks in plasma cell differentiation. To this end,
we investigated late B cell differentiation steps in LNs from a
subgroup of CVID patients who underwent LN biopsies. Analyzing samples from five patients, we found that the LN from three
CVID patients contained large hyperplastic GC consisting of
highly proliferating, Ki67⫹ B lymphocytes. A major fraction of
cells in these GC also expressed the transcription factor BCL-6,
which is essential for the proliferation of GC B cells. BCL-6 inhibits genes involved in cell cycle control, B cell activation, and B
cell differentiation, such as p27kip1, Id2, CD69, or Blimp-1,
thereby maintaining the rapid proliferation of GC B cells whereas
terminal differentiation into plasma cells is delayed (24). BCL-6 is
essential for the GC reaction, because the formation of GC in LN
and spleen was absent, and Ab affinity maturation was impaired in
BCL-6-deficient mice (25, 26). Both GC histology and expression
of BCL-6 highly suggest that B cell differentiation is not disturbed
up to the centroblast or centrocyte stage in the LN of these patients.
This was further underscored by the detection of OCT-2 and
BOB-1 expression (data not shown).
Blimp-1 is a “master” regulator of plasma cell formation as it is
both sufficient and required for plasmacytic differentiation (18,
27). Genes repressed by Blimp-1 are associated with proliferation,
BCR signaling, CSR or cell cycle, such as BCL-6, c-myc, btk, or
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FIGURE 2. In vitro stimulation of PBMC. A and B, PBMC were stimulated for 24 h with CD40L ⫹ anti-IgM and expression of CD86 (A) and
CD69 (B) was analyzed on the surface of stimulated B cells from patients
P2 and P3 and a HD representative for four independent controls. C,
PBMC from patients P2 and P3 and one representative HD were stimulated
with CD40L ⫹ IL4 for 13 days. Percentages of CD38⫹ B cells are shown
for each dot plot. All stimulation experiments were reproduced at least
twice depending on the availability of patient samples.
5501
5502
FIGURE 4. A proposed simple model of late plasma cell differentiation
defined by Ki67, BCL-6, Blimp-1, and Syndecan-1 expression.
can-1⫺ subset as a less differentiated population than cells expressing both factors (Blimp-1⫹Syndecan-1⫹). At first glance, the
model put forward by Kallies et al. suggesting a correlation between increasing expression of Blimp-1 and progressive maturation of plasma cells seems to be inconsistent with our results. However, it should be kept in mind that Kallies et al. used as a tracer
for Blimp-1 expression the fluorescence of enhanced green fluorescent protein (EGFP) integrated by homologous recombination
into the Blimp-1 encoding Prdm1 gene. Because EGFP is a highly
stable protein it tends to accumulate within cells resulting in increased light emission (30). Therefore, changes in gene expression
are reliably detected only by destabilized EGFP mutants (31). It is
therefore not unlikely that Blimp-1 and EGFP may differ in halflives and turnover rates during plasma cell development, and
increasing EGFP fluorescence intensities may not reflect proportionally increasing amounts of Blimp-1 protein. Thus, the EGFPpositive cells described by Kallies et al. may correspond to the
Blimp-1lowSyndecan-1high plasma cells detected in the control LN
sections. Further, it is also conceivable that the observed differences are due to species differences and/or might reflect the distinct
tissues examined.
A previous report ascribed the appearance of Syndecan-1⫹ but
Blimp-1⫺ B cells to technical problems arising when the plane of
the section does not pass through the center of the nucleus, thereby
impeding a detection of Blimp-1 (23). Based on the facts that such
cells abundantly surround the GC of control LN and tonsils but
completely lack in LN from patient P3, we are putting forward that
Blimp-1lowSyndecan-1high B cells represent a discrete B lymphocyte subset rather than a technical artifact. Because Blimp-1 is
known to be a transcription factor required for the transition from
GC B cells to plasma cells, it is conceivable that Blimp-1 expression is only high at this point and that the Blimp-1lowSyndecan1high1low cells represent plasma cells of a later stage, in which
Blimp-1 is reduced to a minimum sufficient to maintain the plasma
cell stage.
Terminal plasma cell formation therefore may be impaired even
though Blimp-1 is expressed. The blocked differentiation step varied between patients P1 and P2 vs patient P3 suggesting different
underlying mechanisms. It will be of interest to examine the functional integrity of Blimp-1, especially seen in patients P1 and P2,
where no Syndecan-1⫹ cells were detectable. However, a severe
defect of Blimp-1 is improbable because Blimp-1 also has important functions in cells other than plasma cells rendering a deficiency for Blimp-1 embryonic lethal (32). Besides Blimp-1, the
expression of XBP-1 is also pivotal for terminal plasma cell differentiation (33). Because XBP-1 acts downstream of Blimp-1, it
is conceivable, that the induction of this protein might be impaired
in our patients. However, deletion of XBP-1 is embryonic lethal
too, because it is an important factor in the unfolded protein response and is essential for the development of fetal hepatocytes
(34). Keeping this in mind, it is unlikely that a genetic defect
abrogating the function of XBP-1 accounts for the defective terminal plasma cell differentiation in our CVID patients.
In conclusion, our results define three subsets of plasma cells,
according to their differential Blimp-1 and Syndecan-1 expression,
in control LN. In LN from the three CVID patients that we analyzed here, we were able to unravel distinct blocks during these
stages of plasma cell differentiation. Our data point toward different B cell defects downstream of Blimp-1 that seem to represent
essential factors in plasma cell development. Although different
subgroups of CVID patients may suffer from other defects or mutations (3, 6, 7) defective plasma cell differentiation is a key finding in CVID and it will be of major interest to further investigate
target genes downstream of Blimp-1.
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AID (28, 29). The mutual inhibition of BCL-6 and Blimp-1 represents a double-negative feedback loop that ensures the exclusive
expression of genes necessary at either the GC or plasma cell
stage. In control LN, we could observe three different plasma cell
subsets by analyzing their Blimp-1 and Syndecan-1 expression.
According to these results, we propose a model in which late
plasma cell differentiation can be divided into at least three steps
defined by their Blimp-1 and Syndecan-1 expression (Fig. 4).
Plasma cell precursors up-regulating Blimp-1 further become
Blimp-1/Syndecan-1 double-positive plasma cells and can differentiate into Blimp-1lowSyndecan-1high cells.
Blimp-1-expressing cells were observed in the LN from all three
CVID patients. This finding was unexpected because the serum of
these patients contains no Abs, suggesting a defect in the differentiation of plasma cells. However, only the LN from patient P3
contained Syndecan-1⫹ cells, although at lower numbers compared with the control sections; these cells all coexpressed
Blimp-1. Thus, only subsets representing the first or second stages
of our model of plasma cell differentiation were detected in the LN
of these CVID patients. Further, the numbers of Blimp-1⫹Syndecan-1⫺ cells in the LN of the patients were higher than in the
control LN, which is consistent with our hypothesis of a blocked
plasma cell differentiation.
In addition to the arrested plasma cell differentiation, peripheral
B cells from patient P2 also showed an impaired activation in
terms of CD86 and CD38 up-regulation after in vitro stimulation
via the BCR or CD40L ⫹ IL-4, respectively. Defective isotype
switch was shown in vitro and by cell transfer experiments into
Rag-2/common-␥-chain-deficient mice, where the cells did not secrete IgG but normal levels of IgM. Further, analysis of SHM in
CD19⫹CD27⫹ cells from the peripheral blood of patient P2
showed no mutated V regions. In contrast, the defect of patient P3
seems not to interfere with these processes because the cells revealed intact CSR both in vitro and in transfer experiments and
exhibited no defective up-regulation of CD86 and CD38 and V
regions in CD19⫹CD27⫹ cells were found to be mutated both with
normal frequency and pattern.
The finding of normal proliferation and production of cytokines
such as IFN-␥, IL-2, and IL-10 by T cells (data not shown) corroborates the B cell specificity of the defect in patient P2, whereas
in P3 the defect seems to affect cells other than B or T cells, which
might provide factors necessary for B cell differentiation in LN.
In mice, distinct subsets of plasma cells differentially expressing
Blimp-1 and Syndecan-1 have also been reported by Kallies et al.
(19). The authors describe plasma cells of the Blimp-1⫹Synde-
PLASMA CELL DIFFERENTIATION IN CVID PATIENTS
The Journal of Immunology
5503
Acknowledgments
We gratefully acknowledge the help, suggestions, and advice of Uli Salzer
and Michael Schlesier.
17.
Disclosures
18.
The authors have no financial conflict of interest.
19.
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