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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Division of Rheumatology and Clinical
Immunology, Department of Medicine, and from the Department of Surgery,
University Hospital of Freiburg, Germany.
Hypogammaglobulinemia is the hallmark of common variable
immunodeficiency (CVID) syndrome, a heterogeneous disorder predisposing patients to recurrent bacterial infections. In this study, we investigated the peripheral B-cell compartment of 30 well-characterized CVID patients in comparison to 22 healthy controls. Flow
cytometric analysis of peripheral blood lymphocytes revealed a
reduction of class-switched
CD27+IgM Common variable immunodeficiency (CVID) comprises a
heterogeneous group of humoral immunodeficiencies of unknown etiology, with a prevalence of about 1 in 50 000. It is characterized by reduced
serum levels of all switched immunoglobulin (Ig) isotypes (IgG, IgA,
IgE), predisposing patients to frequent infections of the respiratory
tract with encapsulated bacteria. Splenomegaly as well as malignancies
and autoimmune phenomena may develop in 20% to 30% of the
patients.1 Multiple etiologies are likely to result in
this common clinical phenotype. Depending on the patients analyzed,
primary T-cell defects,2-4 an exaggerated T-cell
suppression,5 and primary B-cell defects,6-8
all leading to a complex failure of T- and B-cell cooperation, have
been reported.
Therefore, a classification of the patients is of primary importance to
acknowledge the heterogeneity of the disease. About 80% of all CVID
cases display normal T- and B-cell numbers in their peripheral blood.
They have been functionally classified by Bryant et al9
according to their capacity to produce IgM, IgA, and IgG in vitro upon
stimulation with Staphylococcus aureus Cowan I
(SAC) plus interleukin-2 (IL-2) or anti-IgM plus IL-2 (see "Patients, materials, and methods"). Peripheral blood
lymphocytes (PBLs) of patients in group A fail to produce any Ig
isotype in vitro, while group B patients produce IgM only and group C
patients are indistinguishable from healthy controls in producing
normal amounts of all isotypes in vitro despite low serum Ig levels in vivo. A minority of CVID patients (5%-10%) have strikingly low peripheral B-cell counts (< 1% of PBLs), suggesting defects at the
early B-cell differentiation stages in bone marrow.10
Another subtype (5%-10%) exhibits noncaseating, sarcoidlike
granulomas in different organs and tends to additionally develop a
progressive T-cell deficiency.11,12 In our studies we
included only CVID patients with normal B-cell counts (> 1% of PBLs)
and without evidence of granulomatous disease.
The recent observation13 that X-linked hyper-IgM syndrome
patients are lacking
CD27+IgD Patients and controls
Cell preparation
Antibodies and flow cytometry PBMCs (2.5 × 105/50 µL RPMI 1640 plus 10% fetal calf serum) were stained for 20' at 4°C with 10 µL of a mixture of the following antibodies at optimal concentrations: CD27-fluorescein isothiocyanate (FITC) (Dako, Glostrup, Denmark) or CD21-FITC (Coulter-Immunotech, Hamburg, Germany), anti-IgD-phycoerythrin (Southern Biotechnology Associates, Birmingham, AL), CD19-PC5 (Coulter-Immunotech), and anti-IgM-Cy5 (Dianova, Hamburg, Germany). Four-color data acquisition was performed with a FACSCalibur (Becton Dickinson, Mountain View, CA). Data analysis (CellQuest, Becton Dickinson) was performed by forward versus side-scatter gating on viable lymphocytes (PBLs) in combination with gating on CD19+ cells.Ig synthesis in vitro For in vitro Ig synthesis, 5 × 105 PBMCs were stimulated for 8 days with the T-cell-independent stimulus SAC (Calbiochem, La Jolla, CA) diluted to 1:10 000 plus 20 U/mL IL-2. Cultures were set up in 500 µL RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin, streptomycin, and L-glutamine (Biochrom). Control cultures were kept in medium without B-cell stimulants. Supernatants were collected after 8 days and stored at 20°C until assayed for IgG, IgA, and IgM content
by enzyme-linked immunosorbent assay.6 In this study we
only report IgG and IgM concentrations and omitted IgA because it
paralleled IgG and provided no additional information. Results are
expressed as nanograms per milliliter of Ig isotype produced in
stimulated cultures minus control cultures. Based on the results of the
Ig synthesis following SAC plus IL-2 stimulation in vitro,
CVID patients were assigned into one of the 3 subgroups defined by
Bryant et al9 (Table 1): group A, no significant synthesis
of IgM (< 500 ng/mL) or IgG (< 500 ng/mL); group B, significant
synthesis of IgM (> 4000 ng/mL) but not of IgG (< 500 ng/mL); and
group C, normal synthesis of IgM (> 4000 ng/mL) and IgG (> 500
ng/mL).
Sorting of CD19+CD27  IgG naive B cells and
CD27+IgGIgM+ memory B cells.
Purified B cells or B-cell subpopulations were cultured at
1 × 105/200 µL in U-shaped microtiter plates and
stimulated with SAC plus IL-2 (20 U/mL). Parallel
0.5 × 105 B cells were cocultured with
2 × 105 CD4+ T cells in flat-bottomed
microtiter plates. CD4+ T cells had been positively
selected by DETACHaBEADs (Dynal) and depleted of contaminating
monocytes by incubation (20', 4°C) with anti-CD14 and anti-CD16
monoclonal antibodies (Coulter-Immunotech) and subsequent treatment
with immunomagnetic beads coated with antimouse IgG (Dynal). The purity
of CD4+ T cells was more than 99%. Supernatants of B-cell
cultures were collected after 8 days and stored at 20°C until
assayed for Ig isotypes by enzyme-linked immunosorbent assay.
Statistical analysis Statistical comparisons of numeric data were made using an unpaired Student t test. Classified data were evaluated by the 2 or Fisher exact test. Differences between groups
were considered significant at P < .05.
Decrease of class-switched
CD27+IgM A recent report of Rajewski's group14 demonstrated a
substantial number of non-class-switched
CD27+IgD+IgM+ memory B cells in the
peripheral blood of humans. When dissecting the B-cell compartment
according to surface Ig and CD27 expression, PBLs of healthy donors
comprised 4.3% ± 1.6% naive B cells
(CD27
Good correlation between the frequency of
CD27+IgM IgD) in the peripheral
blood (r = 0.81, Figure 3).
This correlation was independent of whether the cells were derived from
CVID patients or healthy donors, suggesting a normal intrinsic function
of class-switched memory B cells in CVID patients of group C.
Isolated naive B cells of healthy donors do not produce IgM or IgG in vitro after SAC plus IL-2 stimulation Because more than 90% of B cells from group A CVID patients express a naive CD27 phenotype, we analyzed for more
adequate comparison the capacity of naive B cells of healthy donors to
produce Ig in vitro after stimulation with SAC plus IL-2. Highly
purified CD19+CD27IgG B cells
were stimulated for 8 days with SAC plus IL-2 in the presence or
absence of autologous CD4+ T cells. In supernatants of
T-cell-free cultures, no traces of Igs were detectable, indicating
that SAC plus IL-2 is unable to induce Ig synthesis in isolated
CD27IgM+IgD+ naive B cells (Table
2). However, in the presence of
autologous CD4+ T cells, naive, mature B cells produced
large amounts of IgM, but no class switch to IgG was observed (Table
2). Therefore, the Ig synthesis pattern in vitro of mature
CD27IgM+IgD+ normal B cells
closely resembles that of PBLs of CVID group B patients. Cultures set
up with highly purified
CD27+IgM+IgD+ memory B cells of
healthy donors produced large amounts of IgM even in the absence of T
cells (Table 2). They, however, failed also to produce significant
levels of IgG, confirming the previous finding that SAC plus IL-2 is
not sufficient to induce class switch in vitro.15 The data
presented in Table 2 also explain why in cultures of PBLs stimulated
with SAC plus IL-2 it is impossible to correlate IgM production in
vitro to the IgM memory B-cell pool: In the presence of
CD4+ T cells, both CD27IgM+ naive
B cells and CD27+IgM+ memory B cells are
induced to IgM production.
IgM production of CVID group A patients in vitro is dependent on culture conditions We have applied the CVID classification of Bryant et al9 over the last 10 years in our clinic and found that patients usually stayed in the same group when tested on repeated occasions. However, in 2 patients repeatedly classified as type A, a reversion to type B could be observed. Interestingly, we have never observed the opposite that is, progression from type B to A. To
clarify the validity of a negative IgM production in vitro we studied in a more systematic approach the influence of culture conditions on
the outcome of Ig synthesis in vitro of CVID group A patients. We
therefore coincubated 0.5 × 105/200 µL purified B
cells and 2 × 105/200 µL autologous CD4+ T
cells. In one patient (Table 3, patient
no. 2), this increase of B-cell numbers in the culture normalized the
IgM production, suggesting that a low B-cell frequency of 1.5% (7500 B
cells/200 µL of culture) in the original PBL preparation might have
been a cause for the "defective" IgM production in vitro. Another
type A patient with 10% B cells in the original PBL fraction (Table 3,
patient no. 3) showed normal IgM production in vitro when 0.5 × 105/200 µL isolated B cells were cocultured with
2 × 105/200 µL autologous CD4+ T cells,
but when 2 × 105 PBLs were added to the isolated B cells
the IgM production subsided again. This suggests suppression by a
population other than B cells and CD4+ T cells.
Similar observations have previously been reported.16 Moreover, in a recent study of our group using the "Zubler system" to maximally stimulate B cells17 and the enzyme-linked
immunospot assay technique as the read-out system, all type A patients
produced IgM spots but no or very few IgG spots.18 In
accordance with these and previous data,19 we found a
normal, spontaneous IgM synthesis by 23 of 23 CVID-derived Epstein-Barr
virus lines (data not shown). Taken together, all these findings
indicate that failing IgM production in vitro is not an absolute and
irreversible defect in CVID patients; it may have various causes and
does not seem to be a reliable marker for a specific subgroup of
CVID patients.
A new, easy FACS-based classification of CVID patients with normal B-cell numbers The percentage of class-switched CD27+IgMIgD memory B cells of
the PBL fraction was a highly reliable marker for a classification of
CVID patients. In all healthy donors more than 0.5% (1.6% ± 0.6%)
of PBLs belong to the
CD27+IgMIgD population. In
contrast, PBLs of 77% of our CVID patients with normal B-cell counts
contained less than 0.35% of class-switched memory B cells
(0.1% ± 0.1%, P < .0001). We designated this group of patients as CVID group I (Table 4). A
total of 23% of our patients showed a less significant reduction of
the peripheral switched memory B-cell compartment (0.9% ± 0.4%,
P = .005); they were classified as CVID group II. The
difference to group I was also highly significant
(P < .002) (Table 4). At the same time this group showed
a significant increase of total B cells (12.6% ± 4.7%) compared
with group I (6.5% ± 3.5%, P = .019) and healthy donors (7.7% ± 2.7%, P = .046) (Table 4). Our new
CVID group I comprises only former group A and B patients of the Bryant
classification, while group C patients segregate with the new
group II.
As can be seen in Figure 4,
CD21
Our recent results of an in vivo vaccination study (A. Rump, manuscript
in preparation) with the neoantigen phage
After antigen-independent development in the bone
marrow,21 immature B cells leave the bone marrow and
gather in the longer-lived mature, naive
IgD+IgM+CD27 In the last 2 years CD27 as well as CD148 25 have been
established as reliable markers for human memory B
cells.26,27 In all healthy donors more than 0.5% of PBLs
belong to the CD27+IgM When we compared our results with the classification procedure based on Ig synthesis in vitro,9 we found that, with the exception of 2 group B patients, all A and B patients belong to the new group I. We could show a significant correlation between IgG production in vitro and the percentage of switched memory B cells in vivo and demonstrated a variability of IgM production in vitro depending on the protocol. Thus, low to absent IgM production in vitro may be due to low B-cell numbers (1%-2%) in some patients and may normalize with a proportionate increase of B cells in the cell culture system (Table 3). In other patients of group A, the removal of suppressive cell populations may result in significant IgM production.5,16 Therefore, IgM production is not a reliable marker for a CVID classification. Neither was the percentage of IgM memory B cells, because the size of this population in healthy donors varied considerably. Because the development of memory B cells is essentially linked to the
formation of GCs in secondary lymphoid organs,23 the
finding of a significantly reduced switched memory B-cell compartment
in CVID type I strongly supports the hypothesis that GC reactions are
disturbed in this disease.34 Many possible causes for
disturbed GC reactions have been described. Mutations in the CD40
ligand gene,35,36 defects of the tumor necrosis factor- In contrast to mice, up to 25% of human peripheral B cells express
hypermutated IgM and CD27 on their surface and were therefore classified as IgM memory B cells.14 The origin of the IgM
memory population is discussed controversially. Patients of group Ib exhibit an expansion of mature B cells and a concomitant reduction of
switched and IgM memory B cells (Table 4), suggesting defects in the
formation of both memory compartments, while the IgD A different pathogenesis seems to apply for the CVID group II patients. This group comprises all former C patients and 2 additional patients of group B. On average, PBLs of these patients contained a higher percentage of total B cells compared with patients in group I and healthy donors, suggesting an increased proliferation, an augmented lifespan, or decreased apoptosis of B cells. Their IgM memory B cells especially seem to be expanded compared with healthy donors. The switched memory B cells were only slightly decreased, and quantitative flow cytometry of memory B-cell subsets as well as the synthesis of Ig in vitro could not reliably distinguish them from healthy controls. CVID group II patients probably have a normal GC reaction but somehow fail to produce substantial amounts of antibodies in vivo or have an increased catabolism of Ig. The presence of functional GC reactions in some CVID patients with switched memory B cells has additionally been supported by the finding of normal hypermutation rates in 6 of 8 tested CVID patients by Levy et al.44 The hypogammaglobulinemia in group II patients may be due to impaired terminal plasma cell differentiation, a disturbed homing of plasma cell precursors,45 or a shortened lifespan of plasma cells in vivo.46 Many interactions like CD27/CD70,47,48 CD134/CD134L,49,50 and IL-6 with its receptor51,52 promote the terminal differentiation of B cells into plasma cells. Interestingly, one report indicated an increase in IL-6 in CVID-derived PBLs.53 In 2 recent papers, Brouet et al28 and Jacquot et
al29 analyzed the role of CD27 expression and function in
patients with CVID. Both found a substantial decrease in
CD27+ B cells in a subgroup of CVID patients, as similarly
observed for our patients. Brouet et al28 demonstrated for
3 of 5 of these patients a defective up-regulation of CD27 on isolated
B cells after in vitro activation, suggesting a defect in CD27
expression. This finding was confirmed by the report of Jacquot et
al29 describing 2 of 6 patients with defective
up-regulation of CD27 after in vitro activation. However, it does not
distinguish between a primary defect in CD27 up-regulation and a defect
secondary to a disturbed activation signal following SAC plus IL-2
stimulation. Interestingly, both authors describe another subgroup with
normal CD27 expression but impaired in vitro function despite
costimulation with CD70 transfectants. Thus, Brouet et al28
reported that isolated B cells from 2 of these patients failed to
produce Ig when costimulated with CD70 transfectants. Unfortunately,
this finding was not correlated to Ig production in the absence of CD70/CD27 costimulation, thus rendering it impossible to distinguish defects in CD27 function from defects in SAC plus IL-2/CD40
stimulation. The group of Jacquot et al,29 however,
clearly shows for one patient a disturbed costimulatory signaling via
CD27. Unfortunately, neither group correlated their data to extended
clinical phenotypes and the existing classification of Bryant et
al.9 These findings point at an important role of
CD27/CD70 costimulation in the pathogenesis of CVID and deserve further
investigation. Recent results from our group show that naive,
pre-germinal center B cells (CD27 In conclusion, this study proposes a new CVID classification that is based on flow cytometric analysis of different B-cell subsets in peripheral blood. It accommodates the previous functional classification of Bryant et al9 and confirms and extends reports by Brouet et al28 and Jacquot et al29 on low CD27+ B-cell counts in a subset of severely immunocompromised CVID patients. It shows a correlation with clinical findings and may permit the prediction of successful vaccination in some CVID patients. A consensus on this classification system will provide a powerful tool to rapidly define and compare homogeneous subgroups of CVID for functional studies and genetic linkage analysis.
Submitted July 18, 2001; accepted October 19, 2001.
Supported by the Landesstiftung Baden Wuerttemberg. C.G. is a recipient of an award of the Hans-Hench Stiftung.
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: H. H. Peter, Div of Rheumatology and Clinical Immunology, Hugstetter-Strasse 55, 79106 Freiburg, University Hospital of Freiburg, Germany; e-mail: peter{at}mm61.ukl.uni-freiburg.de.
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IgD
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Abstract
Introduction
X174 
family