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V-region gene analysis of locally defined synovial B and plasma cells reveals selected B cell expansion and accumulation of plasma cell clones in rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 1, January 2011, pp 63–72
DOI 10.1002/art.27767
© 2011, American College of Rheumatology
V-Region Gene Analysis of Locally Defined Synovial B and
Plasma Cells Reveals Selected B Cell Expansion and
Accumulation of Plasma Cell Clones in Rheumatoid Arthritis
Tobias Scheel,1 Angelika Gursche,2 Josef Zacher,2 Thomas Häupl,3 and Claudia Berek1
Objective. To elucidate the development of synovial tissue–specific B cell immune responses, the clonality of individual naive B cells, memory B cells, and
plasma cells and their organization and histologic localization in the inflamed tissue were investigated in
patients with rheumatoid arthritis (RA).
Methods. B and plasma cells were isolated by
laser capture microdissection (LCM) from the synovial
tissue of patients with RA. In addition, single naive B
cells, memory B cells, and plasma cells were sorted from
synovial tissue cell suspensions. RNA was extracted
from the cells, and Ig VH genes were amplified, cloned,
and sequenced.
Results. Both LCM and single cell sorting analyses showed that naive and memory B cells infiltrated the
RA synovial tissue. Comparison of the V-gene repertoire
of B and plasma cells suggested that synovial plasma
cells were generated, by and large, from locally activated
B cells, indicating that a selected population of memory
B cells differentiates into large plasma cell clones that
then accumulate in the inflamed tissue. Clonally related
plasma cells were isolated from separate and distinct
localized areas of the tissue, suggesting that the newly
generated plasma cells have a high migratory capacity.
Conclusion. These results support the idea of a
continuous activation of selected B cell clones, and
hence a massive accumulation of plasma cells, in RA
synovial tissue. As B cells and their secreted antibodies
are an important factor in controlling inflammatory
processes, patients with RA displaying intensive synovial tissue lymphocytic infiltrations might benefit from
B cell depletion therapy. Early treatment will prevent
accumulation of pathogenic plasma cells.
Rheumatoid arthritis (RA) is a chronic inflammatory process that leads to swollen joints and, ultimately, to destruction of the joints. Normally, the synovial lining layer consists of a thin membrane of
synoviocytes attached to an extracellular matrix, known
as the sublining layer, in which blood vessels, fat cells,
and fibroblasts are embedded. In patients with RA, a
massive infiltration of macrophages, T cells, and B cells
into the sublining layer is observed, and lymphocyte
accumulation is supported by intensive vascularization
(1,2).
A striking histologic feature of RA is the formation of lymphocytic aggregates in the synovial tissue,
which may resemble tertiary lymphoid structures (3–6).
Whereas small aggregates consist mainly of T lymphocytes, in large aggregates, B cells are the dominant cell
type. The importance of B cells in the immunopathologic processes of RA is still controversial (7,8), although
there is an increasing body of evidence showing significant clinical improvement when patients with RA are
treated with rituximab, a B cell–depleting anti-CD20
monoclonal antibody (9–11).
B cells may contribute to the pathogenesis of RA
in multiple ways. B cells are the precursors of plasma
cells, which, through the production of autoantibodies,
enhance the inflammatory processes. Furthermore, B
cells, acting as antigen-presenting cells, support the
polyclonal activation of T cells and thus enhance the
production of cytokines and other proinflammatory molecules. B cells also play a key role in the development of
Supported by the Bundesministerium für Bildung und Forschung (grant NGFN2). The Deutsches Rheuma-Forschungszentrum,
a Leibniz Institute, is supported by the Berlin Senate of Research and
Education.
1
Tobias Scheel, PhD, Claudia Berek, PhD: Deutsches
Rheuma-Forschungszentrum, Berlin, Germany; 2Angelika Gursche,
MD, Josef Zacher, MD: Helios-Kliniken Berlin-Buch, Berlin, Germany; 3Thomas Häupl, MD: Charité–University Medicine Berlin,
Berlin, Germany.
Address correspondence to Claudia Berek, PhD, Deutsches
Rheuma-Forschungszentrum, Charitéplatz 1, 10117 Berlin, Germany.
E-mail: berek@drfz.de.
Submitted for publication March 9, 2010; accepted in revised
form September 21, 2010.
63
64
SCHEEL ET AL
Table 1. Demographic and clinical characteristics of the 7 patients with rheumatoid arthritis (RA)*
Patient
Age, years
Sex
Age at onset of RA, years
Disease duration, years
Steroids/NSAIDs/analgesics
Glucocorticoids, mg/day
DMARD
RF
Anti-CCP
DAS28
ESR, mm/hour
CRP, mg/dl
1
2
3
4
5
6
7
69
F
61
8
Yes
5
MTX
⫹
⫹
5.19
70
3.62
73
M
67
6
Yes
5
LEF
⫹
⫹
4.48
10
1.54
62
F
58
4
No
No
MTX
⫹
⫹
5.39
24
2.5
54
M
37
17
Yes
5
LEF
⫹
ND
5.58
24
0.26
70
F
47
23
Yes
5
LEF
⫹
⫹
6.49
44
10.42
67
F
61
7
No
7.5
MTX ⫹ etan.
⫹
⫹
4.05
17
0.13
51
F
38
13
Yes
10
HCQ
⫹
ND
4.92
20
0.28
* NSAIDs ⫽ nonsteroidal antiinflammatory drugs; DMARD ⫽ disease-modifying antirheumatic drug; MTX ⫽ methotrexate;
LEF ⫽ leflunomide; etan. ⫽ etanercept; HCQ ⫽ hydroxychloroquine; RF ⫽ rheumatoid factor; anti-CCP ⫽ anti–cyclic
citrullinated peptide; ND ⫽ not determined; DAS28 ⫽ Disease Activity Score in 28 joints (range 0–10); ESR ⫽ erythrocyte
sedimentation rate; CRP ⫽ C-reactive protein.
organized lymphoid tissue, and in RA, they are a
prerequisite for the development of ectopic lymphoid
tissue (12,13). By inducing lymphoid neogenesis, which
is associated with more severe synovial and systemic
inflammation (14,15), B cells may play an important role
in establishing and maintaining the processes of chronic
inflammation (16).
A number of approaches to the analysis of B cell
responses in the synovial tissue of patients with RA have
been described. In general, studies of enzymatically
digested synovial tissue have analyzed the V-gene repertoire of synovial B cells (17–19). Microdissection of B
cells from ectopic germinal centers has shown that
synovial B cells take part in an antigen-specific immune
response involving somatic hypermutation and, possibly,
affinity-selected differentiation into plasma cells (5,19–
21). Previous studies utilizing a modified VH-gene fingerprinting method, in which the VH-gene repertoire of
B cells was compared between the peripheral blood,
synovial fluid, and synovial tissue of patients with RA,
yielded findings suggesting that clonal B cell expansion is
a characteristic feature of RA (19,22,23).
In the present study, we analyzed the B cell
immune responses in synovial tissue that lacked germinal center formation. B cells were microdissected directly from large B cell infiltrates and/or multiple plasma
cell aggregates in the inflamed tissue of patients with
RA. B and plasma cells were microdissected from
distinct and separate locations, and their VH-gene repertoire was compared. The analysis showed that local B
cell activation and direct differentiation into plasma cells
may occur in RA synovial tissue. Intensive expansion of
selected B cell clones was confirmed by an independent
approach in which single naive B cells, memory B cells,
and plasma cells were sorted from synovial cell suspensions. Our findings demonstrate that there is a continuous activation of B cells as well as differentiation of
these cells into plasma cells, leading to an accumulation
of large plasma cell clones, in RA synovial tissue. In
patients with RA, B cell depletion, which interferes with
plasma cell development and Ig secretion, may thus help
to control the chronic inflammatory processes of this
disease.
PATIENTS AND METHODS
Patients. Synovial tissue was obtained from 16 patients
with a diagnosis of RA according to the American College of
Rheumatology criteria (24). Synovial tissue samples from 3
patients with B cell and/or plasma cell infiltrates (samples from
patients 1–3 [P1–P3]) were analyzed by laser capture microdissection (LCM). In addition, single B cells and plasma cells
were sorted from synovial cell suspensions (samples P4–P7).
The demographic and clinical features of all 7 of these patients
with RA are summarized in Table 1.
Immunohistochemistry. Frozen tissue sections were
fixed in acetone at ⫺20°C for 10 minutes. Endogenous biotin
was blocked using an avidin–biotin blocking kit (Vector),
followed by a 20-minute blocking step with phosphate buffered
saline/3% bovine serum albumin. To identify cell types and
their location in the synovial tissue, tissue sections were stained
with the following mouse anti-human antibodies: anti-CD20
(L26; Dako), anti-CD4 (MT310; Dako), anti-CD8 (DK25;
Dako), anti-CD68 (EBM11; Dako), anti–HLA–DR (L243;
DRFZ), anti-CD55 (MCA1614; Serotec), anti-CD31 (JC/70A;
Dako), and anti-human FDC (8D6; BD PharMingen). To
detect mouse primary antibodies, an alkaline phosphatase–
CLONAL ACCUMULATION OF SYNOVIAL B AND PLASMA CELLS IN RA
Table 2.
65
Clonal diversity of microdissected synovial B cells*
No. of
areas
No. of
caps
Agg
7
22
157
33
Ass
Agg
2
2
6
14
18
38
139
142
310
55
48
85
9
5
6
37
25
18
12
147
139
173
93
1,153
67
61
44
393
Patient, cell population
Patient 1
Plasma cell
Patient 2
B cell Inf
Plasma cell
Plasma cell
Patient 3
B cell Inf
Plasma cell
Plasma cell
Total
No. of
sequences
No. of different
V–D–J rearrangements
No. of
clones
10
35
11
Ass
Agg
56
* Populations of B cell infiltrates (Inf), plasma cells associated with B cell infiltrates (Ass), and plasma cell
aggregates (Agg) were isolated from rheumatoid arthritis synovial tissue by laser capture microdissection.
labeled polymer (Dako) was used and visualized with the
Alkaline Phosphatase Substrate I kit (Vector). Plasma cells
were detected by staining with the biotinylated monoclonal
antibody Wue-1 (DRFZ) (25), followed by detection with
peroxidase-coupled streptavidin (Sigma) and visualization using Fast diaminobenzidine (Sigma). Hematoxylin (Merck) was
used to stain the nuclei.
LCM analysis. Frozen tissue sections (kept frozen at
⫺70°C) were immediately transferred to acetone at ⫺20°C for
3 minutes. To protect against RNA degradation, sections were
stained using the HistoGene LCM Immunofluorescence Staining kit (Arcturus). Plasma cells were labeled with Alexa
488–Wue-1 antibodies (DRFZ) for 4 minutes at room temperature and dehydrated using 70% ethanol for 30 seconds, 95%
ethanol for 30 seconds, 100% ethanol for 30 seconds, and a
final step of xylene for 5 minutes. Clusters of B cells were
identified on consecutive sections by staining with the mouse
anti-human CD20 antibody L26 (Dako) and Alexa 488–labeled
goat anti-mouse (Invitrogen) as a secondary antibody.
For microdissection, the laser spot diameter was adjusted to 7 ␮m, and cell samples were collected onto CapSure
HS caps (Arcturus). B cell infiltrates were isolated as small
areas of ⬃10–20 B cells. Two-to-eight adjacent areas (depending on the size of the B cell infiltrate) were dissected from each
of the follicles analyzed. Plasma cells were obtained by microdissecting neighboring single cells to avoid contamination of
juxtaposed B cells. For further analysis, ⬃10 cells were collected onto one cap. To remove co-isolated cells, the cap
surface was pressed onto a sterile tissue preparation strip
(Arcturus). RNA was immediately extracted using the PicoPure RNA isolation kit (Arcturus), and integrity of the RNA
was controlled with the use of the Agilent 2100 Bioanalyzer.
When RNA was prepared from the remaining tissue sections
after 2.5 hours of microdissection, RNA integrity numbers
between 5.7 and 6.9 were obtained.
The complementary DNA (cDNA) of rearranged Ig
genes was synthesized using the SensiScript reverse transcription (RT) kit (Qiagen) with primers specific for the different Ig
isotypes: for C␥, 5⬘-GAGGGCGCCAGGGGGAAGAC; for
C␣, 5⬘-GAGGCTCAGCGGGAAGACCTTG; and for C␮, 5⬘ACGGGGAATTCTCACAGGAGAC. V-region genes were
similarly amplified using a mix of VH primers (26) together
with the isotype-specific primers. The amplifications were
done with 1.25 units of AmpliTaq Gold enzyme (Applied
Biosystems). The cycle program consisted of an initial denaturation step at 95°C for 10 minutes, followed by 50 cycles of
95°C for 30 seconds, 68°C for 30 seconds, and 72°C for 40
seconds, and a final extension step of 72°C for 10 minutes.
Amplified Ig cDNA was separated on a 2% low-melting
agarose gel, and bands in the range of 350 bp were excised
and purified using the NucleoSpin Extract II kit (MachereyNagel).
Enzymatic digestion of synovial tissue and single cell
sorting. Samples of synovial tissue were cut into small pieces
(maximum size 1 mm3) and digested using Collagenase D
(Roche) for 90 minutes. After Ficoll centrifugation, cells were
stained using the monoclonal antibodies anti-CD19–
allophycocyanin-Cy7 (BD PharMingen), anti-CD27–Cy5
(DRFZ), and anti-CD38–fluorescein isothiocyanate (BD
PharMingen). Before staining, Fc receptors were blocked
using anti-CD16/anti-CD32 (Miltenyi). Single CD27⫺CD38⫺
naive B cells, CD27⫹CD38⫺ memory B cells, and
CD27highCD38⫹ plasma cells were sorted into 96-well plates,
and cDNA was synthesized using a Qiagen one-step RT–
polymerase chain reaction (PCR) kit. For amplification, primers specific for the constant-region genes of H- and L-chain
sequences (for CH1, C␮ 5⬘-GGGAATTCTCACAGGAGACGA,
C ␥ 5⬘-GGAAGGTGTGCACGCCGCTGGTC, and C␣
5⬘-TGGGAAGTTTCTGGCGGTCACG; for C␬, 5⬘-GTTTCTCGTAGTCTGCTTTGCTCA; and for C ␭ , 5⬘-CACCAGTGTGGCCTTGTTGGCTTG) together with primers specific for the different V-region genes (26) were used. Using the
CH1 C␥ primer, the heavy-chain isotypes IgG1 and IgG2 could
not be distinguished. In addition, using the CH1 C␣ primer, the
IgA1 and IgA2 isotypes were indistinguishable. Cells showing
positive reactions on PCR were purified using ExoSAP-IT
(USB).
Sequencing of the immunoglobulin V-region genes.
PCR products were cloned into the TOPO TA cloning vector
(Invitrogen). Randomly picked clones were sequenced with an
automated DNA sequencer (ABI Prism 310 Genetic Analyzer;
Applied Biosystems) using the BigDye Terminator Cycle Sequencing kit (version 1.1; Applied Biosystems). Putative germline gene sequences were identified using the V BASE Se-
66
SCHEEL ET AL
Table 3. Clonal diversity of sorted single synovial B cells
Patient, cell
population
Patient 4
Plasma cells
Patient 5
Naive B cells
Memory B cells
Plasma cells
Patient 6
Plasma cells
Patient 7
Naive B cells
Memory B cells
Plasma cells
Total
RESULTS
No. of
No. of
No. of
No. of
sorted cells VH sequences VL sequences clones
46
19
9
96
96
96
46
40
72
35
32
51
1
96
55
34
6*
96
96
96
718
31
30
60
353
19
21
36
237
7
* VH and VL gene sequences derived from clones P6-1 and P6-2
differed by somatic diversity.
quence Directory (provided by I. Tomlinson, Cambridge, UK).
The third complementarity-determining region (CDR3) length
of the VH region was determined according to the sequences
described by Kabat et al (27). The sequences can be found in
GenBank, under the accession numbers HM994882–
HM996558.
Statistical analysis. Statistical significance was calculated using the chi-square test (for differences in VH-gene
repertoire and in Ig-class distribution), the Mann-Whitney test
(for differences in numbers of somatic mutations), or Student’s
t-test (for differences in CDR3 length). P values less than 0.05
were considered significant.
Heterogeneous phenotype of the chronically inflamed synovial tissue of patients with RA. Synovial
tissue samples were obtained during surgery from 16
patients with established RA (Table 1). All samples were
assessed by immunohistologic staining, and only 7 of the
16 tissue samples were found to have substantial numbers of B and/or plasma cells. In samples P1 and P6, only
plasma cell aggregations were found, whereas in samples
P2–P5 and P7, large follicle-like structures surrounded
by plasma cells were also seen. However, none of the
characterized tissue samples showed evidence of ectopic
germinal centers. The synovial B cell immune response
was investigated in the synovial tissue of those 7 patients,
using 2 different approaches. In samples P1–P3, B cells
and plasma cells were isolated by LCM, while samples
P4–P7 were used to sort single naive B cells, memory B
cells, and/or plasma cells from synovial tissue cell suspensions (Tables 2 and 3).
A typical example of B and plasma cell distribution in the inflamed synovial tissue is shown in Figure 1
(for sample P2). B and plasma cells were separately
microdissected from the different locations. Plasma cells
associated with B cell infiltrates (Figure 1, arrowheads)
and plasma cells found in small aggregates along the
vasculature (Figure 1, arrows) were isolated, and the
VH-gene repertoire of the different cell populations was
determined.
Figure 1. Immunohistologic assessment of a representative synovial tissue sample from a patient
with rheumatoid arthritis (sample P2). Tissue sections were stained with hematoxylin, and plasma
cells (black staining) were labeled with the Wue-1 antibody. B cell infiltrates (Inf), associated
plasma cells (Ass) (arrowheads), and plasma cell aggregates (Agg) (arrows) were isolated by laser
capture microdissection. Insets, Higher-magnification views of the boxed areas. Bar ⫽ 100 ␮m;
original magnification of insets ⫻ 200.
CLONAL ACCUMULATION OF SYNOVIAL B AND PLASMA CELLS IN RA
Migration of naive and memory B cells into the
synovial tissue. The analysis of B cells microdissected
from the lymphocytic infiltrates showed that 55% of the
synovial B cells expressed IgM, while 29% expressed
IgG, and 16% expressed IgA (Figure 2A). Only one-half
of the IgM sequences were unmutated. Naive B cells are
defined as cells expressing unmutated immunoglobulins
of the IgM isotype. On this basis, only a minority (24%)
of the B cells in the lymphocytic infiltrates were of the
naive phenotype. Similarly, when naive and memory B
cells were sorted from synovial cell suspensions, again
only a minor fraction (⬍10%) of B cells were of the
naive phenotype (in samples P4, P5, and P7).
A different result was found in plasma cells, since
the analysis showed that practically all of the plasma
cells expressed IgG and IgA (Figure 2A). Overall, the
distribution of the Ig classes reflected the antibody levels
in the serum, as previously described (28,29). Only 9%
of the plasma cells expressed IgM, and with the exception of 2 sequences, all of them carried nucleotide
exchanges, suggesting that synovial plasma cells are
derived from memory B cells rather than from naive B
cells. The finding that, independent of their localization,
practically all synovial plasma cells expressed a memory
phenotype is evidence against the possibility of polyclonal activation of naive B cells and direct differentiation into antibody-secreting cells.
Somatic diversity of B and plasma cells. The
mean mutation frequency in B cells was 9.1 nucleotide
exchanges per mutated VH-gene segment. In plasma
cells, on average, a slightly higher mutation frequency
was found (Figure 2B). Sample P2 showed an average of
10.1 mutations per VH segment, while samples P1 and
P3 had average mutation frequencies of 17.1 and 17.8,
respectively. When the somatic diversity was compared
between sequences derived from plasma cells associated
with B cell infiltrates and sequences derived from
plasma cell aggregates, no significant difference was
seen (Figure 2B).
Expression of a selected VH-gene repertoire by
synovial plasma cells. A sequence analysis showed that
the VH-gene repertoire of synovial B cells was not
significantly different from that of peripheral blood B
cells in healthy donors (30) (Figure 2C). Both in naive B
cells and in memory B cells, the usage of VH-, D-, and
JH-gene segments and the length of the CDR3 (Figure
2D) were comparable.
In contrast, plasma cells differed from synovial B
cells in their VH-gene repertoire. An overrepresentation
of VH1 rearrangements and an underrepresentation of
VH4 rearrangements were observed in plasma cells
67
Figure 2. A–C, Sequence analysis of microdissected B and plasma
cells in synovial tissue from patients with rheumatoid arthritis, showing
A, the Ig class distribution, B, the number of somatic mutations, and C,
the VH-gene repertoire of B cell infiltrates (Inf) and plasma cell
subsets (associated plasma cells [Ass] and aggregates [Agg]). ⴱ ⫽ P ⬍
0.05. In C, the solid black bars indicate the VH-gene repertoire of
peripheral blood naive B cells (as reported in ref. 30). D, Length of the
third complementarity-determining region (CDR3) of the VH genes,
determined for all V–D–J rearrangements that were isolated (samples
P1–P3; shaded bars). In contrast, sequences isolated from plasma cell
aggregates in sample P3 (solid line) showed a different distribution of
the VH-CDR3 length. aa ⫽ amino acids.
(Figure 2C). The alterations in the VH-gene usage were
more pronounced in the plasma cell aggregates, with
differences reaching statistical significance (P ⬍ 0.001).
Evidence of selection was also seen in the analysis of the
CDR3 of plasma cell VH sequences. Although there was
68
SCHEEL ET AL
Figure 3. Sequence alignments (left) and schematic presentation of the sequence analysis (right) of microdissected B and plasma cells
in rheumatoid arthritis synovial tissue sample P2, showing clonal B cell expansion and V-gene diversification. Right, Only those V–D–J
rearrangements that were isolated repeatedly from independent polymerase chain reactions are depicted (●). Thin lines connect the
different locations in which sequences with an identical rearrangement were found (B cell infiltrates [Inf; white ovals], associated
plasma cells [Ass; grey ovals], and plasma cell aggregates [Agg; grey rectangles]). The indicated areas correspond to those described
in Figure 1. Left, Sequences from clones P2-1 and P2-2 (indicated by thick lines on the schematic), both of which showed intraclonal
diversity, are compared. All triplets differing from the nucleotide sequences of the putative VH-germline genes are shown (the third
complementarity-determining region [CDR3] is given in full length). The name of the sequence indicates the area (Agg1, Agg2, or
Agg4, and Inf1 or Inf2), the cap, and the sequence number. In addition, the Ig class is indicated. Numbering and position of the CDRs
were determined according to the sequences described by Kabat et al (27).
no significant difference in the usage of amino acids in
the CDR3 of B cells compared with that in the CDR3 of
plasma cells, the length of the CDR3 region was prolonged in plasma cell VH sequences of sample P3
(Figure 2D). These findings support the idea that
antigen-specific activation of B cells and selective accumulation of plasma cells are present in RA synovial
tissue.
Clonal expansion of activated synovial B cells. Ig
heavy-chain sequences are determined by a specific
V–D–J rearrangement and, hence, a unique CDR3. This
feature can be used as a clonal marker of B cell
development. The analysis of the VH-gene repertoire
showed that 10 V–D–J rearrangements (30.3%) in sample P1, 35 (18.6%) in sample P2, and 11 (6.4%) in
sample P3 were isolated repeatedly (Table 2). Clonally
related cells were found when B cells were dissected
from different areas of the same infiltrate and their
cDNA was amplified independently. They were also
seen when B and plasma cells were prepared from
separate locations, as shown by the schematic presentation of B cell expansion (Figure 3). Clonally related B
and plasma cells were found when plasma cells were
isolated from areas directly associated with a B cell
infiltrate (Inf1 and Ass1 and Inf2 and Ass2) (Figure 3).
In addition, clonally related cells were detected in
separate and distinct plasma cell aggregates (Inf1 and
Agg4). In some areas, plasma cell aggregates were in the
direct neighborhood of the B cell infiltrate (Inf1 and
Agg2). However, B and plasma cells with an identical
V–D–J rearrangement were also detected in aggregates
that were more than 1 mm apart from the B cell
infiltrate (Inf1 and Agg1 and Agg3) (Figures 1 and 3).
These findings show that newly generated plasma cells
have the capacity to migrate deeply into the synovial
tissue, survive, and thus accumulate at these sites.
Evidence of intensive clonal expansion was also
seen in sample P1, in which only plasma cells were
detected. Microdissection showed that within one cluster
of plasma cells, multiple cells with an identical V–D–J
rearrangement were found, and similar to that observed
in sample P2, descendents of the same B cell clone were
detected in areas far apart from each other. One such
V–D–J rearrangement was isolated from 3 different
plasma cell aggregates.
In addition, single naive B cells, memory B cells,
and plasma cells were sorted from synovial cell suspensions, and the V-gene repertoire was determined. In
CLONAL ACCUMULATION OF SYNOVIAL B AND PLASMA CELLS IN RA
samples P5 and P7, no identical V–D–J rearrangements
were seen when naive and memory B cells were compared, and none of the plasma cells showed an identical
rearrangement (Table 3). However, within the plasma
cell population, clonally related cells were detected. Two
plasma cells in sample P7 did express the same V–D–J
rearrangement.
Even more surprising was the result obtained in
sample P6. In this patient, ⬃100 pieces of synovial tissue
were obtained during wrist surgery. After the tissue was
digested, only 96 plasma cells were randomly sorted
from the cell suspension, and therefore the chance to
find clonally related cells was extremely low. Nevertheless, within this small group of plasma cells, 6 V–D–J
rearrangements were isolated repeatedly. Sequences belonging to clone P6-1 and also sequences belonging to
clone P6-2 were both isolated from 4 single sorted
plasma cells (Table 3). In summary, using 2 different
approaches, we obtained evidence of intensive B cell
activation and accumulation of large plasma cell clones
in RA synovial tissue.
Switching of immunoglobulin class and intraclonal
diversity. In general, sequences with an identical V–D–J
rearrangement did not differ in their Ig class. Evidence
of Ig class switching was found in only a few clones
isolated from sample P2 (Figure 3) and sample P3. In
the majority of cases, a switch to IgG1/2 and IgG4 was
evident. However, class switching to IgG1/2 and IgA was
also documented, as was that from IgM to IgG3 and
IgG1/2. In addition, in most cases, sequences with an
identical rearrangement showed an identical pattern of
somatic mutations. In only 7 (11%) of 63 isolated clones
was there evidence of somatic diversification, suggesting
that B cell activation in synovial infiltrates does not, by
and large, induce hypermutation. Somatic diversification
seems to require the microenvironment of the germinal
center.
To some extent, intraclonal diversity was seen in
sequences from sample P2 (Figure 3). Cells belonging to
clone P2-1 were found in 3 of 6 plasma cell aggregates
analyzed (Figure 3, indicated by bold line connecting
Agg1, Agg2, and Agg4). A comparison with the putative
germline gene VH1-2 showed that these V-region sequences had only 6 nucleotide exchanges in common but
differed by up to 20 somatic mutations (as shown by the
upper sequence alignment in Figure 3). Two sequences
with an identical V-region were isolated as IgG1/2 and
IgG4 (Figure 3). Similarly, cells belonging to clone P2-2
were isolated from 2 separate B cell infiltrates and, in
addition, from an area of plasma cells (Figure 3, indicated by bold line connecting Inf1, Inf2, and Agg4). The
69
B cell–derived sequences differed by up to 9 somatic
mutations from the putative germline V-region gene
VH1-69 (as shown by the lower sequence alignment in
Figure 3). In contrast, one plasma cell sequence
(Agg1.4.1 in clone P2-2) was identical to one of the B
cell sequences, although it differed in Ig class. Similar
results were demonstrated in sample P3.
Long-term reactivation of memory B cells. Extreme intraclonal diversity in the V-gene repertoire of
single plasma cells was found in sample P6 (supplementary results available from the corresponding author at
http://www.drfz.de/index.php?id⫽848). Altogether, in
this sample, 96 plasma cells were sorted, and 55 VH
sequences, 33V␬ sequences, and 1 V␭ sequence were
determined (Table 3). The sequence analysis showed
that 55 of the plasma cells analyzed could be grouped
into 44 independent clones. Although identical V–D–J
sequences were found repeatedly, the possibility of
contamination was excluded, because identical rearrangements were seen for both VH and V␬ genes, and because
the sequences differed substantially in their pattern of
somatic mutations (supplementary results available
from the corresponding author at http://www.drfz.de/
index.php?id⫽848). Thus, VH regions from clones P6-1
and P6-2 differed by more than 70 nucleotides from the
respective germline gene sequences of VH4.39 and VH349. Similarly, the V␬ sequences differed by 23–52 nucleotides from the respective germline sequences.
Only a continuous activation of B cells and
generation of plasma cells over a long period of time
could explain the accumulation of these exceedingly high
numbers of somatic mutations in the V-region genes.
The intensive somatic diversification of the underlying B
cell clones may have taken place in the peripheral
secondary lymphoid organs. It is also possible that these
large plasma cell clones were derived from previous
ectopic germinal center reactions.
DISCUSSION
Using 2 different approaches, B and plasma cells
were isolated from synovial tissue samples, and their
V-gene repertoire was determined. Both the LCM protocol and the single cell sorting protocol revealed an
intensive expansion of selected B cell clones within RA
synovial tissue. With the LCM technique, which has the
advantage that cells of defined localization can be
isolated, we directly demonstrated activation of B cells,
their local differentiation into plasma cells, and accumulation of plasma cells within the inflamed synovial tissue
over time.
70
Both techniques showed the presence of naive
and memory B cells in synovial lymphocytic infiltrates.
Proportions of ⬃25% naive B cells and ⬃75% memory
B cells were found, which supports the idea that there is
a specific recruitment of memory B cells into the
synovial tissue. Enhanced responsiveness to inflammatory chemokines may favor migration of memory B cells
(31,32). In the synovial tissue, memory B cells are more
likely to be activated by local self antigens, which thus
allows them to receive signals that promote their maintenance within the synovial tissue. In addition to selective migration, there may be a preferential expansion of
memory B cells, an interpretation that was supported by
the finding of clonally related memory B cells within the
synovial infiltrates (Figure 3).
B and plasma cells with an identical rearrangement and an identical pattern of somatic mutations were
found not only in plasma cells in close association with
infiltrates but also in plasma cells isolated from independent aggregates. In some cases, clonally related plasma
cells were microdissected from areas more than 1 mm
apart, which suggests that within the inflamed synovial
tissue, the locally generated plasmablasts and/or mature
plasma cells have a high migratory capacity. Thus,
accumulation of plasma cells within the synovial tissue is
supported by migration of plasmablasts from the peripheral lymphoid organs and, in addition, by generation
of new plasma cells within the inflamed synovial tissue.
The comparison of the VH-gene repertoire of B and
plasma cells suggested that there is an antigen-dependent
activation of synovial B cells and an antigen-selected
differentiation into plasma cells in RA synovial tissue.
This interpretation is in line with recent evidence showing that memory B cells are not activated by bystander
inflammatory signals, and furthermore, in vivo experiments have shown that self-renewal and differentiation of
memory B cells requires antigen-dependent signals (33).
In some synovial tissue, chronic inflammation
supports the development of organized lymphoid structures and allows the formation of germinal centers. The
analysis of germinal center B cells microdissected from
synovial tissue samples showed B cell proliferation and
somatic diversification of the V-gene repertoire in these
structures (5,19,20). In the present study, however, tissue
samples that did not show evidence of ectopic germinal
center formation were analyzed, and in general, sequences with an identical V–D–J rearrangement showed
the same pattern of somatic mutations, even when the
V-region gene was joined to a different C-region gene.
In a single infiltrate, only rarely were we able to isolate
the sequences, such as Inf1.3.1 and Inf1.5.1 from clone
SCHEEL ET AL
P2-2, that showed intraclonal diversity (Figure 3). These
data suggest that in synovial tissue without ectopic
germinal centers, B cells will not accumulate substantial
somatic mutations, as has been found in murine models
of RA (34). Indeed, the few sequences with different
patterns of somatic mutations may result from germinal
center reactions in the peripheral lymphoid organs or
from previous, no longer detectable, synovial germinal
centers.
Nevertheless, although there was little evidence
of B cell proliferation in the absence of synovial germinal center structures, it is possible that there are short
phases in which B cells intensively proliferate and differentiate into plasma cells. If the synovial tissue supports long-term plasma cell survival, then one would
expect accumulation of these cells over time. This interpretation is supported by the fact that the frequency of
clonally related cells was much higher within the plasma
cell population than in memory B cells. Furthermore, a
comparison of Ig classes expressed in clonally related B
and plasma cells indicated that the plasma cells that
were present at the time point of the analysis may have
been the result of previous phases of B cell activation.
With knowledge of the organization of the CH-region
genes, one would expect cells expressing IgG1 or IgG2
to switch to IgG4 and then to IgA. The finding of plasma
cells expressing IgG1 and the corresponding B cells
expressing IgG4 supports the notion that plasma cells
have not been generated only recently but rather have
accumulated over a long period of time. In synovial
tissue without B cells, such as RA synovial tissue samples
P1 and P6, plasma cells may be generated within the
synovial tissue and may reflect previous phases of synovial B cell activation.
The high degree of somatic diversity found in
plasma cells supports the idea that there is a continuous
activation of selected B cell clones over a long period of
time. It would be important to identify the specificity of
these clones and to determine whether autoantigens are
responsible for the continuous activation of synovial B
cells and their differentiation into plasma cells. The
method of sorting single cells has the advantage in that
VH and VL sequences from individual B and plasma cells
can be cloned into expression vectors, and thus recombinant antibodies can be generated. Preliminary results
show that the highly mutated plasma cell clones P6-1
and P6-2 are not rheumatoid factors and do not have
specificity for citrullinated peptide antigens. However,
these B cells may have lost their autospecificity by the
accumulation of excessive numbers of somatic mutations.
CLONAL ACCUMULATION OF SYNOVIAL B AND PLASMA CELLS IN RA
When the sequences belonging to clones P6-1
and P6-2 were analyzed, it was found that the frequency
of somatic mutations in the V-region genes was of the
same order of magnitude as that seen in highly mutated
IgD sequences isolated from tonsillar germinal center B
cells. It has been suggested that these hypermutated IgD
sequences are unable to mature into circulating memory
B cells (35). In our study, in contrast, we found that
hypermutated B cells had differentiated into plasma
cells, whose V-region amino acid sequences showed less
than 60% homology to the corresponding germline
sequence. Thus, the chronic activation of the immune
system in patients with RA might be seen as a consequence of B cell activation and differentiation of these
cells into plasma cells. B cell depletion is known to
interrupt the continuous generation of new plasma cells
and hence prevents their accumulation in the synovial
tissue. Therefore, the presence of large B cell infiltrates
and/or high frequencies of synovial plasma cells could
thus define a profile that would identify those patients
who might benefit from therapy that targets the B cell
compartment.
6.
7.
8.
9.
10.
11.
12.
13.
14.
ACKNOWLEDGMENTS
We are grateful to T. Kaiser, G. Steinhauser, and S.
Schürer for providing technical support. We also thank R. S.
Jack for critical reading of the manuscript.
15.
16.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Berek had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Scheel, Häupl, Berek.
Acquisition of data. Scheel, Gursche, Zacher, Häupl.
Analysis and interpretation of data. Scheel, Berek.
17.
18.
19.
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regions, reveal, locally, cells, expansion, defined, selected, arthritis, analysis, genes, synovial, plasma, clones, rheumatoid, accumulation
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