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. Author manuscript; available in PMC: 2015 Jun 24.
Published in final edited form as: Arthritis Rheum. 2013 Apr;65(4):1085–1096. doi: 10.1002/art.37828

Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome associated lymphoproliferation

D Saadoun 1,2,*, B Terrier 1,*, J Bannock 3, T Vazquez 1, C Massad 3, I Kang 4, F Joly 5, M Rosenzwajg 1, D Sene 2, P Benech 5, D Klatzmann 1, E Meffre 3,†, P Cacoub 1,2,†
PMCID: PMC4479193  NIHMSID: NIHMS503142  PMID: 23279883

Abstract

Background

Primary Sjögren's syndrome (pSS) is the autoimmune disease associated with the higher risk of developing non-Hodgkin lymphoma.

Objective

To determine the nature of B cells driving lymphoproliferation in pSS.

Methods

B cell subsets and function were analyzed in peripheral blood from 66 adult patients with pSS [including 14 patients with B-cell lymphoproliferative disorder (LPD)] and 30 healthy donors, using flow cytometry, calcium mobilization, and gene array analysis. We tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from pSS-LPD.

Results

We report here the expansion of an unusual CD21-/low B-cell population which correlates with lymphoproliferation in pSS patients. A majority of CD21–/low B cells from pSS patients expressed autoreactive antibodies, which recognized nuclear and cytoplasmic structures. These B cells belonged to the memory compartment because their immunoglobulin genes were mutated. They were unable to induce calcium flux, become activated, or proliferate in response to B-cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. However, CD21–/low B cells from pSS remained responsive to TLR stimulation. Gene array analyses of CD21–/low B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage.

Conclusion

pSS patients who display high frequencies of autoreactive and unresponsive CD21-/low B cells are susceptible for developing lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.

Keywords: Sjögren's syndrome, CD21-/low B cells, lymphoproliferation, autoimmunity, anergy

Introduction

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease primarily characterized by chronic inflammation of the exocrine glands, in particular the salivary and lachrymal glands. Extraglandular manifestations occur in many patients and may involve almost any organ. B-lymphocyte hyperactivity in pSS is manifested by the presence of anti-SS-A and anti-SS-B antibodies, rheumatoid factor, cryoglobulins, and hypergammaglobulinemia. The threat that hangs over patients with pSS is the development of a lymphoma. Unfortunately, we have thus far no sufficient data to deal with such pertinent concerns. Prolonged B-cell survival and excessive B-cell activity, probably related to increased production of B-cell activating factor (BAFF) (1-3), may lead to lymphomas occurring in 5% of Sjögren's syndrome (SS) patients (4, 5). Significant predictors of lymphoproliferative disease in pSS include parotid, lymph node and/or splenic enlargement, monoclonal gammapathy, hypogammaglobulinemia, mixed cryoglobulinemia, palpable purpura, CD4+ T cell lymphopenia and/or reduced levels of C4 (6-9).

It is proposed that the first event of lymphomagenesis in Sjögren's syndrome is the chronic stimulation of polyclonal B cells secreting autoreactive antibodies, such as rheumatoid factor. Such autoreactive B cells may become monoclonal, leading to the occurrence of lymphoproliferations. The following step would be a chromosomal abnormality, which would confer to these cells low grade B cell lymphoma comportment (10). The non-random utilization of VH and VL by Sjögren's syndrome associated lymphoma B cells (11, 12) and the demonstration that these lymphoma B cells may display rheumatoid factor activity (13) support the hypothesis that these lymphomas grow through an auto-antigen driven process.

We report here that an unusual CD21-/low B cell population correlates with the lymphoproliferative status in pSS patients. Because CD21 augments B-cell receptor (BCR)-mediated signaling as part of the B-cell coreceptor complex, its down-regulation may confer a state of anergy to these cells, as has been demonstrated among CD21-/low B cells in patients with rheumatoid arthritis (RA), common variable immunodeficiency (CVID) or hepatitis C associated cryoglobulinemia patients (14-16). These CD21-/low B cells are enriched in autoreactive clones that are unresponsive to BCR stimulation, suggesting that these cells are controlled by the tolerizing mechanism of functional anergy. Gene array analyses of CD21–/low B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. These B cells belonged to the memory compartment because their immunoglobulin genes were mutated as reported in hepatitis C associated cryoglobulinemia patients (15, 16). Taken together, our data suggest that the induction of an unresponsive program in autoreactive B cells may represent risks to develop B cell lymphoproliferation in pSS.

Materials and methods

Study subjects

We recruited 66 patients with primary Sjögren syndrome (pSS) according to the European-American consensus criteria (17) (59 women and 7 men; mean ± SD age: 55.9 ± 14.5 years) including 14 patients with B-cell lymphoproliferative disorder (LPD). B-cell lymphoproliferation was defined by an overt B-cell non-Hodgkin lymphoma (B-NHL) according to the WHO classification (18) or as the presence of type II mixed cryoglobulinemia (7, 8) sometimes associated with lymph node or splenic enlargement. Seven patients had B-NHL including 4 marginal zone lymphomas, 1 lymphocytic and 2 undetermined. Among the 7 remaining patients with type II mixed cryoglobulinemia, 2 had lymph node enlargement and 2 had splenomegaly. Blood samples from 30 healthy donors (HD) were obtained from Etablissement Français du Sang (Hôpital Pitié-Salpêtrière). The study was performed according to the Declaration of Helsinki. All study subjects provided informed consent, in accordance with the Institutional Review Board.

Phenotypic analysis

Peripheral blood mononuclear cells (PBMCs) were obtained by density-gradient centrifugation. Phenotypic analyses were performed with anti–human monoclonal antibodies: anti–human CD10, C19, CD20, CD21, CD22, CD44, CD69, CD86, HLA-DR, IgM, and Annexin V were obtained from Beckman Coulter; anti–human CD1c, CD25, CD27, CD38, CD84, CD95, BR3, Kappa light chain and IgD were obtained from BD Biosciences. FACS analyses were performed on a Navios flow cytometer using CXP analysis software (Beckman Coulter). CD19+ B lymphocytes counts (cells/μl) were established from fresh blood samples using CYTO-STAT tetraCHROME kits with Flowcount fluorescents beads as an internal standard and tetra CXP software with a Navios cytometer according to the manufacturer's instructions (Beckman Coulter, Villepinte, France).

B cell enrichment and cell sorting

CD27- B cells were enriched from total PBMCs by negative magnetic bead selection using a cocktail of biotinylated antibodies against CD2, CD14, CD16, CD23, CD27, CD36, CD43 and Glycophorin A, followed by anti-Biotin MicroBeads (Naïve B cell isolation kit II, Miltenyi Biotec, Paris). The purity of CD27- B cells was typically >95%. For proliferation assays, CD27- B cells were separated into CD21+ and CD21-/low fractions using a one-step magnetic bead-based selection process. Cells were fractionated by CD21 with anti-CD21-PE (BD Biosciences), followed by anti-PE Microbeads (Miltenyi Biotec). The purity of CD21+ and CD21-/low fractions was typically >85%.

For gene expression profile and single cell sorting, PBMCs were sorted by staining the cells with monoclonal anti-human antibodies against CD45-VioBlue, anti-CD27-PE (Miltenyi Biotec), CD21-FITC, CD19-ECD, and CD10-APC (Beckman Coulter). For single-cell PCR, CD10-CD27-CD21-/lowCD19+ B cells from pSS patients were sorted on a FACSAria (Becton Dickinson) into 96-well PCR plates.

B cell activation, survival and proliferation

Enriched CD27- B cells, which contained CD21+ and CD21-/low B cells from pSS patients, were plated at 500,000 cells per well in a 48-well plate in RPMI 10% serum and 20 μg/mL polyclonal F(ab)'2 goat anti-human IgM (Jackson Immunoresearch), 1 μg/mL anti-human CD40 (Invitrogen), and/or 1 μg/mL CpG (Invivogen) for 48 hours. To assess B cell activation and survival, cells were stained with anti–human monoclonal antibodies: CD19, CD21, CD25, CD27, CD40, CD69 and CD95. The proportions of apoptotic and dead cells were assessed by flow cytometry to measure binding with annexin V using Annexin V-PE and 7-AAD (BD Pharmingen). To assess B cell proliferation, CD21+ and CD21-/low MZ B cells were plated at 1 × 105 cells per well of a 96-well flat-bottom plate with various combinations of the following reagents: 20 μg/mL polyclonal F(ab)'2 goat anti-human IgM; 1 μg/mL anti-CD40; 1 μg/mL CpG; 1 μg/mL of TLR3 agonist (name, company?) and 1 μg/mL of TLR7 agonist (name, company?). Cells were incubated for 48 hours and pulsed for 8 hours with tritiated thymidine.

Calcium Mobilization

Cells were resuspended in RPMI 1640 (GIBCO, Invitrogen) and stained with anti-CD21-PE, anti-CD19-ECD (Beckman Coulter), and anti-CD27-APC (BD Biosciences) antibodies for 30 min at 4°C. Cells were washed twice in RPMI and resuspended at 1×106 cells/mL. Cells were loaded with Fluo-4 AM (Invitrogen) at a final concentration of 5μM in the presence of 0.2% Pluronic F-127 (Sigma) for 30 min at room temperature. Cells were washed twice in RPMI-SVF 5% and resuspended at 1×106 cells/mL. [Ca2+]i was monitored over time by flow cytometry on gated CD19+CD27-CD21+ and CD19+CD27-CD21-/low B cells. Baselines were read for 30 seconds, after which the cells were removed and stimulated with 20 μg/mL of F(ab)'2 anti-IgM (Jackson ImmunoResearch Laboratories) then Ionomycin at 1mg/mL (Sigma-Aldrich).

Antibody production, ELISAs, and Immunofluorescence assays

Cloning strategy, expression vectors, and antibody reactivity against specific antigens were as described (19). Highly polyreactive ED38 was used as positive control in HEp-2 reactivity and polyreactivity enzyme-linked immunosorbent assays (ELISAs) (19). Antibodies were considered polyreactive when they recognized at least 2 and usually all of the 3 analyzed antigens that include double-stranded DNA (dsDNA), insulin, and lipopolysaccharide (LPS). All recombinant antibodies were also tested for rheumatoid factor reactivity (anti-IgG) as previously described (20). For indirect immunofluorescence assays, HEp-2 cell–coated slides (Bion Enterprises Ltd) were incubated in a moist chamber at room temperature with purified recombinant antibodies at 50 to 100 μg/mL. FITC-conjugated goat anti–human IgG was used as detection reagent. Pictures were taken with an Axioskop (Zeiss) using a Plan-Neofluar 40×/.75 objective and Axiovision 3.1 acquisition software.

Microarray gene expression profile analysis

RNA was extracted from 105-3.105 batch-sorted conventional IgM+CD27-CD21+CD19+ B cells and IgM+CD27-CD21-/lowCD19+ B cells using the NucleoSpin RNA II kit (Macherey-Nagel, Hoerd, France). Each sample contained 50-200 ng of RNA, and the quality of the purified RNA was assessed with the 2100 Bioanalyzer from Agilent. Using the Illumina TotalPrep RNA Amplification Kit (Applied Biosystems), 50 ng of RNA was amplified and labeled to produce cDNA. Labeled cDNA was hybridized on whole human genome chips (HumanHT-12 v3 Expression BeadChip Kit, Illumina).

PredictSearch™, a powerful bioinformatic solution dedicated to identifying relevant correlations between genes and concepts, was used to generate functional networks. This tool is updated daily with the whole NCBI Pubmed database and seeks relevant correlations between gene-gene or gene-concept within abstracts. It also enables an oriented search for co-citations between genes with action words such as “activation” or “repression” and their semantic variants. PredictSearch™ software was also implemented with the whole set (>18000) of transcriptional signatures deposited in the NCBI GEO database and extracted with the DBF-MCL algorithm using TranscriptomeBrowser tool (21). Furthermore PredictSearch™ provides direct access to already published gene pathways.

Statistical analyses

Data are expressed as the mean plus or minus standard deviation (SD) or median. Categorical variables were compared using the Fisher's exact or Chi-square tests, and continuous variables were compared using the t-test or Mann-Whitney U test when appropriate. All tests were 2-sided at a 0.05 significance level. Graphing and statistical analyses were performed using Prism software (GraphPad Software, Inc.).

Results

pSS patients display an unusual expansion of peripheral CD27-CD21-/low B cells

We first examined B cell subpopulations in pSS patients. We found an increase of the CD27-B cell subset in pSS and pSS-LPD patients compared to HD (78.1% and 76.8% vs. 64.6% of cells among CD19+ B cell subset, P<0.001 and P<0.01 respectively) (Figures 1A and 1B) but not in absolute number in which CD27- B cells were decreased in pSS and pSS-LPD (108.4 and 96.4 cells/μL vs. 129.2 cells/μL, P<0.0001 for both) (Figures 1A and 1B). The analysis of CD21 expression on B cells from pSS and pSS-LPD patients and HD showed an increased of CD27-CD21-/low B cells both in percentage-wise (medians) and in absolute numbers in pSS and pSS-LPD patients compared to HD (8.7% and 25.9% vs. 3.1% of cells among CD19+ B cell subset, P<0.0001 for both; and 12.8 and 42.7 cells/μL vs. 6.2 cells/μL, P<0.0001 for both) (Figures 1C and 1D).

Figure 1. Expansion of an unusual CD27-CD21-/low B cell population in pSS patients.

Figure 1

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(A, B) Increased proportion of CD27- B cells in pSS patients compared with HD. (C, D) Increased proportion of CD27-CD21-/low B cells in pSS-LPD patients compared with pSS and HD (data are represented as median values in figure 1 D). (E, F) CD27-CD21-/low B cells (gate II) display decreased expression of IgD and IgM compared to CD27-CD21+ B cells (gate I). (G) CD27-CD21-/low cells display a unique B cell phenotype. Cells were gated on CD27-CD19+ B cells in order to delineate phenotype differences between CD21-/low and CD21+ CD27- B cell populations for B cell markers (CD19, CD20, BAFF-R, Kappa light chain), maturity markers (CD10, CD38, CD22, CD40) and activation/proliferation markers (CD69, CD86, CD95, HLA-DR).

* P<0.05, ** P<0.01, *** P<0.001.

CD27-CD21-/low B cells were significantly higher in pSS-LPD patients compared to pSS patients without LPD (P<0.001). The expression of IgD and IgM was analyzed on CD27-CD21+ and CD27-CD21-/low B cells from pSS patients. CD27-CD21-/low B cells displayed decreased expression of IgD and IgM compared to conventional CD27-CD21+ B cells. While CD27-CD21-/low B cells comprised IgD+ and IgD- cells, 92.5% of cells were positive for surface IgM (Figure 1E). CD27-CD21-/low B cells expressed similar MFI intensity of IgD than CD27-CD21+ B cells. The proportion of IgD+ cells was lower in CD27-CD21-low cells as compared to CD27-CD21+ B cells (Figure 1E). In addition, CD27-CD21-/low B cells expressed lower levels of surface IgM (MFI 27.9 vs. 46.6, P<0.001) when compared with CD27-CD21+ B cells from the same patients (Figure 1F). Four pSS-LPD patients had a low C4 complement level but there were no correlation between CD27-CD21-/low B cells expansion and complement deficiency. We conclude that CD27-CD21-/low B cells are expanded in pSS patients, especially those displaying lymphoproliferation features.

CD27-CD21-/low cells display a unique B cell phenotype

In order to provide a more precise definition of the phenotype of this expanded population, CD27-CD21-/low B cells were screened by flow cytometry for characteristic B cell markers, maturity markers and activation/proliferation markers, allowing a comparison with conventional CD27-CD21+ B cells from the same patients. CD27-CD21-/low B cells expressed higher levels of CD19 and CD20 when compared with CD27-CD21+ B cells from the same patients, whereas they expressed lower levels of BAFF receptor (BAFF-R) and κ light-chain (Figure 1G). CD27-CD21-/low B cells did not express markers found on immature B cells such as the B cell progenitor marker CD10 and, like mature cells, expressed only low levels of the development marker CD38. These markers are commonly used to distinguish new emigrants immature/transitional B cells (22). Furthermore, the CD27-CD21-/low and conventional CD27-CD21+ B cell populations were both positive for the BCR-associated regulator CD22 that is expressed on mature B cells (23), and for other molecules found only on mature circulating B cells including CD40 and CD44. Lastly, analysis of CD27-CD21-/low B cells for signs of recent activation in vivo demonstrated an increased expression of CD69, CD86 and CD95 compared to conventional CD27-CD21+ B cells of the same patients (Figure 1G). Furthermore, CD27-CD21-/low B cells were not proliferating as assessed by the Ki67 expression. Hence, CD27-CD21-/low B cells show a midly activated B cell phenotype potentially associated to chronic stimulation.

CD21-/low B cells in pSS express highly autoreactive antibodies

CD27-CD21-/low B cells with a similar phenotype have been reported in several autoimmune.diseases (14, 15, 24, 25). CD27-CD21-/low B cells from RA and CVID patients expressed mostly unmutated IgMs whereas those from patients with hepatitis C virus associated with mixed cryoglobulinemia harbor mutated Ig genes (14-16). To assess if CD27-CD21-/low B cells from pSS patients belong to the naïve or memory B cell fraction, we analyzed the immunoglobulin repertoire and reactivity of these B cells using a single B cell cloning approach (19). We found that most CD27-CD21-/low B cells from 4 pSS patients expressed diverse IgM antibodies, the majority of which were mutated suggesting that these B cells belonged to the memory compartment (Tables S1, S2, S3 and S4). In addition, somatic hypermutations were mostly located in Ig CDRs as in conventional memory B cells, suggesting that CD27-CD21-/low B cells were antigen selected (Supplemental Figure 1) (Reference Wardemann, Immunity IgG reactivity and SHM in CDR3s). Many CD27-CD21-/low B cells expressed HEp-2-reactive and polyreactive antibodies (Figure 2, A and B), including some with rheumatoid factor (anti-IgG) reactivity (Figure 2 C). CD27-CD21-/low B cells often expressed antibodies recognizing cytoplasmic and to a lesser extent nuclear structures with diverse staining patterns (Figure 2, D and E). We conclude that CD27-CD21-/low B cells from pSS patients are enriched in autoreactive B cell clones that may have been selected by self-antigens..

Figure 2. CD21-/low B cells in pSS express highly autoreactive antibodies.

Figure 2

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(A) Antibodies from CD27-CD21-/low B cells from four pSS-LPD patients were tested by ELISA for anti-HEp-2 cell reactivity (A) and against double-stranded DNA (dsDNA), insulin and lipopolysaccharide (LPS) (B) and for rheumatoid factor (anti-IgG) reactivity (C). CD27-CD21-/low B cells often expressed antibodies recognizing cytoplasmic and to a lesser extent nuclear structures (D and E).

CD27-CD21-/low B cells reveal defects in BCR and CD40-mediated activation

We analyzed the function of CD27-CD21–/low B cells after BCR (using F(ab)'2 anti-IgM), CD40 (using CD40L) and/or TLR9 (using CpG) triggering by studying the expression of CD25, CD69, CD40 and CD95 on CD27-CD21–/low and conventional CD27-CD21+ B cells from pSS patients (Figure 3). BCR, CD40, and/or TLR3, TLR7 and TLR9 triggering induced the expression of CD25, CD69, CD40 on CD27-CD21+ B cells from pSS patients, but only resulted in a slight expression of CD95. In contrast, BCR and/or CD40 triggering of CD27-CD21–/low B cells from the same patients failed to properly induce the expression of CD25, CD69 and CD40, while the use of an alternative pathway through TLR3, TLR7 and TLR9 triggering strongly induced the expression of CD25 and CD69 compared with CD27-CD21+ B cells. In addition, CD27-CD21–/low B cells exhibited increased CD95 expression after BCR, CD40, or TLR3, TLR7 and TLR9 stimulation compared with CD27-CD21+ B cells. Thus, CD27-CD21–/low and CD27-CD21+ B cells display distinct B-cell responses after stimulation, and CD27-CD21-/low B cells reveal defects in BCR and CD40-mediated activation but not after TLR triggering (Figure 3).

Figure 3. CD27-CD21-/low B cells reveal defects in BCR and CD40-mediated activation.

Figure 3

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CD21-/low CD27- B cells failed to up-regulate CD69 (A), CD25 (B) and CD40 (C) expression after stimulation with F(ab′2) anti-IgM and/or recombinant human CD40L but not after stimulation with with TLR3 and TLR7 agonists and/or TLR9 agonist CpG; they also exhibited an increased CD95 expression compared with CD21+ CD27- B cells (D). Data are representative of at least four independent experiments. * P<0.05 compared to stimulation wit anti-IgM and/or CD40L.

We next analyzed the ability of CD27-CD21–/low B cells upon BCR stimulation to induce intracellular calcium flux compared with conventional CD27-CD21+ B cells. In contrast to CD27-CD21+ B cells from pSS patients, BCR triggering induced only a weak elevation of intracellular calcium concentration in response to BCR stimulation in CD27-CD21-/low B cells. Hence, CD27-CD21-/low B cells do not exhibit proper calcium flux after BCR stimulation (Figure 4).

Figure 4. CD21-/low B cells fail to increase [Ca2+]i in response to BCR triggering.

Figure 4

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(A, B) CD21-/low B cells had attenuated calcium responses compared with CD21+ B cells after BCR cross-linking. (C) Slightly higher amount of mean fluorescence of Fluo-4 in the CD21-/low B cell population. (D) Higher increase of maximal normalized peak fluorescence of Fluo-4 in the CD21+ B cell population. Data are representative of five independent experiments. * P<0.05, ** P<0.01.

CD27-CD21-/low B cells are prone to dying by apoptosis and do not proliferate after BCR and CD40 triggering

We assessed apoptosis of CD27-CD21-/low B cells by analyzing annexin V and 7AAD staining. We found that freshly isolated CD27-CD21-/low B cells from pSS patients contained a higher frequency of annexin V+7AAD- early apoptotic cells and annexin V+7AAD+ late apoptotic and necrotic cells compared with conventional CD27-CD21+ B cells (Supplemental Figure 2A and 2B). Compared with medium, BCR, CD40 and/or TLR9 triggering was not able to rescue CD27-CD21-/low B cells from apoptosis. In contrast, stimulated CD27-CD21+ B cells contained lower levels of annexin V+ expressing cells (Supplemental Figure 2A and 2B). Thus, CD27-CD21-/low B cells are more susceptible to apoptosis than CD27-CD21+ B cells and are poorly rescued by BCR, CD40 and/or TLR9 triggering. We next studied the proliferation induced by BCR and TLR triggering using the incorporation of tritiated thymidine. CD27-CD21-/low B cells showed decreased cell proliferation compared with CD27-CD21+ B cells, mainly after BCR triggering and to a lesser extent after TLR9 stimulation (Supplemental Figure 2C). Thus, CD27-CD21-/low B cells exhibit increased apoptosis and decreased proliferation after stimulation, features that are commonly associated with an anergic phenotype.

Gene expression profiles of CD27-CD21-/low B cells in pSS patients reveal the selective up-regulation of an anergic pathway

In order to define gene signatures associated with expansion, phenotype and function of CD27-CD21–/low B cells, we compared gene expression profiles expressed in CD27-CD21–/low B cells versus conventional CD27-CD21+ B cells isolated from 4 pSS patients (Figure 5A). A total of 2510 transcripts were found to be differentially expressed between the two CD27- B-cell subpopulations, including 1934 up-regulated (> 1.5-fold change) and 576 down-regulated transcripts (< 0.7-fold change) in CD27-CD21–/low B cells. Focusing on the transcriptional modulations that discriminate CD27-CD21–/low from CD27-CD21+ B cells in the same patients, we identified transcriptional signatures that are likely to underlie the functional properties of these cells. Hierarchical clustering of differentially expressed genes between CD27-CD21–/low and CD27-CD21+ B cells highlighted an anergy-related transcriptional signature that is specifically up-regulated in CD27-CD21–/low B cells (Figures 5A and 5B). The gene content of this transcriptional module is enriched in genes encoding cell surface and intracellular proteins that are involved in signal transduction and in T cell co-stimulation, such as CD83, CD86/B7-2 and CD72. CBL-B, which is a potent negative regulators of intracellular signaling, is also strongly induced in the CD27-CD21–/low versus CD27-CD21+ B cells. CD72 was shown to function as a regulator of peripheral B cell tolerance, by regulating and being associated with CBL-B, leading to the down-regulation of BCR signaling. Similarly, CD27-CD21–/low B cells up-regulated the transcription of a whole set of immunoreceptor tyrosine–based inhibition motif (ITIM) receptor genes, which are likely to inhibit B-cell activation and proliferation, including CD72, SIGLEC, CD22 and FCRL2 genes. We also found a selective up-regulation of FCRL3, which was suggested to play a pivotal role in autoimmunity. Finally, up-regulation of RFNT1/Raftlin, the B cell-specific major raft protein that is necessary for the integrity of lipid rafts and BCR signal transduction, further highlights the related functional involvement of the genes of this transcriptional signature. Through flow cytometry, we confirmed at the protein level that CD27-CD21–/low B cells up-regulated FCRL2, FCRL3, CD72, CD22, CD11c, and down-regulated CD1c (Figure 6). Taken together, transcriptomic analysis of CD27-CD21–/low B cells highlights the selective up-regulation of genes that belong to an inhibitory pathway, which could explain in part the functional anergy of CD27-CD21–/low B cells.

Figure 5. Gene array comparisons of CD21+ and CD21–/low CD27- B cells from pSS patients using the Illumina Human Whole Genome Array.

Figure 5

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(A) Over-expressed transcripts differentially expressed by CD21–/low and CD21+ CD27- B cells from 4 pSS patients are shown. Up- and down-regulated transcripts are indicated in red and green, respectively. (B) Over-expressed transcripts involved in an anergy and tolerance breakdown cluster are presented.

Figure 6. FACS analysis of proteins differentially expressed between CD21+ and CD21–/low CD27- B cells from pSS patients.

Figure 6

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FACS plot analysis of surface markers overexpressed and downregulated in CD21-/low CD27-B cells. FCRL2, FCRL3, CD72, CD11c and CD22 are overexpressed at the protein level in CD21–/low CD27- B cells from pSS patients, as found in transcriptomic analysis. In contrast, CD1c marker is downregulated at the protein level in CD21–/low CD27- B cells from pSS patients.

Discussion

We report herein on the identification of a unique autoreactive B cell population in pSS lacking CD21 expression and refractory to B-cell stimulation that correlates with the lymphoproliferative status. Unresponsive CD21-/low B cells expressing IgMs have been identified in RA and CVID patients as well as in patients with hepatitis C virus-associated mixed cryoglobulinemia (14-16). The analysis of the immunoglobulin repertoire of CD21-/low B cells from pSS patients demonstrated that these B cells belonged to the memory compartment because their immunoglobulin genes were mutated. Hence, CD21-/low B cells from pSS patients are similar to anergic CD21-/low B cells from patients with hepatitis C virus-associated mixed cryoglobulinemia, which also expressed mutated IgMs (15, 16), whereas most CD21-/low B cells from RA and CVID patients expressing unmutated immunoglogulins likely belong to the naïve B cell compartment (14, 24). However, CD21-/low B cells from pSS patients differ from their counterpart in patients with hepatitis C virus-associated mixed cryoglobulinemia, in that they do not express CD27 (15, 16). Nonetheless, CD21-/low B cells from all patients are refractory to most stimulation, suggesting that autoreactive CD21–/lo B cells may use a common anergic program to be tolerized. Indeed, the phenotype of CD21-/low B cells revealed the modulation of the expression of many molecules that may prevent the activation of these B cells. The down-regulation of the complement receptor CR2/CD21 previously reported on mouse and human anergic B cells is likely to result in an increased BCR signaling threshold and compromise the ability of CD21-/low B cells to be activated (14-16, 26, 27). In addition, CD21 is able to break anergy in mouse B cells, further suggesting that its down-regulation contributes to an anergic stage in mice and humans (14-16, 26, 28, 29). In pSS, CD21-/low B cells were highly enriched with autoreactive clones. In addition, gene array experiments analyzing CD21-/low B cells from pSS patients affected the expression of genes such as CD19, CD22, CD72, and FCRL3, genes that have been found to be involved in the development of many autoimmune diseases (30-32). These genes encoded molecules belonging to important pathways leading to B cell activation, including those initiated by the BCR. The upregulated transcription of CD86, CD72, GRB2, CBL-B, and FCRL2 genes is consistent with an anergy-like phenotype. CD86 as well as CD69 and CD95 expression normally induced on activated B cells may reflect the chronic stimulation status of autoreactive CD27-CD21–/low B cells in pSS patients. CD72 was shown to function as a regulator of peripheral B cell tolerance, by regulating and being associated with CBL-B, leading to the down-regulation of BCR signaling (33). Interestingly, CBL-B expression was shown to be a key protein required for induction of anergy in T cells (34). The upregulated transcription of immunoreceptor tyrosine–based inhibition motif (ITIM) receptor genes such as CD72, CD22, FCRL2 and SIGLEC are also likely to inhibit B-cell activation and proliferation. The validation of many gene upregulations by flow cytometry further demonstrates the functional unresponsiveness of CD27-CD21–/low B cells in pSS. Indeed, CD21-/low B cells from pSS patients showed impaired calcium-mediated signaling, did not up-regulate activation markers, and did not proliferate in response to BCR triggering. CD21-/low B cells were also prone to die faster than their CD21+ counterparts, suggesting that they have a shorter half-life. In addition, the increased CD95 expression CD21-/low B cells could be responsible for their increased susceptibility to cell death. Most of these features are characteristic of mouse anergic B cells (35) and of more recently reported anergic human B cells (14-16).

Lastly, we observed that autoreactive CD27-CD21–/low B cells were functionally attenuated after BCR and/or CD40 triggering but not after TLR stimulation as reported in patients with hepatitis C virus-associated mixed cryoglobulinemia (16). Leadbetter et al. showed that activation of autoreactive RF+ B cells was mediated by immune complexes and requires the synergistic engagement of the BCR and members of the MyD88-dependent TLR family (TLR7, TLR8 and TLR9); this thus establishes a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease (36). Activation in this mode is therefore likely to be a fundamental event in the loss of peripheral B-cell tolerance in a wide variety of settings, and these data establish a critical role for endogenous TLR ligands in aberrant activation of the adaptive immune system in autoimmunity. The chronic stimulation of polyclonal B cells secreting autoreactive antibodies, such as rheumatoid factor is proposed to be the first event of lymphomagenesis in Sjögren's syndrome (10). Interestingly, pSS and hepatitis C virus-associated mixed cryoglobulinemia share increased production of B-cell activating factor (BAFF) (2, 3), and an increased risk of lymphoma, particularly mucosa-associated lymphoid tissue lymphomas (4, 5, 37). Recalling what has been observed in hepatitis C associated mixed cryoglobulinemia, the hypothesis of a viral trigger has long been suspected in pSS. Different studies reported that Epstein-Barr virus (EBV) genome and proteins were detected in salivary glands of patients with pSS (38-40). EBV encoded small RNA (EBER), a double strand RNA, complexes with SSB to induce TLR3-related secretion of type I interferon (IFN) and other proinflammatory cytokines (40). Although it is unclear at this point if infectious agents or self-antigens may favor the emergence of CD21-/low B cells in pSS patients, their pattern of somatic hypermutations with replacement mutations located in CDRs interacting with antigens highly suggests that these B cells followed an antigen-driven selection as reported for conventional memory B cells (Ref Wardemann Immunity IgG reactivity and repertoire). Then, these autoreactive B cells may give rise to the emergence of a preferential clone, leading to the occurrence of lymphoproliferations. The demonstration that these lymphoma B cells may display rheumatoid factor activity (13) supports the hypothesis that Sjögren's syndrome associated lymphoma B cells grow through an auto-antigen driven process. However, our sequence analysis of the immunoglobulin repertoire of CD21-/low B cells from the 4 pSS patients was not successful so far at identifying such monoclonal expansion, which may initially develop in the salivary gland of these patients and may not be identified in the patient's blood before later stages of the disease.

Taken together, elevated numbers of highly autoreactive unresponsive CD21-/low B cells remain in the blood of pSS patients instead of being eliminated, and immune reactions may create a favorable environment to break tolerance and eventually activate these CD21-/low B cells, potentially leading to the development of B cell lymphoproliferation.

Supplementary Material

Supplemental Figure 2

Supplemental Figure 1. Somatic hypermutation patterns in CD21-/low B cells from primary Sjogren syndrome patients reveal their antigen mediated selection.

The frequency of replacement and silent mutations was assessed in the complementary determining regions (CDRs) compared to framework regions (FWRs). Replacement mutations were more frequent than silent mutations in heavy and light chain V gene CDRs compared to FWRs, suggesting antigen mediated selection of CD21-/low B cells. The frequency of mutations in VH, Vκ, and Vλ genes in CD21-/low B cells was calculated from the number of replacement (R; black bar) and silent (S; white bar) nucleotide exchanges per base pair in FWRs and CDRs. The R/S ratio for each region is indicated. Non-mutated sequences were excluded from this analysis.

Supplemental Figure 2. CD21-/low CD27- B cells are prone to dying by apoptosis and exhibiting decreased proliferation after BCR and CD40 triggering.

(A) Increased percentages of annexin V+7AAD- and annexin V+7AAD+ cells in CD21-/low CD27- B cells compared with their CD21+ counterparts. (B) Histograms show increased percentages of annexin V+7AAD- and annexin V+7AAD+ cells in CD21-/low CD27- B cells. (C) CD21-/low CD27- B cells exhibit decreased proliferation compared with CD21+ CD27- B cells. Data are representative of three independent experiments.

Acknowledgments

We thank Drs. L. Devine and C. Wang for single cell sorting. We thank Nathalie Ferry, Audrey Peres-Lascar, Catherine Blanc, Fabien Pitoiset and Cesar Prucca for technical assistance. This study was supported by the Agence Nationale pour la Recherche sur le Sida et les Hépatites (ANRS) and by Grant Number AI061093, AI071087, AI082713 and AI095848 from NIH-NIAID (to E. M.).

Footnotes

Author contributions: D.S., B.T., J.B., T.V., C.M., F.J., W.C., M.R., P.B., D.K., E.M., and P.C. devised and performed experiments; D.S., D.Se., and P.C. provided patient referrals; D.S., B.T., F.J., E.M., and P.C. interpreted results; and D.S., B.T., E.M., and P.C. designed the research and wrote the paper.

Conflict of interest disclosures: The authors declare no competing financial interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure 2

Supplemental Figure 1. Somatic hypermutation patterns in CD21-/low B cells from primary Sjogren syndrome patients reveal their antigen mediated selection.

The frequency of replacement and silent mutations was assessed in the complementary determining regions (CDRs) compared to framework regions (FWRs). Replacement mutations were more frequent than silent mutations in heavy and light chain V gene CDRs compared to FWRs, suggesting antigen mediated selection of CD21-/low B cells. The frequency of mutations in VH, Vκ, and Vλ genes in CD21-/low B cells was calculated from the number of replacement (R; black bar) and silent (S; white bar) nucleotide exchanges per base pair in FWRs and CDRs. The R/S ratio for each region is indicated. Non-mutated sequences were excluded from this analysis.

Supplemental Figure 2. CD21-/low CD27- B cells are prone to dying by apoptosis and exhibiting decreased proliferation after BCR and CD40 triggering.

(A) Increased percentages of annexin V+7AAD- and annexin V+7AAD+ cells in CD21-/low CD27- B cells compared with their CD21+ counterparts. (B) Histograms show increased percentages of annexin V+7AAD- and annexin V+7AAD+ cells in CD21-/low CD27- B cells. (C) CD21-/low CD27- B cells exhibit decreased proliferation compared with CD21+ CD27- B cells. Data are representative of three independent experiments.

NIHMS503142-supplement-Supplemental_Figure_2.doc (250KB, doc)

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