Complement receptor 2/CD21- human naive B cells contain mostly autoreactive unresponsive clones

doi:10.1182/blood-2009-09-243071Prepublished online March 15, 2010;

Jonathan Samuels, Jessica Berman, Jane H. Buckner, Charlotte Cunningham-Rundles and Eric MeffreIsabelle Isnardi, Yen-Shing Ng, Laurence Menard, Greta Meyers, David Saadoun, Iva Srdanovic, autoreactive unresponsive clonesComplement receptor 2/CD21-negative human naive B cells mostly contain

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1

Complement receptor 2/CD21-negative human naive B cells mostly contain

autoreactive unresponsive clones

Isabelle Isnardi1, Yen-Shing Ng1,2, Laurence Menard1,2, Greta Meyers1,2, David

Saadoun1, Iva Srdanovic1, Jonathan Samuels1,3, Jessica Berman4, Jane H. Buckner5,

Charlotte Cunningham-Rundles6, and Eric Meffre1,2*

1Laboratory of Molecular Immunology, Hospital for Special Surgery, New York, NY

10021, USA, 2Current address: Department of Immunobiology, Yale University School of

Medicine, New Haven, CT 06509, USA, 3Current address: Division of Rheumatology,

Hospital for Joint Diseases, NYU Langone Medical Center, New York, NY 10003, USA;

4Division of Rheumatology, Hospital for Special Surgery, New York, NY 10021, USA,

5Translational Research Program, Benaroya Research Institute, Seattle WA 98105,

USA, 6Department of Medicine and Pediatrics, Mount Sinai Medical Center, New York,

NY 10029, USA.

* Correspondence : [email protected]

Short Title: Characterization of human anergic B cells

Blood First Edition Paper, prepublished online March 15, 2010; DOI 10.1182/blood-2009-09-243071

Copyright © 2010 American Society of Hematology




2

Abstract

Complement receptor 2 (CR2/CD21)-negative B cells have been found enriched in

patients with autoimmune diseases and in common variable immunodeficiency (CVID)

patients who are prone to autoimmunity. However, the physiology of CD21-/lo B cells

remains poorly characterized. We found that some rheumatoid arthritis (RA) patients

also display an increased frequency of CD21-/lo B cells in their blood. A majority of CD21-

/lo B cells from RA and CVID patients expressed germline autoreactive antibodies, which

recognized nuclear and cytoplasmic structures. In addition, these B cells 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. Moreover,

gene array analyses of CD21-/lo B cells revealed molecules specifically expressed in

these B cells and which are likely to induce their unresponsive stage. Thus, CD21-/lo B

cells mostly contain autoreactive unresponsive clones, which express a specific set of

molecules that may represent new biomarkers to identify anergic B cells in humans.




3

Introduction

Random V(D)J recombination generates a large number of autoreactive B cells, which

can be silenced in the bone marrow by three main tolerance mechanisms: deletion,

receptor editing, and anergy 1-3. Deletion results in the removal of autoreactive clones by

apoptosis whereas receptor editing allows autoreactive B cells to alter their self-reactive

B cell receptor (BCR). This process may rescue immature B cell clones from deletion

and allow their differentiation to resume. In contrast to deletion and receptor editing,

anergy does not remove autoreactive B cell clones from the total B cell population but

renders them irresponsive to antigenic stimulation 4-7. Anergic autoreactive B cells

remain in the periphery but they have a short life span, which ultimately result in their

elimination 8,9.

Initial reports have demonstrated that deletion is mainly used to eliminate B cells, which

express highly autoreactive BCRs against membrane-bound antigens 10,11. However,

receptor editing has since been shown to be the major B cell tolerance mechanism

against these antigens, and clonal deletion appears to be a default mechanism when

receptor editing fails to silence autoreactive B cells 12. Alternatively, anergy seems to be

preferentially induced in B cells that express moderately autoreactive BCRs toward

soluble antigens 11. Using transgenic mouse models, anergic B cells have been

described as being unable to become activated, proliferate or secrete antibodies upon

BCR triggering (reviewed in 7). Indeed, BCR signaling is abnormal in these cells and

BCR aggregation fails to induce an increased concentration of intracellular calcium

(Ca2+)i or tyrosine phosphorylation cascades. It is believed that this irresponsive state

results from chronic BCR exposure to self-antigens, which desensitizes BCR signaling

abilities 13,14. The characterization of unresponsive B cells in unmanipulated mice and in




4

humans showed that anergic B cells represent a small percentage of circulating B cells

15,16.

We report here that an unusual B cell population, which downregulate the complement

receptor CR2/CD21 and was previously reported in SLE and common variable

immunodeficiency disease (CVID) patients, develops in some RA patients 17,18 ,19-21,22 ,23.

These CD21-/lo B cells are enriched in autoreactive clones that are refractory to most

stimulation, suggesting that human CD21-/lo B cells use an anergic mechanism to be

tolerized.

Methods

Patients and healthy donor controls

CVID and RA patients are described in Table S1-S2. Healthy donors include a 36-year-

old Caucasian male (HD10) and 24-year-old Caucasian female (HD11). Additional

blood leukocyte preparations from control donors were obtained from the New York

Blood Center. Samples were collected after individuals signed informed consent in

accordance with Hospital for Special Surgery institutional review board-approved

protocols and the Declaration of Helsinki.

B cell purification, single cell sorting, cDNA and RT-PCR

Peripheral B cells were purified from the blood of patients and control donors by

negative selection using RosetteSepTM procedure (StemCell Technologies).

Alternatively, mature naïve B cells were enriched from peripheral blood mononuclear

cells using the Naïve B Cell Isolation Kit II (Miltenyi). B cells were stained with

fluorescein isothiocyanate (FITC) anti-human CD27, phycoerythrin (PE) anti-human




5

CD10, and either anti-human IgM biotin and allophycocyanin (APC) anti-human CD19,

or PE-Cy7 anti-human CD19 and APC anti-human CD21 (Pharmingen, Beckton

Dickinson). Biotinylated antibodies were revealed using streptavidin-PECy7 (Caltag

Laboratories). Single CD21loCD10+IgMhiCD27- new emigrant, CD19+CD10-CD21+CD27-

conventional mature naive and CD19+CD10-CD21-/loCD27- B cells from patients and

control donors were sorted on a FACSVantage (Becton Deckinson) into 96-well PCR

plates containing 4 μl of lysis solution (0.5X PBS containing 10 mM DTT, 8 U RNAsin

(Promega), and 0.4 U 5’-3’ RNase Inhibitor (Eppendorf)) and immediately frozen on dry

ice. All samples were stored at -70°C. RNA from single cells was reverse-transcribed in

the original 96 well plate in 12.5μl reactions containing 100U of Superscript II RT (Gibco

BRL) for 45 min at 37°C. RT-PCR reactions and primer sequences were as described

24. Immunoglobulin sequences and mutation status were analyzed by Ig BLAST

comparison with GenBank using the National Center for Biotechnology Information

IgBlast server (http://www.ncbi.nlm.nih.gov/igblast/). Heavy chain CDR3 was defined as

the interval between the conserved cysteine at position 92 in the VH framework 3 and the

conserved tryptophan at position 103 in JH segments.

Antibodies

Additional flow cytometry analyses were performed using anti-BAFF-R-FITC, CD86-

FITC, MHC class II/HLA-DR-FITC, CD44-FITC, CD62L-FITC, CD25-FITC, Fas/CD95-

FITC, ICOSL-PE, TACI-PE, CD80-PE, MHC class I/HLA-A,B,C-PE, CD11c-PE, CD32-

PE, CD93-PE, CD40-Alexa Fluor 647, CXCR4-APC (Biolegend), CD22-FITC, CD72-

FITC, CD21-PE, CD38-PE, CD69-PE, CD85j-PE, IgD-PE, CD19-PECy7, CD21-APC

(Pharmingen, Beckton Dickinson), CCR7-FITC, IL-4R-PE (R&D Systems), and CD23-

FITC (Serotec). Unconjugated anti-CD58 (Pharmingen, Beckton Dickinson) and CD85d




6

(Abcam) mouse monoclonal antibodies were coupled using the Alexa Fluor 488

Monoclonal Antibody Labeling Kit (Molecular Probes).

Microarray gene expression profile analysis

RNA was extracted from 105-3.105 batch sorted CD19+CD10-CD21+CD27- conventional

mature naive and CD19+CD10-CD21-/loCD27- B cells using the Absolutely RNA

microprep kit (Stratagene). 100-200 ng of RNA was obtained per sample, and the

quality of the purified RNA was assessed by the Bioanalyzer from Agilent. Using the

Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to

produce cDNA. Labeled cDNA was hybridized on chips containing the whole human

genome (Human Genome U133 2.0 from Affymetrix). The data discussed in this

manuscript have been deposited in National Center for Biotechnology Information’s

Gene Expression Omnibus and are accessible through GEO Series accession number

GSE13917

(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=hzgjvycoecikyrc&acc=GSE13917)

.

B cell activation, proliferation and survival

Naïve B cells, which contained CD21+ and CD21-/lo B cells from control donors and

patients were plated at 150,000-200,000 cells per well in a 96 well plate in RPMI 10%

serum and 20 μg/mL polyclonal F(ab)’2 rabbit anti-human IgM (Jackson

Immunoresearch), 1 μg/mL multimeric soluble recombinant human CD40L (Alexis

Biochemicals), and/or 1 μg/mL CpG (Invivogen) for 48 hours. To assess B cell

proliferation, naïve B cells were labeled with CFSE (Molecular Probes-Invitrogen), plated

and stimulated as described above. The cells were harvested on day 3 and labeled for




7

CD19 and CD21 expression before determining the division number by decreasing

CFSE fluorescence in flow cytometry experiments. The proportions of apoptotic and

dead cells were assessed by flow cytometry to measure binding with annexin V and

propidium iodide using the Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen).

Calcium Mobilization

To measure free intracellular calcium concentration [Ca2+]i, naïve B cells were first

labeled with anti-CD19 and anti-CD21 antibodies, and loaded with 5 mM Fluo-3 AM

(Molecular Probes-Invitrogen) in the presence of 0.2% Pluronic F-127 (Sigma) for 30 min

at room temperature. Cells were washed, resuspended at 106 cells/mL and [Ca2+]i was

monitored over time by flow cytometry on gated CD19+CD21+ and CD19+CD21-/lo B cells.

After the baseline was established, cells were stimulated with 20 μg/ml F(ab')2 mouse

anti-human IgM. Mean [Ca2+]i was analyzed using the FCS Assistant 1.2.9 software (BD

Biosciences).

Antibody production, ELISAs and IFAs

Cloning strategy, expression vectors, and antibody reactivity against specific antigens

were as described 24. Highly polyreactive ED38 was used as positive control in HEp-2-

reactivity and polyreactivity ELISAs 24. Antibodies were considered polyreactive when

they recognized at least 2 and usually all of the 4 analyzed antigens that include single-

stranded DNA (ssDNA), double-stranded DNA (dsDNA), insulin and lipopolysaccharide

(LPS). 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 50-100 μg/mL. FITC-conjugated goat anti-human IgG was

used as detection reagent. Pictures were taken with an Axioskop (Zeiss) using a Plan-




8

Neofluar 40x/.75 objective and Axiovision 3.1 acquisition software.

Statistics

Differences between healthy donor and patients populations were analyzed for statistical

significance with unpaired Student’s t-tests, using SigmaPlot software (Systat). A P

value of less than 0.05 was considered significant.

Supporting Information

Supplemental material includes eight figures and nineteen tables.

Results

CVID and RA patients may display unusual peripheral CD21-/lo B cells

CVID comprises a heterogeneous group of patients suffering from reduced serum levels

of several Ig isotypes resulting in an increased susceptibility to bacterial infections (Table

S1) 25. Indeed, CVID patients frequently show severely decreased frequencies of CD27+

memory B cells, especially those expressing switched isotypes 21,26,27. In addition, some

CVID patients display an unusual population of B cells that lack complement receptor 2

(CR2/CD21) 21-23. The presence of 20% or more CD21-/lo B cells in the blood of CVID

patients was proposed to be a criteria for the classification of these patients as CVID

group Ia who are prone to develop autoimmunity (Figure 1A-B) 21,22. Since CD21-/lo B

cells were also reported in the blood of patients with autoimmune diseases including

SLE 17,18 ,19,20, we analyzed the frequency of CD19+CD27-CD21-/lo B cells in healthy

donors and untreated active RA patients (Table S2). We found that the proportion of

CD21-/lo B cells was low in peripheral CD27-depleted B cells from healthy donors (Figure




9

1C-D) 17. In contrast, a subgroup of RA patients displayed a high frequency of CD21-/lo B

cells (Figure 1C-D). Similarly to CVID patients, CD19+CD27-CD21-/lo B cells in RA

patients were not new emigrant/transitional B cells because they expressed neither

CD10 nor CD38 (data not shown). Moreover, the frequency of CD19+CD10-CD27-CD21-

/lo (from now on referred to as CD21-/lo) B cells was stable in the same RA patients over a

4 year-period. Thus, both CVID and RA patients may display an increased proportion of

CD21-/lo B cells in their blood.

CD21-/lo B cells express highly autoreactive antibodies including ANAs

The physiology of CD21-/lo B cells has not been thoroughly investigated and the

involvement of these B cells in the development of autoimmunity is unknown. BCR

expression in CD21-/lo B cells was assessed by flow cytometry after depletion of CD27+

memory B cells. In agreement with previous reports, we found that the majority of CD21-

/loCD27- B cells from healthy donors expressed isotype-switched B cells (Figure 2A) 28-30.

In contrast, CD21-/loCD27- B cells from RA and CVID patients preferentially expressed

IgM and/or IgD (Figure 2A). Indeed, patients’ CD21-/lo B cells were enriched in IgMloIgD+

B cells, which represented about 30% of their CD21-/lo B cells whereas this population

only accounted for 10% of CD21-/lo B cells in healthy donors (Figure 2A).

To determine whether unswitched CD21-/lo B cells from RA and CVID patients, as well as

from healthy donors, expressed autoreactive BCRs, we compared the reactivity of

antibodies expressed by single CD21-/lo and single conventional CD19+CD10-CD27-

CD21+ (referred to as CD21+) mature naïve B cells from the same individuals. Most Ig

genes expressed by CD21-/lo B cells from RA and CVID patients were devoid of somatic

hypermutations, thereby demonstrating that these B cells belonged to the naïve

compartment. IgM genes amplified from CD21-/lo B cells from two healthy donors

displayed unmutated and mutated sequences, revealing that these cells contain both




10

naïve and memory B cells (Tables S3-S18 and data not shown). Therefore, we only

expressed BCRs encoded by germline Ig genes to compare naïve B cell populations.

We found that the frequency of polyreactive and HEp-2-reactive clones was increased in

CD21-/lo compared to CD21+ B cells in all individuals (Figure 2B and S1). Differences in

HEp-2 reactivity between CD21-/lo and CD21+ B cells reached significance for both

healthy donors and patients (Figure S2). In addition, CD21-/lo B cells often expressed

highly HEp-2 reactive antibodies (arbitrarily defined by ELISA OD405>2) compared to

their CD21+ B cell counterparts (Figure 2C). Highly HEp-2 reactive antibodies often

displayed heavy chain complementarity determining regions containing many positive

charges favoring autoreactivity and were mostly kappa clones, which suggested that

receptor editing using lambda light chains was not induced to silence these B cells

(Figure 2D and Tables S3-S18) 24,31,32. Indeed, CD21-/lo B cells showed a statistically

significant decrease in upstream Vκ gene usage combined to an increase in Jκ1 gene

usage compare to conventional CD21+ B cells, further suggesting decrease secondary

recombination events in CD21-/lo B cells despite their autoreactive features (Figure S3).

Thus, CD21-/lo B cells are enriched in polyreactive and strongly autoreactive clones,

which are normally silenced during B cell development in the bone marrow.

Immunofluorescence assays revealed that 5.6%-22.2% of naïve CD21-/lo B cells

expressed anti-nuclear antibodies (ANAs) (Figure 3A-B). ANAs expressed by CD21-/lo B

cells showed a diversity of nucleolar and speckled anti-nuclear staining patterns (Figure

3B). In contrast, ANAs were rarely detected in CD21+ B cells from healthy donors and

RA patients but could be fairly frequent in CD21-/lo B cells from CVID patients (Figure 3A

and data not shown). In addition, CD21-/lo B cells expressed antibodies that often

recognized specific cytoplasmic structures, which included mitochondria, the golgi

apparatus, and cytoskeleton components (Figure 3C-D, and Tables S3-S18). These




11

antibodies mostly corresponded to the highly HEp-2 reactive clones identified by ELISA.

In contrast, antibodies expressed by CD21+ B cells rarely recognized well-defined

structures in HEp-2 cells. We conclude that CD21-/lo B cells differ from CD21+

conventional B cells in that they often express autoreactive antibodies that may

recognize nuclear or cytoplasmic antigens.

CD21-/lo B cells fail to get activated through BCR and CD40 triggering

We analyzed the physiology of CD21-/lo B cells by studying the induction of CD25, CD69,

CD23, TACI, ICOS-L and Fas by flow cytometry on CD21+ and CD21-/lo gated B cells

after BCR, CD40 and TLR9 stimulations for 2 days. CD21+ B cells from RA01, RA19,

CVID218 and CVID321 patients upregulated CD69 and CD25 after BCR, CD40 or TLR9

triggering (Figure 4A-B and data not shown). In contrast, CD21-/lo B cells from the same

patients failed to properly induce cell surface expression of CD25 and CD69 after BCR

and CD40 triggering (Figure 4A-B). The induction of CD25 and CD69 in CD21-/lo B cells

by TLR9 stimulation with CpG, although decreased compared to conventional B cells,

were less affected than those induced by BCR and/or CD40 triggering (Figure 4A-B).

Defects in the induction of CD23 on CD21-/lo compared to CD21+ B cells were also

observed after BCR, TLR9, or especially CD40 stimulations (Figure S4-S5). These

defects were not the result of a global inability of CD21-/lo B cells to get activated,

because CD21-/lo B cells upregulated cell surface expression of ICOS-L, TACI and Fas

after BCR, CD40 or TLR9 stimulation (Figure S4-S5). In addition, similar results were

obtained when CD21+ and CD21-/lo B cells were initially fractionated and then stimulated

in vitro for 2 days (data not shown). Thus, CD21+ and CD21-/lo B cells display distinct B

cell responses after BCR, CD40 or TLR9 stimulation.

BCR triggering does not induce calcium flux in CD21-/lo B cells




12

The inability of CD21-/lo B cells to upregulate some activation markers upon BCR

triggering could result from an inability to properly induce intracellular calcium flux. We

analyzed [Ca2+]i after BCR stimulations in these B cells compared to conventional CD21+

B cells. BCR triggering induced a strong [Ca2+]i increase in CD21+ conventional B cells

from healthy donors as well as RA and CVID patients (Figure 4C and data not shown).

In contrast, CD21-/lo B cells from RA01, RA19, RA26, CVID321, and CVID218 patients

only induced a weak elevation of [Ca2+]i in response to BCR stimulation (Figure 4C and

data not shown). We conclude that CD21-/lo B cells do not exhibit a proper calcium

response following BCR stimulation.

CD21-/lo B cells do not proliferate after BCR and CD40 triggering

Since CD21-/lo B cells showed an impaired activation compared to CD21+ B cells in

response to several stimulations, we investigated their ability to proliferate after antigenic

stimulation. Carboxyfluorescein succinimidyl ester (CFSE) labeled CD21+ and CD21-/lo B

cells from 5 RA and 4 CVID patients and healthy donor controls were stimulated through

their BCR or CD40 for 3 days and CFSE dilution, which reflects B cell proliferation, was

then assessed by flow cytometry (Figure 5A and data not shown). Anti-IgM F(ab’)2

stimulation induced the proliferation of a few CD21+ B cells in all individuals whereas

CD40L did not (Figure 5A). BCR and CD40 co-triggering resulted in the proliferation of a

majority of CD21+ B cells from both healthy donors and patients and some of these B

cells underwent up to two divisions (Figure 5A). In contrast, RA and CVID CD21-/lo B

cells failed to proliferate after BCR and CD40 costimulation. We conclude that unlike

CD21+ conventional B cells, CD21-/lo B cells are unable to proliferate after antigenic

stimulation, further suggesting their unresponsive stage.

CD21-/lo B cells are prone to die by apoptosis




13

We assessed the survival potential of CD21-/lo B cells by analyzing the binding of

Annexin V to their surface, which along with propidium iodide (PI) staining can define

Annexin V+PI- early apoptotic cells and Annexin V+PI+ late apoptotic and dead cells. We

found that freshly isolated CD21-/lo B cells from RA and CVID patients contained a higher

frequency of both Annexin V+PI- early apoptotic and Annexin V+PI+ late apoptotic and

dead B cells compared to conventional CD21+ B cells (Figure 5B). When naïve B cells

from RA and CVID patients were left unstimulated for 12 hours, CD21-/lo B cells

contained about 35% of Annexin V+PI+ dying cells whereas there were less than 10% in

CD21+ B cells (Figure 5C). Moreover, triggering through BCR, CD40 and/or TLR9 only

partially rescued these B cells from cell death. In contrast, stimulated CD21+ B cells

contained virtually no Annexin V+PI+ B cells (Figure 5C). In agreement with their

increased susceptibility to die, CD21-/lo B cells also contained a higher proportion of

Annexin V+PI- early apoptotic cells than CD21+ B cells, regardless of the stimulation

(Figure S6). Hence, CD21-/lo B cells are more susceptible to apoptosis and cell death

than CD21+ B cells and their ability to be eliminated is poorly rescued by BCR

stimulation in combination with either CD40 or TLR9 triggering.

The transcriptome of CD21-/lo B cells shows an inhibitory gene signature

We intended to further characterize CD21-/lo autoreactive unresponsive B cells by

performing gene array profiling and compare transcripts expressed in CD21-/lo versus

CD21+ mature naïve B cells isolated from 2 RA and 3 CVID patients (Figure 6). 1,079

transcripts were found expressed with a greater than twofold statistically significant

difference by the 2 B cell subpopulations and included 656 upregulated and 423

downregulated transcripts in CD21-/lo B cells (Figure 6A). Some of those differentially

expressed genes, and a few others of specific interest, are shown on Figure 6B and

Table S19. Correlating with a decreased expression of CD21 on their cell surface, we




14

found that CD21-/lo B cells down-regulated the transcription of CR2/CD21, as well as

other complement binding molecules CR1/CD35, CD55 and CD59 (Figure 6B). Many

genes encoding survival and activation molecules like CD40, OX40L and BCMA, as well

as the cytokine receptors IL-4R and IL-13R that stimulate B cell proliferation, were all

downregulated in CD21-/lo B cells (Figure 6B). In contrast, CD21-/lo B cells upregulated

the transcription of a whole set of immunoreceptor tyrosine-based inhibition motif (ITIM)

receptor genes likely to inhibit B cell activation and proliferation. They included CD72,

Fc receptors (FcR) CD32/FCGR2B and FCRL5/IRTA2, as well as LILRB and SIGLEC

genes (Figure 6B). Genes encoding phosphatases potentially mediating the inhibitory

effect of these ITIM-receptors where also found upregulated in the CD21-/lo B cells

(Figure 6B). In addition, these B cells downregulated transcripts encoding the chemokine

receptors CCR7, CXCR4 and CXCR5 as well as lymphotoxin A which regulates B cell

trafficking (Figure 6B). Lymphocyte homing capacities are also likely to differ between

CD21-/lo and CD21+ B cells because transcripts for many integrins (ITG), including

ITGAX, which encodes CD11c molecules, were upregulated in CD21-/lo B cells, while the

transcripts for selectin CD62L were downregulated (Figure 6B). The increased amounts

of growth arrest-specific 7 (GAS7) and CDKN1A/p21 combined to decreased cyclin-

dependent kinase 6 (CDK6) and MYC transcripts in CD21-/lo B cells are likely to result in

the observed defective proliferation of these cells. In addition, the upregulation of

Fas/CD95 and caspase genes in CD21-/lo B cells could be responsible for their increased

susceptibility to cell death. Finally, we found that some transcription factor genes were

upregulated in CD21-/lo B cells compared to CD21+ B cells, such as BCL11B, EGR2,

HOXA3, HOXB7, HOXC4, NFATC2, SOX4, TBET, and TOX, while a few transcription

factor genes were found downregulated, such as FOXP1 (Figure 6B). In conclusion, the

transcriptome of CD21-/lo B cells is characterized by the upregulation of many genes

encoding molecules likely to inhibit B cell activation, proliferation and survival and the




15

downregulation of B cell activating genes, supporting the observation that CD21-/lo B

cells are refractory to antigenic stimulation.

CD21-/lo B cells display a specific B cell phenotype

The expression of many genes found either upregulated or downregulated in gene array

experiments was validated by flow cytometry in RA01, RA19, CVID214, CVID218 and

CVID321 after B cell surface staining with specific monoclonal antibodies.

Representative results for RA and CVID patients, as well as for healthy controls, are

shown in Figure 7 and S7 and S8 respectively. In agreement with gene array analysis,

CD21-/lo B cells were CD19hiCD40loCD86+CD58hiIL-4RloCD11c+CCR7-

/loCXCR4loCD44loCD62L-/loCD72hiCD32hiCD85j+CD85d+Fas+ (Figure 7 and S7-S8).

Moreover, we also found that BAFF-R was downregulated and MHC class I and ITIM

receptor CD22 was upregulated in CD21-/lo B cells, whereas changes in their gene

expression were not detected in gene array experiments (Figure 7 and S7-S8). Human

CD21+ and CD21-/lo B cells expressed TACI, ICOS-L, CD80 and MHC class II at similar

levels and did not expressed CD93, which was found on the cell surface of mouse

anergic transitional 3 (T3) B cells (Figure 7 and S7-S8, and data not shown). Hence,

CD21-/lo B cells display a unique phenotype, which is characterized by the

downregulation of receptors that normally favor B cell survival and/or activation, and the

upregulation of molecules inhibiting B cell functions.

Discussion




16

We report herein the identification in humans of a unique autoreactive B cell population,

which lacks CD21 expression and is refractory to B cell stimulation. Indeed, naïve

CD21-/lo B cells express a diverse germline BCR repertoire, which is enriched in

autoreactive clones. In addition, CD21-/lo B cells showed impaired calcium mediated

signaling, did not upregulate some activation markers, and did not proliferate in

response to BCR triggering. CD21-/lo B cells were also prone to die faster than CD21+

conventional B cells, suggesting they have a shorter half-life. Because most of these

features are characteristic of mouse anergic B cells, autoreactive CD21-/lo B cells, which

escaped central B cell tolerance deletion and remain in the periphery in an irresponsive

stage, therefore represent a population of anergic B cells in humans 4,14,15,33-37.

B cells which express intrinsically autoreactive BCRs encoded by the VH4-34 gene have

been suggested to be anergic because they fail to induce normal calcium flux after BCR

triggering 38. In addition, VH4-34 B cells are enriched in mature naïve B cells and

remain excluded from germinal centers (GCs) in healthy donors 38,39. However, we did

not find any increase in VH4-34 gene usage in CD21-/lo B cells, which expressed a very

diverse immunoglobulin repertoire. The abundance of self-antigens expressed on

erythrocytes and recognized by VH4-34 BCRs may induce a unique anergic program in

these cells that differs from that of CD21-/lo B cells, which may recognize a broader set of

self-antigens. Another population of irresponsive B cells which downregulated IgM

expression was recently reported in humans 16. These B cells also express autoreactive

antibodies and fail to induce calcium flux and tyrosine phosphorylation cascade after IgD

triggering but could get activated and proliferated in vitro 16. By analogy to anergic

mouse models, which express different autoreactive transgenic antibodies and develop

different anergic B cell programs, we would like to propose that VH4-34-expressing, IgM-




17

negative and CD21-/lo B cells represent different populations of anergic B cells in

humans.

The existence of some CD21-/lo B cells have been previously reported in CVID and SLE

patients, in individuals infected with either human immunodeficiency virus (HIV) or

hepatitis C virus combined with mixed cryoglobulinemia, as well as in healthy donors 17-

23,28-30,40. All these CD21-/lo B cells, with the exception of those in CVID patients, belong

to the memory B cell compartment because they frequently express mutated isotype

switched antibodies 28-30. In agreement with these reports, we found that memory B cells

represented the majority of CD21-/lo B cells in healthy donors. In contrast, CD21-/lo B

cells from RA and CVID patients were often naïve B cells because they produced

germline encoded IgM and IgD antibodies. However, both naïve and memory CD21-/lo B

cells displayed a similar gene expression profile and expressed common cell surface

molecules, which differed from conventional CD21+ B cells 28,30. It is unknown whether

memory CD21-/lo B cells from healthy donors are anergic but CD21-/lo B cells from HIV-

infected viremic individuals and CVID patients poorly respond to antigenic stimulation

23,28. Chronic antigen exposure, such as in HIV-infected viremic individuals, may lead

antigen-reactive B cells to an anergic phenotype similar to CD21-/lo naïve B cells and

disable them to potentially secrete antibodies.

The phenotype of CD21-/lo B cells revealed the modulation of the expression of many

molecules that may prevent the activation of these B cells. The downregulation of the

complement receptor CR2/CD21 previously reported on mouse anergic B cells is likely

to result in an increased BCR signaling threshold and compromise the ability of CD21-/lo

B cells to be activated 34,37. In addition, CD21 is able to break anergy in mouse B cells,

further suggesting that its downregulation contribute to an anergic stage in mice and




18

humans 34,37,41. The downregulation of CD40, OX40L, IL-4R and IL-13R, which play an

important role in B cell activation and proliferation, may also be responsible for the

anergic responses of CD21-/lo B cells. Moreover, increased amounts of many ITIM

receptors on CD21-/lo B cells, including CD22, FcγR2B/CD32, and CD72 that was

already reported in mouse anergic B cells, can further dampen the activation of these B

cells 42-45. The overexpression of many phosphatases in CD21-/lo B cells is also likely to

decrease their antigenic responses. Chemokine receptors including CCR7, CXCR4, and

CXCR5 as well as selectin CD62L have been shown to play a major role in B cell

trafficking in secondary lymphoid organs and GCs 46,47. The downregulation of these

receptors on CD21-/lo B cells is likely to exclude them from B cell follicules and being

recruited in germinal centers 9,48. In addition, the upregulation of many integrins also

involved in B cell homing may direct these autoreactive B cells to specific compartments

where they may be eventually eliminated. Moreover, the increased expression of

molecules favoring cell death and blocking cell cycle entry may further prevent the

expansion of CD21-/lo B cells and favor their removal. Altogether, CD21-/lo B cells

express a specific set of molecules that may prevent the activation of these B cells.

Some of these molecules may also serve as biomarkers to identify human unresponsive

autoreactive naïve B cells and distinguish them from the other CD21-/lo B cells belonging

to the memory compartment.

Unresponsive autoreactive CD21-/lo B cells express a specific B cell phenotype, which

may be regulated by transcription factors. However, transgenic mouse models did not

identify a specific transcription factor whose expression would be responsible for the

establishment of an anergic B cell program 42. Indeed, it has been postulated that

because the maintenance of an anergic stage requires constant BCR triggering by self-

antigens, the anergic phenotype may not depend on a genetic program ensured by




19

transcription factors 13,14. Gene array comparisons between CD21-/lo and CD21+ naïve

B cells revealed the induction of a set of transcription factors in human unresponsive

autoreactive CD21-/lo B cells. It is unknown whether this transcription factor signature is

dependent on constant BCR triggering by self-antigens, but it unveils new leads in the

identification of factors that may control the expression of the molecules resulting in the

inability of CD21-/lo B cells to respond to antigenic stimulations.

Three mechanisms normally ensure the silencing of developing autoreactive B cells.

Receptor editing mediated by secondary recombination has been reported to be very

efficient at silencing autoreactive B cells reacting against membrane bound antigens and

deletion seems to be used when receptor editing failed to silence autoreactive B cells 12.

Anergy may be preferentially induced in B cells that express moderately autoreactive

BCRs toward soluble antigens 11. In agreement with this hypothesis, we found that

CD21-/lo B cells that express unmutated IgM in healthy donors display weakly

autoreactive BCRs and may represent anergic B cells. Healthy donors also showed low

frequencies of CD21-/lo B cells, suggesting that anergy is rarely used to silence

developing autoreactive B cells in physiological conditions. Factors that favor the

development or the maintenance of CD21-/lo B cells in the blood of some RA and CVID

patients remain to be determined. The serum of CVID group Ia but not RA patients

contains elevated concentrations of B-cell activating factor (BAFF), which is known to

inhibit the counterselection of anergic B cells in mice and likely to contribute to the large

accumulation of CD21-/lo B cells in these CVID patients (49 and data not shown).

The induction of an unresponsive program in highly autoreactive B cells may represent

risks to develop autoimmunity. Indeed, elevated numbers of highly autoreactive

unresponsive CD21-/lo B cells remain in the blood of RA and CVID patients instead of




20

being eliminated and immune reactions may create a favorable environment to break

tolerance and eventually activate these CD21-/lo B cells. In RA, all patients develop

autoimmunity, whether or not they display CD21-/lo B cells, and it is unclear how these

B cells contribute to the initiation and/or progression of the disease. However, CVID

group Ia patients who display high frequencies of CD21-/lo B cells are more susceptible

to develop autoimmune syndromes, suggesting that CD21-/lo B cells may contribute to

the development of autoimmunity in humans.

Acknowledgments

We are very much indebted to the patients and their families. We thank Dr. S.

Rudchenko and S. Semova for help with the single cell sorter, Dr. D. Orange, D.

Goldenberg and K. Warnatz for providing RA and CVID patient samples and Dr G.

Charvin for microscopy analysis. This publication was made possible by Grant Number

AI061093 and AI071087 from NIH-NIAID.

Authorship Contributions

I. I performed the experiments, with help from E. M. for cell isolation and sorting, and

from Y.-S. N., L. M., G. M., D. S. and I. S. for antibody cloning and reactivity testing. E.

M. initiated the collaboration with J. S., J. B., J. H. B. and C. C.-R. who provided patient

blood samples. I. I. and E. M. designed the research and wrote the manuscript.




21

Disclosure of Conflicts of Interest

The authors declare no competing financial interests.

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Figure legends

Figure 1. CVID and RA patients may display an increased frequency of CD21-/lo B

cells in their blood. (A) CVID group Ia patients were previously defined as patients

displaying a frequency of CD21-/lo naïve B cells > 20% of total B cells 21. Each diamond

represents an individual and (B) dot plots show CD19 and CD21 expression on CD27-

gated peripheral B cells from representative CVID group Ia patients. (C) Increased

CD21-/lo B cell frequency in RA patients. (C) Dot plots show CD19 and CD21 expression

on CD27-depleted peripheral B cells from a representative healthy donor (HD10) and RA

patients. (D) The frequencies of CD21-/lo naïve B cells are compared between healthy

donors and RA patients. Each diamond represents an individual and statistically

significant differences are indicated.

Figure 2. CD21-/lo naïve B cells express autoreactive BCRs. (A) A majority of CD21-/lo

B cells from RA and CVID patients express IgM and IgD. Dot plots show IgM and IgD

expression gated on CD19+CD21+(left) and CD19+CD21-/lo (right) CD27-depleted B cells.

(B) Most CD21-/lo naïve B cells express autoreactive antibodies. Antibodies cloned from




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single CD21+CD27-CD10- and CD21-/loCD27-CD10- B cells from two healthy donors

(HD10 and 11), 3 RA and 3 CVID patients were tested by ELISA for reactivity against

HEp-2 lysates. Dotted lines show ED38-positive control 24. Horizontal lines show cut-off

OD405 for positive reactivity. For each B cell population, the frequency of HEp-2-reactive

and non HEp-2-reactive clones is summarized in pie charts with the number of

antibodies tested indicated in the centers. The frequency of HEp-2 reactive B cells was

higher in CD21-/lo than in CD21+ B cells in all individuals. (C) CD21-/lo B cells express

highly autoreactive antibodies. The frequency of highly HEp-2-reactive antibodies,

arbitrarily defined by ELISA OD405>2, expressed by CD21+ and CD21-/lo B cells from HD,

RA and CVID patients is represented. A large fraction of CD21-/lo B cells from most

individuals were highly HEp-2-reactive. P values calculated using a paired T-test are

indicated when significant. (D) A majority of highly autoreactive antibodies from CD21-/lo

B cells express kappa light chains. The frequency of highly HEp-2-reactive CD21-/lo B

cells from HD, RA and CVID patients is represented subdivided among kappa and

lambda clones. Many highly HEp-2-reactive CD21-/lo B cells express kappa light chains

suggesting that receptor editing using lambda light chains was not induced to silence

these clones.

Figure 3. CD21-/lo B cells contain anti-nuclear and anti-cytoplasmic clones. The

frequencies of anti-nuclear (A) and anti-cytoplasmic (C) clones in CD21+ and CD21-/lo B

cells from HD, RA and CVID patients are shown. P values calculated using a paired T-

test are indicated when significant. (B) ANAs expressed by CD21-/lo B cells showed a

diversity of nucleolar (RA01 21- κ181, RA01 21- κ137, RA14 21- κ15, and RA14 21- κ83),

nucleolar and speckled (RA19 21- κ127) and speckled (CVID 21- κ12) anti-nuclear




29

staining patterns. (D) Anti-cytoplasmic CD21-/lo B cells can react with well-defined

intracellular structures which include mitochondria (RA01 21- κ140, and CVID321 21-

κ82), the golgi (RA14 21- κ18), and cytoplasmic fibers (RA19 21- κ61, RA14 21- κ62, and

RA01 21- κ157 that is likely to recognize actin).

Figure 4. CD21-/lo naïve B cells fail to properly respond to BCR triggering. CD21-/lo

naïve B cells from (A) RA01 and (B) CVID321 patients fail to upregulate CD69 and

CD25 expression after stimulation with F(ab’2) anti-IgM, recombinant human CD40L,

and TLR9 agonist CpG. Total naive B cells containing both CD21-/lo and CD21+ B cells

were plated and dot plots show CD69 and CD25 expression after 2 days according to

gated CD21 expression. (C) BCR triggering in CD21-/lo naïve B cells does not properly

increase [Ca2+]i. [Ca2+]i was assessed by flow cytometry in CD21-/lo and CD21+ naive B

cells from a healthy donor (HD), RA01 and CVID321 patients. Both B cell populations

were loaded with Fluo-3 and expressed similar levels of surface IgM expression when

experiments were performed. Arrow indicates when F(ab’)2 anti-IgM was added. Data

from each group of individuals are representative of two-three independent experiments.

Figure 5. CD21-/lo naïve B cells do not proliferate after BCR and CD40 co-

triggering and are prone to cell death. (A) Total naive B cells from healthy donors

(HD), RA and CVID patients containing both CD21-/lo and CD21+ B cells were labeled

with CFSE and stimulated with F(ab’2) anti-IgM and/or recombinant human CD40L. Dot

plots show CD21 expression according to CFSE dilution after 3 days. CFSE dilution in

CD21+ but not in CD21-/lo B cells stimulated with F(ab’2) anti-IgM and recombinant

human CD40L reveals that some CD21+ B cells underwent up to two divisions whereas

CD21-/lo B cells did not divide. (B) Increased frequency of Annexin V+ propidium iodide




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(PI)- early apoptotic, and Annexin V+ PI+ late apoptotic and dead cells in freshly isolated

CD21-/lo naïve B cells from RA and CVID patients compared to their CD21+ B cell

counterparts. (C) BCR, CD40 and/or TLR9 triggering do not prevent the apoptosis and

cell death of CD21-/lo naïve B cells. Total naive B cells containing both CD21-/lo and

CD21+ B cells from RA01 or CVID321 patients were plated and Annexin V and PI

staining was assessed after 12 hours. BCR, CD40 and TLR9 stimulation prevent late

apoptosis and cell death in CD21+ but not in CD21-/lo B cells.

Figure 6. Gene array comparisons of CD21-/lo and CD21+ naive B cells from RA

and CVID patients using the Affimetrix Human Genome U133 Plus 2.0 Array. (A)

Transcripts differentially expressed by CD21-/lo and CD21+ naive B cells from 2 RA and 3

CVID group Ia patients are shown. (B) Selected transcripts well known to B cell biology

or of potential interest are presented. Up- and down-regulated transcripts are indicated

in red and green, respectively. The magnitude of expression is depicted by the color

bar.

Figure 7. CD21-/lo B cells from RA and CVID patients display a phenotype

reflecting gene array profiling data. Overlays for the indicated markers were obtained

when CD27-depleted B cells isolated from the blood of patient RA01 were gated on

either CD19+CD21-/lo (bold line) or CD19+CD21+ (dashed line) B cells. The similar

phenotypes of CD21-/lo B cells from CVID patients and healthy donors are shown in

supplemental Figure S7-S8.




Complement receptor 2/CD21- human naive B cells contain mostly autoreactive unresponsive clones

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