elimination of autoreactive b cells in humanized scid mouse model of sle

Elimination of autoreactive B cells in humanized SCIDmouse model of SLE

Nikola S. Kerekov1, Nikolina M. Mihaylova1, Ivan Grozdev2,

Todor A. Todorov3, Milena Nikolova4, Marta Baleva5, Maria Nikolova6,

Jozsef Prechl7, Anna Erdei7 and Andrey I. Tchorbanov1

1 Department of Immunology, Stefan Angelov Institute of Microbiology, Bulgarian Academy of

Sciences, Sofia, Bulgaria2 Department of Dermatology, Medical Faculty, Medical University, Sofia, Bulgaria3 Department of Pathology, Sofia Medical School, Sofia, Bulgaria4 Department of Nephrology, University Hospital Alexandrovska, Medical University, Sofia,

Bulgaria5 Department of Clinical Immunology, University Hospital Alexandrovska, Medical University,

Sofia, Bulgaria6 National Reference Laboratory of Immunology, National Center of Infectious and Parasitic

Diseases, Sofia, Bulgaria7 Research Group of the Hungarian Academy of Sciences at the Department of Immunology,

Eotvos Lorand University, Budapest, Hungary

Although the exact etiology of systemic lupus erythematosus (SLE) remains elusive, B-cell

hyperactivity and production of autoantibodies directed to components of the cell nucleus

are a well-established pathogenetic mechanism of the disease. Therefore, the targeted

inhibition of DNA-specific B cells is a logical therapeutic approach. The complement

receptor type 1 (CR1, CD35) has been shown to suppress human B-cell activation and

proliferation after co-cross-linking with the BCR, and may serve as a mediator for negative

signal delivery. In order to evaluate this therapeutic approach in a human-like system, we

used immune-restricted SCID mice transferred with PBMCs from SLE patients. The toler-

ance of these humanized SCID mice to native DNA was re-established after administration

of a chimeric molecule consisting of a CR1-specific mAb coupled to the decapeptide

DWEYSVWLSN that mimics dsDNA. The generated protein-engineered chimera was able

to co-cross-link selectively native DNA-specific BCR with the B-cell inhibitory receptor CR1,

thus delivering a strong inhibitory signal.

Key words: Chimeric molecules . Inhibitory B-cell receptors . SCID models of SLE

Introduction

The pathogenesis of systemic lupus erythematosus (SLE) is

characterized by the formation of autoantibodies against a wide

range of self-antigens. Development of this multi-system

autoimmune syndrome in many severe cases leads to mortality.

SLE patients develop high-titered anti-nuclear antibodies

(ANA), the majority of them against double-stranded (ds)

DNA. Multiple organs can be involved in human SLE (skin, lung,

heart, arteries, nervous system), but kidneys being most

severely affected. The pathological changes are provoked by

deposition of immune complexes into renal glomeruli, followed

by kidney structure damages and development of nephritis and

proteinuria [1].Correspondence: Dr. Andrey Tchorbanove-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2011. 41: 3301–3311 DOI 10.1002/eji.201141439 Immunomodulation 3301

The pathogenetic role of self-specific B cells has been attrib-

uted not only to the generation of autoreactive antibodies, but

also to their antigen-presenting functions [2, 3]. A number of

studies have clearly documented the strong correlation between

the level of these B cells and disease severity. The efficacy of

B-cell depletion therapy further supports the key role of B cells in

the pathology of SLE. Activity of any self-specific B cells is regu-

lated by the interplay of multiple factors controlling B-cell

proliferation and differentiation. BCR-mediated activation may

be counteracted by a number of inhibitory receptors: CD32

(FcgRIIb), CD22, CD5, CD72, CD66a, ILT2, PIR-B, CD279 (PD-1),

LAIR-1, as well as the complement receptor type 1 (CR1, CD35).

While a reduced expression of these receptors can result in

uncontrolled B-cell activation [4, 5] and autoimmunity, their

cross-linking inhibits B-cell activation and proliferation and may

serve for targeted suppressive signal delivery [6, 7].

Non-specific immunosuppressive drugs are by now the most

frequent therapy for autoimmune diseases, including SLE. Their

effects are purely symptomatic while most of them are associated

with severe side effects [8–11]. Recently, mAbs have been

introduced into practice as a targeted therapeutic approach.

B-cell depletion by means of anti-CD20 mAb [12–15], or inhibi-

tion of B-cell activation and proliferation by anti-CD22 mAb

Epratuzumab have been applied to lupus patients [16]. The

human Lymphostat-B mAb that blocks another important co-

stimulatory receptor, B-cell activating factor (BAFF), has been

also successfully introduced [17–19].

An alternative approach for specific targeting of B-cell

response is the delivery of negative signals. The cross-linking of

surface immunoglobulin receptors with the inhibitory Fc-gamma

IIb (FcgIIb) receptors by IgG-containing immune complexes is a

natural negative feedback mechanism of Ab production [20–22].

We constructed several chimeric antibodies by coupling the

dsDNA-mimicking peptides or peptides, parts of histone 1 mole-

cule to a rat anti-mouse FcgRIIb-binding mAb. When admini-

strated in lupus-prone MRL/lpr mice they reduced the levels of

anti-histone1 and anti-dsDNA IgG antibodies and proteinuria,

and prolonged the overall survival [23, 24].

The human CR1 (CD35) negatively regulates the proliferation

and differentiation of activated B cells after binding to its

natural ligand: the complement activation fragment C3b [6, 7,

25]. King-Konert et al. reported a fast reduction of the titer

of anti-dsDNA autoantibodies in SLE patients treated with the

chimeric molecule ETI-104, a construct of dsDNA, coupled

covalently to a murine human CR1-specific mAb [26]. However,

a chimeric mAb based on foreign DNA can hardly be standar-

dized, which restricts its therapeutic application. To circum-

vent this problem, we employed a dsDNA-mimicking decapeptide

DWEYSVWLSN, which is recognized by the pathogenic anti-

dsDNA antibodies and may be used instead of native DNA [27].

A protein chimeric molecule containing copies of the DNA-

mimotope peptide bound to anti-human CD35 mAb was

constructed, able to cross-link cell surface BCR with the inhi-

bitory CR1 on self-reactive DNA-specific B cells from SLE

patients [28].

The mouse strains ((NZB�NZW)F1, MRL/lpr, BXSB) spon-

taneously develop anti-dsDNA antibodies and an SLE-like disease,

which evokes some of the symptoms of human SLE like protei-

nuria and glomerulonephritis [29–34]. However, these animal

models do not reproduce the severe complicated clinical mani-

festation found in humans [35–38]. SCID mice suffering from

combined T and B immunodeficiency are unable to mount an

adaptive immune response. Therefore, they can be easily trans-

ferred with lymphocytes from SLE patients or from lupus-prone

mice. Further on, the successfully humanized SCID mice develop

many symptoms of the human lupus (anti-dsDNA antibodies, skin

lesions, proteinuria, and glomerulonephritis, etc.) and are a

suitable model to investigate the efficiency of therapeutic B-cell

depletion [39–41]. In the present study, we established a

humanized SCID model to investigate the effect of the treatment

with DNA-like chimera on the pathogenesis of human SLE.

Results

Chimera preparation and binding to human CR1

The successful chemical coupling of peptides to the immunoglo-

bulin molecule has been already tested by SDS-PAGE and

Western blot [28]. The exact number of DNA mimotope peptides

coupled to the single Ab was determined by mass-spectral

analysis [42]. It showed that 14–16 peptides were bound per

IgG molecule on the single DNA-like chimera (data not shown).

Using flow cytometry, we have previously demonstrated the

ability of the DNA-like chimera to bind CD35 on human B cells

[28]. In order to study the signaling pathway engaged by CR1 on

the human cell line U937, we assessed the effect of the 3D9-DNA-

like chimera in competition with the FITC-conjugated anti-CD35

Ab (Fig. 1A, upper panel) or – with an FITC-conjugated anti-

mouse IgG Ab (Fig. 1A, lower panel).

The DNA-like chimera triggers signal through CR1

The potential of the constructed chimeric molecule to provide

signals through the inhibitory CR1 on cell surface was tested

using CR11 cellline. The signal transduction was confirmed by

the observed tyrosine phosphorylation of the intracellular part of

the inhibitory receptor in CR1-expressing cell line U937 after

incubation with 3D9-DNA-like chimeric molecule (Fig. 1B).

Inhibition of cell proliferation in vitro

The ability of the 3D9-DNA-like peptide chimera to inhibit human

PBMC proliferation was studied. The three chimeric constructs:

one DNA-like and two control chimeras as well as the pure 3D9 Ab

were used for the experiment and their effects were estimated by

adding them at different concentrations to cultured PBMCs

isolated from lupus patients. Cell proliferation was evaluated by


Eur. J. Immunol. 2011. 41: 3301–3311Nikola S. Kerekov et al.3302

pulsing with 1.0mCi [3H] thymidine for the last 18 h of a 4-day

culture period and measuring the amount of DNA-incorporated

thymidine. A significant inhibition of cell proliferation was

observed using 500 ng/mL DNA-like chimera while the control

chimeras did not affect the cell growth with the same effectiveness

(Fig. 2A, upper panel). FACS analyses performed at the end point

of the experiments showed predominant increased T-cell number

in the proliferating control samples while the number of B cells

remained almost constant (data not shown). The same experiment

was reproduced using PBMCs isolated from healthy donors and no

significant differences among the groups were found (Fig. 2A,

lower panel).

Anti-dsDNA IgG Ab-secreting plasma cells are affectedby DNA-like chimera

We found that cultivation of PBMCs from lupus patients in the

presence of the human DNA-like peptide chimera resulted in a

lower number of IgG anti-dsDNA-producing plasmocytes. Our

data demonstrated that co-cross-linking of BCR and the inhibitory

CR1 on disease-associated autoreactive B lymphocytes selectively

suppressed autoantibody production (Fig. 2B, upper panel). The

strongest reduction of the number of plasmocytes secreting

dsDNA-specific IgG was observed by incubation of SLE–PBMCs

with 1000 ng/mL 3D9 DNA-like chimeric molecule. As a control,

we performed the same experiment using PBMCs isolated form

healthy donors. The presence of dsDNA-specific plasmocytes was

not detected (Fig. 2B, lower panel).

Effects of the 3D9-DNA-like chimera on SCIDstransferred with SLE–PBMCs

Human PBMCs obtained from SLE patients with high levels of

anti-dsDNA IgG antibodies or from healthy donors were

transferred to female SCID mice (Fig. 3). The successfully

engrafted SCID mice were treated either with DNA-like chimera,

irrelevant-peptide chimera or PBS, to assess in vivo the effects of

the dsDNA-specific B-cell elimination treatment.

Figure 1. DNA-like chimera binds inhibitory receptor CR1 and triggers signal transduction. (A) The constructed 3D9-DNA-like chimera binds CR1expressed on human cell line U937 (CR11) and competes with FITC-conjugated anti-CR1 Ab for the same receptor. U937 cells were incubated withDNA-like chimera (c, g), purified 3D9 Ab (d, h), or PBS only (a, b, e, f) for 30 min at 41C followed by incubation with either FITC-conjugated anti-CD35(b–d) or FITC-conjugated goat anti-mouse Ab (f–h). After washing, the cells were analyzed by flow cytometry. Data are representative of at least fiveindependent experiments. (B) 3D9-DNA-like chimeric molecules trigger signal transduction through CR1. Human CR11 cell line U937 wasincubated in the presence of the DNA-like chimera at 371C for 10, 5, 2 or 0 min. Cells cultivated in medium only were used as controls. The cellswere lysed, subjected to immunoprecipitation using anti-CR1 antibodies followed by western blotting with an anti-phosphotyrosine Ab (upperpanel) or with the anti-CR1 (3D9) Ab (lower panel). Data shown are representative of three independent experiments.


Eur. J. Immunol. 2011. 41: 3301–3311 Immunomodulation 3303

The injection of DNA-like chimera to SCID mice transferred

with PBMCs from lupus patients for 10 wk of treatment was

enough to suppress the limited number of dsDNA-specific

pathological B cells. It resulted in decrease of the IgG anti-DNA

Ab levels as compared with the irrelevant chimera-treated or PBS-

treated groups transferred with the same cells (Fig. 4A, left

panel). The control group of SCID mice transferred with PBMCs

from healthy donors was not affected (Fig. 4A, right panel).

Differences in proteinuria were also observed. In line with these

observations, the level of albuminuria in the PBS-treated or

irrelevant chimera-treated SLE PBMCs-transferred SCID mice

rapidly increased (Fig. 4B, left panel).

The group injected with DNA-like chimera had low IL-10

serum levels during the treatment, while increased amounts were

observed in the PBS and irrelevant chimera-injected animals after

the transfer (Fig. 5, left panel). The DNA-like chimera-treated

SCID mice transferred with SLE–PBMCs produced much lower

levels of IFN-g as compared with the groups of transferred SCID

mice treated with PBS and irrelevant-peptide chimera (Fig. 5,

right panel). The IL-4 production was not detected in all groups

of animals.

Administration of the DNA-like chimera significantly

decreased the immune complex depositions and improved the

kidney histology findings in the treated SLE-transferred SCIDs. At

the same time, all PBS-treated SLE-transferred SCID mice had

massive mesangial glomerular depositions of IgG-containing

immune complexes and presented massive mononuclear cell

accumulations surrounding the blood vessels (Fig. 6). In contrast,

the renal histology of chimera-treated mice was preserved even

though some infiltrate was present.

Untreated SCID mice transferred with PBMCs from healthy

donors did not develop kidney damages and IgG immune

depositions.

Discussion

The immune system protects self-structures from potentially

harmful foreign substances. Autoimmunity is an exaggerated

immune response erroneously directed against self-molecules

and therefore affecting healthy cells and tissues [1]. SLE is an

autoimmune disease characterized by the development of

Figure 2. Treatment of PBMCs from SLE patients with DNA-like chimera suppresses disease-associated B and T cells in vitro. (A) The 3D9-DNA-mimotope chimera inhibits the spontaneous T-cell proliferation in vitro. PBMCs from SLE patients or healthy donors were isolated and cultured inthe presence of either 3D9 Ab, DNA-like chimera, or control chimeras (lrrPeptide chimera and lrrAb chimera) at different concentrations. Controlsamples were cultured in ConA or in medium only. Proliferation was evaluated by [3H] thymidine incorporation. An individual test for each SLEpatient (n 5 8) and healthy donor (n 5 4) was performed. The results are expressed as the mean cpm value1SD of triplicates. ��po0.01; ��po0.002;Student’s t-test. Data are representative of at least eight independent experiments. (B) The co-crosslinking of BCR and CR1 on disease-associatedB lymphocytes down-modulates the autoreactive B-cell activation. PBMCs from SLE patients or healthy donors were cultured for 4 days in thepresence of the DNA-like chimera or control chimera. Cells cultured in medium alone were used as control. The number of plasma cells secretinganti-dsDNA-specific IgG antibodies was determined using ELISpot assays. The results are expressed as the mean value1SD of triplicates. PBMCsfrom each SLE patient and healthy donor were tested individually. The numbers of spots in the test-wells were compared with control wellscontaining irrelevant chimera-treated or medium only-cultured splenocytes (Student’s t-test; �po0.05). Data are representative of at least fiveexperiments.



autoantibodies to ds DNA and a wide range of other nuclear

antigens. These high-titer autoantibodies participate in the forma-

tion of circulating or in situ immune complexes affecting the

glomerular basal membrane. The pathophysiological role of anti-

dsDNA antibodies has been demonstrated directly by their elution

from the glomeruli of lupus patients and MRL/lpr lupus-prone mice.

DNA-specific B cells play a key role in the pathophysiology of

human and murine lupus due to three basic functions. First of all,

autoreactive B cells present self-DNA/protein complexes to

T cells, and initiate an auto-immune response thus breaking

T-cell tolerance [2, 3]. Second, they produce auto-antibodies

against the proper healthy tissues of the organism. In addition,

autoreactive B cells secrete different cytokines with regulatory

functions.

The current therapy of SLE includes non-steroid anti-inflam-

matory drugs in milder forms [8, 9], or corticosteroids in the

more severe cases. Although these treatments are effective in

terms of symptom relief, they are associated with multiple

undesirable side effects, such as internal hemorrhages and

secondary immune deficiency [10, 11].

The physical or functional depletion of B cells is a possible

way to study the contribution of B cells to the pathogenesis of

SLE. B cells express a number of inhibitory receptors suitable for

therapeutic targeting. Rituximab is a pan-B humanized mAb

Figure 3. Schematic of cell transfer from SLE patients and healthy donors to SCID mice. 1� 107 PBMCs were transferred into each SCID mouse.



Figure 4. Administration of DNA-like chimera decreases anti-dsDNA IgG Ab production and proteinuria in humanized SLE-SCID mice. IsolatedPBMCs from 18 untreated SLE patients and 8 healthy donors were used for cell transfer into SCID mice. Each group of SLE- or healthy donor-transferred SCID mice was separated into three subgroups, that were treated weekly with either DNA-like chimera (50 mg/mouse i.v.), the sameamount of irrelevant-peptide chimera, or PBS alone. (A) Anti-dsDNA IgG antibodies and (B) proteinuria were evaluated. The data are represented asmean1SEM of 16-25 mice per group; p-values are calculated using the Mann–Whitney U test (�po0.05; ��po0.01; ��po0.002), in comparison toPBS-treated PMBC-transferred controls.

Figure 5. Reduction of serum IL-10 and IFN-g in DNA-like chimera-treated SLE-transferred SCID mice. 1� 107 PBMCs from SLE patients weretransferred into SCID mice, which were then treated as in Fig. 4. Serum levels of human (A) IL-10 and (B) IFN-g were measured weekly by sandwichELISA. The data are represented as mean1SEM of 16–25 mice per group; p-values are calculated using the Mann–Whitney U test (�po0.05; ��po0.01;��po0.002), in comparison to PBS-treated PMBC-transferred controls.



(anti-CD20) that has been applied for total B-cell depletion in

multiple autoimmune diseases. However, in spite of a certain

beneficial effect in SLE patients, Rituximab cannot eliminate the

CD20� long-lived plasma cells [12–15].

The specific elimination of disease-associated B cells alone is a

more ambitious task. The co-cross-linking of an inhibitory receptor

with BCR on the auto-reactive B cells could provide the requested

specificity. To this end, we have engineered a chimeric Ab

consisting of histone 1-peptides or dsDNA-mimicking peptides

coupled to a rat anti-mouse FcgRIIb-specific mAb [23, 24]. Immu-

nization of lupus-prone MRL/lpr mice with these chimeric mole-

cules reduced the levels of dsDNA- and histone-specific

autoantibodies, prevented the proteinuria rise and kidney damages.

Using a similar approach, King-Konert et al. applied dsDNA

coupled to a murine anti-human CR1 mAb (ETI-104) for treat-

ment of SLE patients [26]. In this case, the chimeric mAb binds

simultaneously to circulating anti-dsDNA antibodies and the CR1-

expressing erythrocytes of the patients, leading to the formation

of immune complexes, and their enhanced elimination in the

liver or spleen. In a humanized SCID model of SLE, however, the

murine erythrocytes would not be recognized by anti-human CR1

mAbs. Therefore, the only plausible mechanism of the treatment

effect we observed could be a CR1-mediated B-cell-inhibitory

signaling. The chimeric molecule is able to bind human CR1, and

provide signal transduction via this receptor, thus suppressing the

proliferation and differentiation of dsDNA-specific B cells at any

CR11 stage of their differentiation.

To demonstrate the specificity of the DNA-like chimera, we

tested its suppressive effect on tetanus or diphtheria toxoid-

specific B cells, isolated from the same SLE and healthy donors

(with corresponding immunization history) and boosted in vitro

with the respective antigen. No inhibition of plasmocytes secret-

ing anti-tetanus or anti-diphtheria toxoid IgG antibodies was

detected with ELISpot (data not shown).

As far as B cells play an antigen-presenting role, T-cell acti-

vation is also affected. The performed in vitro experiments have

proven the therapeutic potential of this chimeric molecule. The

ability of the DNA-like chimera to affect T cells to which it does

not bind directly could be explained as follows. The role of

disease-associated B cells in lupus is not limited to them being

precursors of pathological autoantibody-producing plasma cells.

The group of M. Schlomchik has constructed lupus-prone mice

unable to produce circulating immunoglobulins while still

developing SLE symptoms. Only strains completely deprived of

Figure 6. Administration of DNA-like chimera preserves renal histology and inhibits immune-complex deposition in kidneys of humanized SLE-SCID mice. (A) Analysis of glomerular IgG depositions in the kidneys of untreated SCID mice (a); PBS-treated SCID mice transferred with SLE-PBMCs (b); DNA-like chimera-treated SCID mice transferred with SLE-PBMCs (c); Irrelevant chimera-treated SCID mice transferred with SLE-PBMCs(d); PBS-treated SCID mice transferred with PBMCs from healthy donors (e) and DNA-like chimera-treated SCID mice transferred with PBMCs fromhealthy donors (f). Cryostat sections were stained with FITC-conjugated goat-anti-human IgG. Representative images are shown. Scale bar, 200mm.Original magnification 400�. (B) Histological examination of paraffin-embedded kidney sections from untreated SCID mice (g); PBS-treated SCIDmice transferred with SLE-PBMCs (h); DNA-like chimera-treated SCID mice transferred with SLE-PBMCs (i); Irrelevant chimera-treated SCID micetransferred with SLE-PBMCs ( j); PBS-treated SCID mice transferred with PBMCs from healthy donors (k) and DNA-like chimera-treated SCID micetransferred with PBMCs from healthy donors (l). Standard haematoxylin/eosin staining technique was used. Representative images are shown.Scale bar, 200 mm. Original magnification 250�.



B cells (in the presence of normal T-cell numbers) developed no

signs of SLE, revealing the important additional Ab-independent

(antigen-presenting) role of B cells in SLE [3]. This often

neglected role of disease-associated B cells could explain the

observed inhibition of T-cell proliferation after incubation of

PBMCs with the DNA-like chimera. The silencing of pathological

dsDNA-specific B cells is expected to down-modulate the activa-

tion and number of T cells recognizing nucleosomes (the primary

disease-inducing self-antigen [43]).

It should be noted that both control chimeras contain some

active components with possible suppressive effect. The irrele-

vant peptide chimera contains an anti-CR1 Ab that may suppress

any cell expressing this receptor, while the irrelevant Ab chimera

contains a number of DNA-like peptides with suppressive effect

on anti-dsDNA Ab-secreting B cells [27]. Under in vitro condi-

tions all potential targets are directly accessible which can

explain the partial suppression observed (Fig. 2A and B). The

high specificity of the DNA-like chimera is expected to be

advantageous under in vivo conditions, at low concentrations and

accessibility of the targeted autoreactive B cells.

Our autoimmune model system, i.e. SCID mice reconstituted

with PBMCs from SLE patients, was characterized by high levels

of dsDNA-specific antibodies, increased urine protein concentra-

tion, and human immunoglobulin glomerular deposits. In addi-

tion, the SLE-PBMC transferred animals developed skin lesions.

At the same time, SCID mice reconstituted with healthy donor

PBMCs did not develop any of the above symptoms. The treat-

ment of SLE–PBMC transferred mice with a weekly i.v. injection

of 50 mg anti-human DNA-like chimera prevented the appearance

of the symptoms described. In contrast, a control groups injected

with PBS alone or irrelevant peptide chimera, developed signifi-

cantly elevated levels of anti-dsDNA antibodies, glomerular

depositions of IgG-containing immune complexes and protei-

nuria. The cytokine profiles of the treated animals correlated

with the results obtained. The DNA-like chimera-treated group

had very low IFN-g and IL-10 production as compared with PBS

and irrelevant chimera controls. A weak and short-lasting

suppression of IL-10 and IFN-g production was observed in the

irrelevant chimera–treated group as a result of non-specific T-cell

inhibition.

The presented transferred SCID model of human SLE is a

novel approach exploring the therapeutic effects of specific

autoreactive B-cell elimination. Using this model, we demon-

strated that protein-engineered CR1-specific chimeric mAbs may

signal negatively the disease-associated B-lymphocytes and thus

provide specific pathogenetic therapy for human SLE.

Materials and methods

mAbs

Purified mouse 3D9 IgG1 mAb specific to human complement

receptor type I (CD35), and mouse monoclonal IgG1 Ab with

irrelevant (anti-influenza A virus) specificity were prepared as

described [28]. FITC-conjugated 3D9 (Pharmingen BD, San

Diego, CA, USA) and FITC-conjugated anti-mouse IgG (Sigma-

Aldrich, Taufkirchen, Germany) were used for FACS experiments.

FITC-conjugated anti-human IgG (Sigma) was used for histolo-

gical staining.

Chimeric Ab molecules

A DNA-mimicking peptide (Ac-DWEYSVWLSN-Ahx-K-NH2) and

an irrelevant peptide containing the same aminoacids in a

shuffled order (Ac-WSLDYWNEVS-Ahx-K-NH2) were purchased

from Caslo Laboratory (Lyngby, Denmark). Three chimeric Ab

molecules were constructed from different peptide/Ab combina-

tions as previously described [23], using the classical EDC

(1-ethyl-3(30-dimethylaminopropyl) carbodiimide.HCl), (Fluka

AG, Buchs, Switzerland) cross-linking technique [44]. Briefly,

during the peptide synthesis an Ahx linker with lysine was added

to the peptides C-end. The Ab and peptide were mixed at a 20-

fold molar excess of the peptide and 60-fold molar excess of

carbodiimide. The reaction mixture was stirred overnight at 41C,

dialized against PBS and concentrated by ultrafiltration. The

chimeric molecules were as follows: a DNA-like chimera,

consisiting of the DNA-mimicking peptide and the anti-human

CR1 mAb 3D9, an irrelevant mAb chimera – consisting of the

DNA-mimicking peptide and the irrelevant Ab, and an irrelevant

peptide chimera, comprising the irrelevant peptide bound to the

3D9 mAb.

SLE patients and healthy blood donors

The study comprised 18 recently diagnosed untreated SLE

patients (female to male ratio 14–4; mean (min–max) age

(22–48). Inclusion criteria were: at least four ARA (American

Rheumatism Association) criteria for SLE, combined with high

titers of anti-nuclear and anti-dsDNA IgG antibodies. The control

group consisted of eight age- and sex-matched healthy donors.

Permission from local Ethical Committee and Informed consent

were obtained in all cases.

PBMCs from patients and controls were isolated by the stan-

dard Ficoll-Paque technique (Amersham Bioscience, Sweden)

and cultured in RPMI-1640 (Gibco, Gaithersburg, MD, USA)

containing 10% FCS, 4 mM L-glutamine, 50 mM 2-mercaptoetha-

nol and antibiotics in 5% CO2 at 371C.

Mice

Female SCID mice (8 wk old; Balb/c background) were obtained

from Harlan Farm, (Blackthorn, UK). The animals were raised

under specific-pathogen-free (SPF) conditions. All manipulations

were approved by the Animal Care Commission at the Institute of

Microbiology in accordance with the national regulations.



Flow cytometry analysis

The specificity of the DNA-like chimera was tested on the CR1-

expressing human lymphoma cell line U937 (ATCC CRL-1593.2)

[45] Briefly, 1�106/mL U937 cells were double-washed with

PBS and incubated with the DNA-like chimeric molecule, or with

unconjugated 3D9 mAB (1 mg/106 cells), or with PBS alone for

30 min at 41C, followed by two washes and a second incubation

with FITC-conjugated 3D9 Ab or anti-mouse IgG for 30 min at

41C. Ten thousands cells were analyzed from each sample with a

BD LSR II flow cytometer using the Diva 6.1.1. software (BD

Biosciences, San Jose, CA, USA).

Proliferation assays

PBMCs from lupus patients or healthy donors were isolated and

cultured (1� 106/mL) in complete RPMI 1640 medium in the

presence of increasing concentrations of the DNA-like chimera or the

control chimeras (ranging from 4 to 2500ng/mL) at 371C/5% CO2.

Cells stimulated with 10mg/mL Concanavalin A (Boehringer

Mannheim, Germany) or cultured in medium only were used as

controls. After 3 days of cultivation [3H] thymidine was added (1mCi/

well). Cells were harvested on glass fiber filters and [3H] thymidine

incorporation in DNA was measured in a liquid scintillation counter.

At the end of cultivation the proliferated cells were measured by FACS

using anti-CD19-PE and anti-CD4-FITC mAbs (Pharmingen BD).

ELISpot assay for counting specific anti-DNAAb-secreting cells

PBMCs obtained from lupus patients or healthy donors were

isolated and cultured (2� 106/mL) in complete RPMI 1640

medium (see above). The cells were cultured for 96 h in the

presence of increasing concentrations (ranging from 40 to

1000 ng/mL) of the DNA-like chimera, or of irrelevant peptide

chimera, or of medium alone at 371C.

Later, ELISpot 96-well plates (Millipore, Bedford, MA, USA)

were coated with 10mg/mL calf thymus dsDNA for 30 min at room

temperature, washed with PBS, and blocked with 1% gelatin in PBS.

The pre-cultured cells were transferred to ELISpot plates with the

DNA-coated membranes and were further cultured for 4 h in a

humidified 5% CO2 atmosphere at 371C. Next, the plates were

washed, incubated with an anti-human IgG conjugated with alka-

line phosphatase (Sigma) for 2 h and developed by NBT-BCIP

substrate (Sigma). The number of spots corresponding to cells

producing IgG anti-dsDNA antibodies was counted by C.T.L

Immunospot S5 Versa Analyzer (Bonn, Germany).

Signaling

U937 cells (1� 107/mL) were incubated on ice for 20 min in the

presence of 30mg/mL of the DNA-like chimera followed by

incubation at 371C for 0, 2, 5 or 10 min. Cells cultivated in medium

only were used as controls. The reaction was stopped with cold PBS

and the cells were centrifuged for 3 min at 400� g. The pellet was

treated with lysis buffer (10 mM Tris base pH 7.4, 50 mM NaCl, 1%

Triton X-100, 1 mM sodium orthovanadate, 50 mM NaF, 25 mM

sodium pyrophosphate, 5 mM EDTA, 10mg/mL Pepstatin–protease

inhibitor cocktail tablets) for 45 min at 41C. The lysate was

centrifuged (16 000� g, 15 min at 41C), pre-cleared on Protein

G-Sepharose (Amersham Biosciences, UK) for 1 h on a rotating

wheel at 41C and immunoprecipitated with anti-CR1 antibodies

bound to protein G-Sepharose beads (Sigma) overnight on a

rotating wheel at 41C. Immunoprecipitates were washed three times

with lysis buffer, subjected to 10% sodium dodecyl sulfate

polyacrylamide gel electrophoresis on two parallel gels and

transferred to two nitrocellulose membranes (0.45mm, Sartorius,

Germany). For the detection of phosphorylated tyrosine residues,

the first membrane was blocked for 2 h in TBS, pH 7.4 with 0.05%

Tween 20 and 5% BSA, incubated further with a mouse anti-

phosphotyrosine Ab (from R&D Systems, Minneapolis, MN, USA)

and finally with a goat anti-mouse IgG-HRP Ab (Sigma). Tyrosine

phosphorylation was evaluated using the ECL technique.

The second membrane was blocked overnight at 41C in TBS,

pH 7.4 containing 0.05% Tween-20 and further incubated in an

optimal dilution of the anti-CR1 Ab, followed by goat anti-mouse

IgG Ab, conjugated to alkaline phosphatase (Sigma) and devel-

oped.

Cell transfer

Groups of female SCID mice (n 5 4–6 per donor) were prepared

for human cell transfer. PBMCs, obtained from lupus patients or

healthy donors were isolated as described (see above). The cells

from each donor were separated into equal parts and a total of

1� 107 cells were transferred i.p. to each SCID mice.

Treatment schedule

Each group of SLE- or healthy donor-transferred SCID mice was

separated into three subgroups. The first subgroup was injected

i.v. once a wk in the course of 10 wks with 50mg per mouse of the

DNA-like chimera. The second group was treated with 50 mg per

mouse of the irrelevant-peptide chimera, and the third one was

injected with PBS. Every seven days the animals from all groups

were bled and the sera were kept frozen at �701C.

Detection of anti-dsDNA antibodies

Serum levels of IgG anti-dsDNA antibodies were evaluated by

ELISA as previously described [23]. Briefly, 96-well Maxisorp

immunoplates (Nunc, Roskilde, Denmark) were coated with

methylated bovine serum albumin (from Calbiochem, Darmstadt,

Germany, 10 mg/mL PBS) followed by overnight incubation with



S1-nuclease-treated calf thymus DNA (Sigma) at 2.5 mg/mL at

41C. The plates were further blocked with 1.0% gelatin,

incubated with serial dilutions from murine sera, followed by

peroxidase-conjugated anti-mouse IgG Ab (Pharmingen BD).

Reaction was revealed with ABTS (2,20-azino-bis(3-ethyl benz-

thiazoline-6-sulfonic acid) solution (Sigma) and read at 405 nm.

For anti-DNA IgG Ab determination the OD values obtained from

the separate experiments were normalized to a single standard

SLE-positive control serum used in every assay.

Proteinuria measurement

The levels of proteinuria were determined weekly with Combi-

screen strips (Analyticon Biotechnologies, Lichenfels, Germany)

and classified semi-quantitatively as follows (0 – none; 1 –

30–100; 2 – 100–300; 3 – 300–500 and 4–500 mg/dL).

Cytokine detection

Human IL-4, IL-10, and IFN-g levels were measured in mouse

sera using human High Sensitivity (HS) ELISA kits (Bender

MedSystems, Vienna, Austria).

Evaluation of glomerular IgG depositions

One kidney from each treated mouse was snap-frozen, cryostate

sections were stained with FITC-conjugated anti-human IgG and

analyzed under a fluorescent microscope (Carl Zeiss, Jena,

Germany); paraffin sections from the other kidney were analyzed

using a standard haematoxylin/eosin staining technique.

Statistical analysis

Values in the figures correspond to mean7SD. The unpaired

Student’s t-test was used to determine differences between each

two groups. The two-tailed Mann–Whitney U test was used when

appropriate. A value of po0.05 was considered as statistically

significant.

Acknowledgements: This study was supported by the Bulgarian

National Science Fund – grants VU-704, VUH-11 and TK-317 (all

to A. Tchorbanov).

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: CR1: Complement receptor type 1 (CD35) � SLE: systemic

lupus erithematosus

Full correspondence: Dr. Andrey Tchorbanov, Stefan Angelov Institute of

Microbiology, Acad. G. Bonchev Street, Block 26, 1113 Sofia, Bulgaria

Fax: 1359-2-870-0109

e-mail: [email protected]

Received: 21/1/2011

Revised: 24/6/2011

Accepted: 3/8/2011

Accepted article online: 10/8/2011



elimination of autoreactive b cells in humanized scid mouse model of sle

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