Suppression of dsDNA-specific B lymphocytes reduces disease symptoms in SCID model of mouse lupus

http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, Early Online: 1–11 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2014.883502

ORIGINAL ARTICLE

Suppression of dsDNA-specific B lymphocytes reduces disease symptoms in SCID model of mouse lupus Vera Gesheva1, Nikola Kerekov1, Kalina Nikolova1, Nikolina Mihaylova1, Todor Todorov2, Maria Nikolova3, and Andrey Tchorbanov1 Department of Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria, 2Department of Pathology, Sofia Medical School, Sofia, Bulgaria, and 3National Reference Laboratory of Immunology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria

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Abstract

Keywords

Self-specific B cells play a main role in the pathogenesis of lupus. This autoimmune disease is characterized by the generation of autoantibodies against self antigens, and the elimination of B and T cells involved in the pathological immune response is a logical approach for effective therapy. We have previously constructed a chimeric molecule by coupling a DNA-mimotope peptides to an anti-CD32 antibody. Using this protein molecule for the treatment of lupusprone MRL/lpr mice, we suppressed selectively the autoreactive B-lymphocytes by cross-linking B cell receptors with the inhibitory FcgRIIb receptors. This approach was limited by the development of anti-chimeric antibodies in MRL mice. In order to avoid this problem, we established a murine severe combined immunodeficiency lupus model, allowing a long-term chimera therapy. Elimination of the double-stranded DNA-specific B cells by chimera therapy in MRL-transferred immunodeficient mice resulted in inhibition of T cell proliferation and prevented the appearance of IgG anti-DNA antibodies and of proteinuria.

Chimeric molecules, inhibitory receptors, MRL/lpr mice, SCID mice, SLE

Introduction Systemic lupus erythematosus (SLE) is a polygenic autoimmune disease characterized by B and T cell hyperactivity leading to the generation of pathogenic autoantibodies, immune complexes deposition and clinical involvement of multiple organs. The current therapies of SLE are nonspecific, and more precise approaches targeting the diseaseassociated B and T lymphocytes only are needed. The formation of antibodies binding to double-stranded (ds) DNA plays an important role in the pathogenesis of SLE [1]. The high affinity and complement-fixing capability of these autoantibodies contributes to their pathogenicity. Antibodies to dsDNA have been eluted from the glomeruli of SLE patients and from the glomeruli of severe combined immunodeficiency (SCID) mice transferred with human hybridoma cells secreting anti-dsDNA IgG, supporting their role in tissue damage and strong correlation with the disease activity [2–4]. The B and T cells involved in the development of SLE play diverse roles. Autoreactive B cells have a regulatory function in the pathogenesis of autoimmune diseases, not only as

Correspondence: Dr. Andrey I. Tchorbanov, PhD, Stefan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Block 26, 1113 Sofia, Bulgaria. Tel: + 359 2 979 6357. Fax: +359 2 870 0109. E-mail: [email protected]

History Received 9 October 2013 Revised 18 November 2013 Accepted 11 January 2014 Published online 5 February 2014

potential autoantibody producing cells but also as important autoantigen-presenting cells [5,6]. Interaction of pathological B cells with self antigens provides transmembrane signal transduction through the B cell receptor. As a result, specific genes involved in the activation, anergy or apoptosis of B cells are modulated. Activated autoreactive B cells could promote the loss of T cell tolerance and modify T cell cytokine producing ability. On the other hand, autoreactive CD4+ T cells could interact with self peptides presented by B cells and deliver stimulating factors allowing autoreactive B cell proliferation and differentiation to autoantibody producing plasmocytes [7]. T cell-secreted pro-inflammatory cytokines also mediate disease progression by several B cell-independent mechanisms [8]. Many studies have described the positive effect of B cell depletion in murine and human lupus by treatment with antiCD20 antibodies [9–12]. However, the potential risk of total B cell depletion is still under discussion. The co-crosslinking of the low affinity inhibitory receptor for IgG (FcgRIIb) with B cell receptor (BCR) negatively regulates BCR signaling through the immunoreceptor tyrosine-based inhibitory motifs in the cytoplasmic tail of the former and inhibits BCR-induced cellular proliferation and other downstream biological responses [13]. Depending on its expression level and functionality, FcgRIIb may either enhance or suppress autoimmunity [14,15]. Furthermore, in contrast to CD20, FcgRIIb is expressed on plasma cells, and their activity can also be controlled via this inhibitory

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receptor [16]. Thus, FcgRIIb is an attractive target for downregulation of autoimmunity. Intravenous immunoglobulin (IVIg) preparations are known to modulate autoimmune diseases via several F(ab’)2- and Fc-dependent mechanisms [17,18]. Some of the positive effects are associated with the involvement of inhibitory FcgRIIb [19,20]. We have previously shown that the treatment of B-lymphocytes from lupus-prone and from healthy mice with IVIg results in an increased expression of their surface FcgIIb receptors [21]. In this study, we test the effect of IVIg treatment on autoreactive B cells transferred in SCID mice, and how this treatment affects FcgRIIb as a target molecule. Mouse models of autoimmunity are a tool for understanding human SLE. In the past decades, several murine strains spontaneously developing SLE-like syndrome, such as (NZBxNZW)F1 (New Zealand Black/White), MRL/lpr, pristane-treated Balb/c and BXSB mice, have been used [22]. Among them, lupus-prone MRL/lpr mice develop autoimmune syndromes closely resembling human SLE with multiple organ involvement: presence of different autoantibodies to nuclear components, mononuclear cell infiltration, massive T cell lymphadenopathy, splenomegaly, immune complex deposition and nephritis [23–25]. SCID mice, lacking both T- and B-lymphocytes and tolerating xenogenic cells, have been widely used for transfer of lymphocytes from SLE patients or from lupus-prone mice. SCID mice are perfect recipients and can develop autoimmune syndromes transferred by the pathogenic donor cells [26–28]. Iwai et al. have developed an autoimmune SCID model by injection of MRL/lpr splenocytes [29]. In this study, we established an autoimmune model by transferring purified B and T cells from MRL/lpr mice to SCID mice. The decapeptide DWEYSVWLSN is a dsDNA-mimicking peptide, which is recognized by anti-dsDNA antibodies. When used for immunization of non-autoimmune mice, the peptide induced production of anti-dsDNA IgG antibodies [30,31]. By coupling the dsDNA-mimicking peptides to a rat anti-mouse FcgRIIb monoclonal antibody (mAb), we constructed a chimeric antibody targeting specifically the disease-associated B-lymphocytes. Using this chimeric molecule, we have already demonstrated a successful suppression of dsDNA-specific B cells and prevention of the appearance of autoantibodies in intact MRL/lpr autoimmune mouse model [1]. However, after 8 weeks of treatment, all mice developed anti-chimeric immunoglobulin antibodies, which excluded the further intravenous administration of the chimera to lupus-prone mice. Thus, due to the neutralization of chimera, the newly formed mature pathological B cells remained unsuppressed, limiting the investigation of autoreactive B–T cell interactions. Furthermore, in an MRL transferred SCID model, we demonstrate that purified MRL B and T cells alone can provoke autoimmune symptoms. As there is no limit for immunizing SCID mice with mAb of foreign origin, using the chimeric molecule, we were able to suppress all potentially pathogenic cells in the MRL-transferred SCID model. Under these specific conditions, autoreactive B cells are the only antigen-presenting cells; their elimination affects the proliferation of autoreactive T cells and, hence disease progression.

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Materials and methods Antibodies Purified rat 2.4G2 IgG2b mAb, specific to the mouse FcgRII (CD32), and rat I/9 monoclonal anti-idiotype IgG2b antibody were prepared as described [1]. Fluorescein isothiocyanate (FITC)-conjugated anti-CD3 and 2.4G2 and phycoerythrin (PE)-conjugated anti-CD19 mAbs (Pharmingen BD, San Diego, CA) were used for fluorescence-activated cell sorting (FACS) experiments. FITC-conjugated anti-mouse IgG (Sigma-Aldrich, Taufkirchen, Germany) was used for histological staining.

Immunoglobulin preparations Pooled human IgG Sandoglobulin (IVIg) was purchased from ZLB Behring (Bern, Switzerland).

Mice Female 7 weeks old MRL/lpr and SCID (Balb/c) mice were obtained from Harlan Farm (Blackthorn, UK). The animals were kept under specific-pathogen-free conditions, and the manipulations were approved by the Animal Care Commission at the Institute of Microbiology in accordance with the national regulations.

Construction of chimeric molecules The chimeric antibody molecules were constructed as described [1]. Briefly, two synthetic peptides – the DNA mimotope peptide (DWEYSVWLSN) and an irrelevant peptide containing the same aminoacids but in a shuffled order (WSLDYWNEVS) were used in the study. The peptides Ac-DWEYSVWLSN-Ahx-K-NH2 and Ac-WSLDYWNEVSAhx-K-NH2 were purchased from Caslo Laboratory (Lyngby, Denmark). The peptides were purified (98% purity) by high-performance liquid chromatography (HPLC). Copies of DNA-mimicking peptide were coupled to the 2.4G2 mAb (DNA chimera) and to the irrelevant I/9 mAb (control chimera 2). The irrelevant peptide WSLDYWNEVS was coupled to the 2.4G2 mAb (control chimera 1). The coupling of the antibodies to the peptides was carried out using the classical 1-ethyl-3(30 -dimethylaminopropyl) carbodiimideHCl (Fluka AG, Buchs, Switzerland) crosslinking technique [32]. During the synthesis of the peptides, an Ahx linker with lysine was added to their C-end. The antibody was mixed with a 20-fold molar excess of the peptide. The chemical reaction was started by the addition of carbodiimide at 60-fold molar excess over the antibody. The reaction mixture was stirred overnight at 4  C, dialized against phosphate-buffered saline (PBS) and concentrated by ultrafiltration. The endotoxin content in the final chimera solutions was determined by a Limulus amebocyte lysate assay (E-Toxate, Sigma-Aldrich). Proliferation assays Splenocytes from 14 weeks old female MRL/lpr mice were isolated and cultured (1.106 per ml) in complete Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Gaithersburg, MD) containing 10% fetal calf serum (FCS),

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4 mM L-glutamine, 50 mM 2-mercaptoethanol and antibiotics in the presence of increasing concentrations of the DNA-like chimera or the control chimeras (ranging from 4 ng/ml to 2500 ng/ml) at 37  C/5% CO2. Cells stimulated with 10 mg/ml Concanavalin A (Boehringer, Mannheim, Germany) or cultured in medium only were used as controls. After 3 days of cultivation, [3H] thymidine was added (1 mci/ well). Cells were harvested on glass fiber filters and [3H] thymidine incorporation in DNA was measured in a liquid scintillation counter. In a separate experiment, the same cells were incubated for 4 days in the presence of increasing concentrations of the DNA-like chimera or medium only and subsequently with anti-CD19-PE or anti-CD3-FITC mAbs (1 mg/106 cells) for 30 min at 4  C, followed by two washes. Ten thousand cells were analyzed from each sample with a FACSCanto II flow cytometer using the Diva 6.1.1. software (BD Biosciences, San Jose, CA).

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Flow cytometry The level of FcgRII expression on B cells was determined by flow cytometry. Groups of female MRL/lpr or SCID mice (five animals for each group) were injected i.p. with 10 mg (500 mg/kg) IVIg. Control groups were injected with PBS alone. Twelve hours later, the splenocytes were harvested aseptically. In a separate experiment, splenocytes (2  106 cells/ml) from 14 weeks old female MRL/lpr mice with progressive disease or from SCID mice were isolated and cultured in complete RPMI 1640 medium (see above) in the presence of 0.04 mg/ml IVIg at 37  C/5% CO2 for 4 h. The spleen cells from both experiments were washed with ice-cold PBS (containing 2.5% FCS and 0.05% sodium azide) and incubated with the FITC-conjugated 2.4G2 and PEconjugated anti-CD19 mAbs (1 mg/106 cells) for 30 min at 4  C, followed by two washes. Ten thousand cells were analyzed from each sample with a FACSCanto II flow cytometer using the Diva 6.1.1. software (BD Biosciences).

Signal transduction Murine B lymphoma cell line A20 (FcgRII positive) or its FcgRII – negative form IIA1.6 (at 1  107/ml) were incubated on ice for 20 min in the presence of 30 mg/ml of the DNA-like chimera followed by incubation at 37  C (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 ethylenediaminetetraacetic acid and 10 mg/ml pepstatin – protease inhibitor cocktail tablets) for 45 min at 4  C. Supernatants were collected after centrifugation for 15 min at 4  C at 16 000 g. Aliquots of the lysate were pre-cleared on Protein G Sepharose (Amersham Biosciences, Buckinghamshire, UK) for 1 h on a rotating wheel at 4  C and were subjected to immunoprecipitation with antibodies to CD32 bound to protein G Sepharose beads (from Sigma-Aldrich). Immunoprecipitation was performed overnight on a rotating wheel at 4  C; and after that, the beads were washed three times with lysis buffer. Immunoprecipitates were boiled for 10 min in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with b-mercaptoethanol, run on two parallel 10% SDS-PAGE gels and transferred to two nitrocellulose membranes (0.45 mm, Sartorius, Go¨ttingen, Germany). For the detection of phosphorylated tyrosine residues, the first membrane was blocked for 2 h in Tris-buffered saline (TBS), pH 7.4 with 0.05% Tween 20 and 5% bovine serum albumin (BSA), incubated further with a mouse antiphosphotyrosine antibody (from R&D Systems, Minneapolis, MN) and finally with a goat anti mouse horseradish peroxidase antibody (Sigma-Aldrich). Tyrosine phosphorylation was evaluated using the enhanced chemiluminescence technique. The second membrane was blocked overnight at 4  C in TBS, pH 7.4 containing 0.05% Tween-20 and further incubated in an optimal dilution of the 2.4G2 antibody, followed by goat anti-rat IgG antibody, conjugated to alkaline phosphatase (Sigma-Aldrich) and developed.

A transfer model establishment and cell transfer Groups of 20 male or 20 female SCID mice were separated into equal male and female subgroups (10 animals each). Splenocytes isolated from 14 weeks old female or male MRL/ lpr mice were used for further purification. B cells were purified using a mouse B lymphocyte enrichment set DM (BD IMagTM, BD Bioscience) and T cells were enriched by a mouse CD4 + T cell isolation kit (Miltenyi Biotec, Gladbach, Germany). The purified B and T cells from male or female mice were combined in 1:10 ratio and distributed to one male and one female SCID group, respectively. A total of 3  107 cells were transferred i.p. to each SCID mice. Treatment schedule Five groups of female SCID mice (n ¼ 6–10) transferred with 3  107 female MRL/lpr murine splenocytes were constituted. The first group was injected i.p. with 500 mg/kg of IVIg; and 12 h later, the animals were further injected i.v. with 50 mg per mouse of the DNA-like chimera (the treatment was once weekly in the course of 10 weeks). The animals from the second transferred group were immunized with the same amount of DNA-like chimera only without IVIg pretreatment, the third group was treated with IVIg only and the animals from the forth group – with PBS. The control group of SCID mice was immunized with IVIg without being transfer with MRL/lpr cells. Every 7 d the animals from all groups were bled, and the sera were kept frozen at 70  C. Proteinuria measurement Proteinuria was measured every week using Combi-screen strips (Analyticon Biotechnologies, Lichenfels, Germany) and graded semi-quantitatively (0: none; 1: 30–100; 2: 100–300; 3: 300–500 and 4: 4500 mg dl1). Cytokine detection Interleukin (IL)-4, IL-10 and interferon (IFN)-g levels were measured in mouse sera using enzyme-linked immunosorbent assay (ELISA) sets (Bender MedSystems, Vienna, Austria).

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Assays for glomerular IgG deposition One kidney from each treated mouse was frozen, and the cryostat sections were stained with an FITC-conjugated antimouse IgG. The depositions of immune complexes in glomeruli were analyzed under a fluorescent microscope (Carl Zeiss, Jena, Germany). The second kidney of the animals was fixed, included in a paraffin block and stained with haematoxylin/eosin using standard methods for histopathological examination.

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ELISA for anti-DNA antibodies The methodology was performed as previously described [1]. Briefly, 96-well Maxisorp immunoplates (Nunc, Roskilde, Denmark) were coated with methylated BSA (from Calbiochem, Darmstadt, Germany, 10 mg/ml PBS) followed by incubation with S1-nuclease-treated calf thymus DNA (Sigma-Aldrich) at a concentration of 2.5 mg per ml PBS overnight at 4  C. The plates were then blocked with 1.0% gelatin, incubated with sera dilutions and later detected with peroxidase-conjugated anti-mouse IgG antibody (Pharmingen BD). The samples were developed using an 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid solution (Sigma-Aldrich) and read at 405 nm. For anti-DNA antibody determination, a standard anti-dsDNA antibody 10F10 was used. Statistical analysis Values in the figures correspond to mean ± SD. The unpaired Student t-test was used to determine differences between each two groups. The two-tailed Mann–Whitney U test was used when appropriate. Survival significance was determined via analysis of survival curves with Prism software from GraphPad (San Diego, CA).

Results Chemical coupling of peptides to the monoclonal antibodies The successful chimera preparation has already been proven by HPLC and SDS-PAGE [1]. The number of DNA mimotope peptides (1800 Da) coupled to the antibody molecule was determined by mass-spectral analysis [33]. It showed that 14–16 peptides were bound per IgG molecule on the single DNA-like chimera (data not shown).

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To identify the cell population inhibited by the chimeric molecule treatment, spleen cells from sick MRL/lpr mice were incubated for 4 d with the DNA-like chimera or medium only, and gated CD19+ and CD3+ cells were studied. FACS analyses performed at the end-point of the experiments showed increased T cell number in the proliferating control samples, while the number of B cells remained constant (Figure 1b). The DNA-like chimera provides signal through FcgIIb receptors The ability of the constructed chimeric molecules to provide signals through the inhibitory receptors on B cell surface was tested using FcgRIIb-possitive or FcgRIIb-negative B cell lines. The signal transduction was confirmed by the observed tyrosine phosphorylation of the intracellular part of the inhibitory receptor at FcgRIIb-expressing B cell line and its absence at FcgRIIb-negative one (Figure 2a). Modulation of FcgRII Expression on B cells after IVIg pretreatment The in vitro and ex vivo expression levels of FcgRIIb were determined by flow cytometry. For ex vivo expression, CD19+ spleen cells from MRL/lpr or SCID mice pretreated or not with IVIg were analyzed using anti-CD32-FITC mAb. The in vitro expression levels of FcgRIIb were studied after IVIg treatment of the isolated murine splenocytes. The in vitro incubation with IVIg increased the expression of FcgRIIb on MRL/lpr splenocytes (Figure 2b, left panel). An increased FcgRIIb expression was also observed after the in vivo IVIg treatment of MRL/lpr mice (Figure 2b, right panel). Establishment of a mild lupus-like transfer model A developed MRL/lpr lupus-like disease is a very aggressive and sex-dependent form of mouse autoimmunity. Some combinations of cell donors and recipients resulted in a high mortality in the transferred groups, which limited the observation of disease progression and the long-term administration of chimeric molecules (Figure 3). We found out that the transfer of B and T cells from female MRL/lpr mice to female SCID mice is the most acceptable combination for our purposes.

Inhibition of cell proliferation in vitro

Effects of the DNA-like chimera in SCIDs transferred with MRL/lpr mouse cells

The ability of the 2.4G2-DNA-like peptide chimera to inhibit the MRL/lpr cell proliferation was studied. The three chimeric constructs: DNA-like and two control chimeras as well as the pure 2.4G2 antibody were used for the experiment, and their effects were estimated by adding them at different concentrations to cultured MRL/lpr mouse splenocytes. The cell proliferation was evaluated by pulsing with 1.0 mCi [3H] thymidine for the past 18 h of a 4-d culture period and measuring the amount of DNA-incorporated thymidine. A significant inhibition of cell proliferation was observed using 100 ng/ml DNA-like chimera, while the control chimeras had a moderate effect on cell growth (Figure 1a).

Purified B or T cells obtained from female MRL/lpr mice by magnetic separators were transferred to female SCID mice. The cell donors had high levels of IgG anti-dsDNA antibodies and albuminuria. The in vivo effects of the DNA-like chimera, i.e. dsDNA-specific B cell elimination, were studied in SCID mice transferred with B and T cells from MRL/lpr mice. Under the controlled conditions and limited cell transfer, all SCID mice survived after the reconstitution and during the treatment period. We considered a 10-week treatment period sufficient to affect the restricted number of potentially transferred B cell targets. The administration of DNA-like chimera to SCID

DOI: 10.3109/08916934.2014.883502

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Figure 1. 2.4G2/DNA-mimotope chimera inhibits the antigen-induced T cell proliferation in vitro. Spleen cells from female (sick) MRL/lpr mice were cultured in the presence of 2.4G2 antibody, or DNA-like, or control chimeras in several different concentrations. Control samples were cultured in ConA or in medium only. (a) Proliferation was evaluated by [3H] thymidine incorporation, and the samples were compared to the untreated spontaneously proliferating cells. Results are expressed as the mean cpm value ± SD of triplicated assays.**p50.01; ***p50.002; Student t-test. (b) The in vitro treatment of lupus mice splenocytes with theDNA-like chimera (100 ng/ml) resulted in a sharp decrease of CD3+ T cell numbers and did not affect the number of CD19+ cells. Flow cytometry analysis data representative of at least five experiments.

mice transferred with MRL/lpr cells (with or without IVIg pretreatment) resulted in a decrease of the IgG anti-DNA antibody levels as compared to the PBS-treated group transferred with the same cells (Figure 4). Differences in albuminuria were also observed. The IVIg-pretreated SCID mice administrated with DNA-like chimera maintained low proteinuria levels during the treatment. A rapid increase of albuminuria was observed in the PBS-injected control SCID mice (Figure 5). As MRL/lpr mice spontaneously develop severe SLE symptoms, cytokine levels can be determined during the disease progression. The Th1 cytokine IFN-g plays an

important role in lupus progression, and high IFN-g levels in mouse serum correlate with disease severity [34]. As expected, the groups of MRL/lpr cell-transferred SCID mice treated with PBS or with IVIg only produced high serum levels of IFN-g as compared to the chimera-injected transferred SCID mice, whether IVIg treated or not. During disease development, the donor MRL/lpr mice had increased serum IL-10 levels, but surprisingly, the transferred IVIg-pretreated SCID mice injected with DNA-like chimera also manifested high IL-10 serum levels (up to 500 pg/ml) two weeks after the transfer.

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Figure 2. FcgRIIb as an effector target molecule. (a) DNA-like chimeric molecules trigger signal transduction through FcgRIIb receptors. The in vitro exposure of murine FcgRII expressing cells to DNA-mimotope chimera resulted in enhanced tyrosine phosphorylation of the inhibitory receptor. Murine FcgRII-positive cell line A20 (lanes 7–10) or its FcgRII-negative form IIA1.6 (lanes 2–5) were incubated in the presence of the DNA-like chimera at 37  C for 0, 2, 5 or 10 min. Cells cultivated in medium only were used as controls (lanes 1 and 6). The cells were lysed, and the supernatants were immunoprecipitated, subjected to SDS-PAGE and immunobloted with an anti-phosphotyrosine antibody (upper panel) or with the 2.4G2 antibody (lower panel). (b) In vitro and in vivo IVIg pretreatment increased FcgRIIb expression on MRL/lpr B cells. Flow cytometry analysis of FcgRIIb expression on CD19+ spleen cells from MRL/lpr and SCID mice. The in vitro IVIg pretreatment (0.04 mg/ml) increased FcgRIIb expression on MRL/lpr splenocytes and did not affect SCID cells (left panel). To study the in vivo effect of IVIg pretreatment, mice were injected i.v. with a 500 mg/kg of pooled human IgG preparation (IVIg) or PBS only. After 12 h, CD19+ splenocytes were analyzed for FcgRIIb expression (right panel). Data are representative of at least five experiments.

Furthermore, the animal groups treated with DNA-like chimera (whether IVIg pretreated or not) exhibited higher serum IL-4 levels (up to 100 pg/ml) as compared to the PBS-injected group (Figure 6). At the end of the experiment, all PBS-treated transferred SCID mice had massive mesangial glomerular depositions of IgG-containing immune complexes, while the IVIg pretreated animals administered with DNA-like chimera had small depositions only (Figure 7, upper panel). The 10 weeks long administration of the IVIg + DNA-like chimera or DNA-like chimera alone resulted in a significant improvement of kidney histology. The microscopical structure of the organs of chimera-treated mice was preserved even though some infiltration was present. In contrast, PBS-treated transferred controls presented massive mononuclear cell accumulations surrounding the blood vessels (Figure 7, lower panel).

Discussion and conclusions In this study, we report a possible way to restrict the communication between autoimmune B and T cells, leading to suppression of lupus syndrome in MRL/lpr cell-transferred SCID mice. The functional elimination of autoantigenspecific B cells leaves autoreactive T cells alone without a potency of prolonged pathogenetic effects. T cell subsets play an important role in the pathogenesis of SLE, including regulation of autoreactive B cell responses, functional effects and organ expansion. Autoreactive T cells produce proinflammatory cytokines amplifying the disease symptoms [7,8]. B cells are involved in the pathogenesis of autoimmune disorders at several levels: as potential antibodyproducing cells; as auto-antigen presenting cells and as producers of regulatory cytokines [35]. Several approaches have been used to investigate the pathogenetic contribution of B cells in SLE patients and lupus-prone mice. The humanized

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DOI: 10.3109/08916934.2014.883502

Figure 3. Establishment of transferred SCID model of murine SLE. Cell transfer from male or female MRL/lpr mice to male or female SCID mice. The transfer of 3  107 purified (B + T) cells isolated from female MRL/lpr mice to female SCID mice results in a prolonged survival of the host animals as compared to the other three combinations.

Figure 4. Treatment of transferred SCID mice with chimeric molecule suppresses the appearance of IgG anti-dsDNA antibodies. Combined intravenous treatment with IVIg and DNA-like chimera prevents the appearance of high titers of IgG anti-dsDNA antibodies in transferred with MRL/lpr (B + T) cells female SCID mice. Groups of mice (6–10 animals per group) were treated once weekly with the DNA-like chimera (50 mg/mouse i.v.) or with PBS only. One of the DNA-like chimera-treated groups was pretreated with IVIg 12 h before the DNA-like chimera infusion, the other one was treated with IVIg alone. All samples were triplicated, and average values were used for analysis. Mean ± SEM values were calculated for each group; p values were calculated using the Mann–Whitney U test (*p50.05; **p50.01; ***p50.002), in comparison to PBS-treated controls.

Figure 5. Administration of DNA-like chimera prevents the appearance of high levels of proteinuria. MRL/lpr (B + T) cells transferred SCID mice were treated with the DNA-like chimera (with or without IVIg pretreatment), or with IVIg alone, or with PBS only. The IVIg pretreated SCID mice administrated with DNA-like chimera maintained low proteinuria levels during the treatment. All samples were triplicated, and average values were used for analysis. Mean ± SEM values were calculated for each group; p values were calculated using the Mann–Whitney U test (*p50.05; **p50.01), in comparison to PBS-treated controls.

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anti-CD20 mAb Rituximab has been used to evaluate the effect of pan-B-cell depletion. B cell elimination correlated with a significant clinical improvement, though changes in the anti-dsDNA Ab titers were not detected [10,12]. Animal models of SLE permit to study the effect of B cell depletion in mice with intact immune system by using murine anti-CD20 mAbs. In some mouse models of autoimmune diseases, this prevents the early stages of autoimmunity indicating a possible role of B cells during autoreactive T cell activation and expansion [11]. Long-living plasma cells are resistant to treatment with anti-CD20 antibodies as they lack this cell marker. Moreover, it was found that B cells in autoimmune-prone strains are more resistant to anti-CD20 depletion compared to nonautoimmune animals [9,36]. Antihuman CD22 antibodies preferentially affect the activation and proliferation of B cells from lupus patients as compared to healthy controls [37]. Similar results were obtained with anti-CD19 antibodies, leading to pre-B cells depletion in transgenic animal models of autoimmune diseases [38]. Alternative approaches for specific targeting of autoreactive B cells are under investigation. One possible mechanism

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Figure 6. Cytokines measurement. Serum levels of IL-4, IL-10 and IFN-g obtained after transfer of 3  107 (B + T) MRL/lpr cells to SCID mice and measured by sandwich ELISA weekly in the course of eight weeks. Groups of mice (6–10 animals per group) were treated once weekly with the DNA-like chimera (50 mg/mouse i.v.) or with PBS alone. One of the DNA-like chimera treated groups was pretreated with IVIg 12 h before the DNA-like chimera infusion, the other one was treated with IVIg only. The data are represented as mean ± SEM of 6–10 mice per group; p values were calculated in comparison to PBS-treated controls using the unpaired Student t-test.

for B cell down-regulation is the cross-linking of the surface immunoglobulin receptors with the inhibitory FcgIIb receptors by IgG-containing immune complexes [19,39,40]. We have previously demonstrated the selective silencing of

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dsDNA-specific B cells in MRL/lpr mice by administrating a DNA-like chimera targeting their FcgRIIb receptors [1]. The B cell-depleted lupus-prone MRL/lpr mice lacked the most of the classical lupus syndromes as immune-deposit-based nephritis, cellular infiltrates in the organs and skin disease [6]. In this study, we tested the potential of DNA-like chimeric molecule to provide negative signaling to the targeted cells and to inhibit their proliferation. In both cases, the involvement of FcgIIb or BCR receptors was responsible for the results obtained in vitro. A low level of proliferation inhibition was also observed with control chimeras and pure antibody, as all of them might non-specifically affect B cells, and indirectly – T cells. Both control chimeras contain some active components with possible suppressive effect. The irrelevant peptide chimera contains an anti-FcgRIIb antibody 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 [31,41]. Under in vitro conditions, all potential targets are directly accessible, which can explain the partial suppression observed (Figure 1a). The high specificity of the DNA-like chimera is expected to be advantageous under in vivo conditions, at low concentrations and limited accessibility of the targeted autoreactive B cells. It is well possible that the chimeric molecule is also depleting B cells with other auto-antigen specificities, which can explain the suppression effect to a large fraction of the proliferating cells. In another study, we have shown that a similar chimeric molecule containing a Histone 1 epitope suppressed simultaneously the production of anti-Histone 1 antibodies and of anti-DNA antibodies. These effects could be explained by the phenomenon of ‘‘tolerance spreading’’, whereby the suppression of disease-associated lymphocytes results in suppression of pathological B and T cells of different specificities [42,43]. The enlargement of lymphoid organs in MRL/lpr mice is due mainly to the accumulation of T lymphocytes [42]. The ability of the DNA-like chimera to affect T cells without binding to them directly could be explained by the antigenpresenting role of B cells. The silencing of pathological dsDNA-specific B cells is expected to downmodulate the activation and number of auto-reactive T cells, which could explain the observed inhibition of T cell proliferation after incubation of splenocytes with the DNA-like chimera [44]. The absence of FcgRIIb leads to significant proteinuria and glomerular inflammatory responses [45]. The treatment of autoimmune-prone MRL mice with therapeutic IVIg has been shown to up-regulate the expression of the FcgIIb inhibitory B cell receptors [21]. If the increase of this surface receptor on immune/inflammatory cells was universal, it could be used therapeutically to block unwanted immune responses. We have demonstrated the increase of FcgIIb receptor expression after in vitro and in vivo IVIg treatment by flow cytometry (Figure 2) and enzyme-linked immunosorbent spot analysis (data not shown). These results were further supported by the results obtained with IVIg pretreated SCID mice transferred with a combination of B + T MRL/lpr cells and treated with DNA-like chimera. The increased FcgRIIb expression on autoimmune B cells

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DOI: 10.3109/08916934.2014.883502

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Figure 7. Histological and fluorescence kidney analyses. Upper panel. Immunofluorescence analysis of IgG deposition in the glomeruli of untreated SCID mice (A), untreated MRL/lpr mice (B), PBS-treated SCID mice transferred with MRL/lpr (B + T) cells (C) and DNA-like chimera-treated SCID mice transferred with MRL/lpr (B + T) cells pretreated with IVIg (D). The kidney sections from at least five mice were stained with FITC-labeled goat-anti-mouse IgG. Data are representative of at least five experiments. Original magnification  400. Lower panel. Kidney sections from at least five mice stained with haematoxylin/eosin from untreated SCID mice (A), untreated MRL/lpr mice (B), PBS-treated SCID mice transferred with MRL/lpr (B + T) cells (C) and DNA-like chimera-treated SCID mice transferred with MRL/lpr (B + T) cells pretreated with IVIg (D). Data are representative of at least five experiments. Original magnification  250.

provides a greater number of potential targets for DNA-like chimeric molecules, and consequently better treatment efficiency. Animal SCID models are very suitable for evaluating B cell depletion mechanisms and their efficiency. We have already established a pristane-induced autoimmune Balb/cSCID transferred model. Spleen cells from pristane-treated and normal Balb/c mice were used for B and T cells isolation, and groups of immunodeficient SCID mice were injected intraperitoneally with isolated B and T cells. Furthermore, the transferred SCID mice developed lupus symptoms, and administration of the DNA-like chimera resulted in low level of IgG anti-DNA antibodies and of proteinuria during the treatment [46].

In this study, to study the effects of specific B cell elimination on the activity of T cells in murine MRL-SLE, we established a transferred MRL-SCID model and used DNAlike chimera, which is a proven instrument for specific inhibition of pathological B cells [1,46]. Groups of immunodeficient SCID mice were injected intraperitoneally with isolated B and T cells from sick MRL/lpr mice. A critical number of cells (1–4  107) should be injected in order to obtain stable reconstitution and production of immunoglobulins [28,47]. Therefore, spleen cells from female MRL/lpr mice were used for B and T cells isolation and a combination of purified T and B cells (3  107) was transferred to the SCID mice. Using this restricted transfer model, we proved that autoreactive B and T cells are responsible for the

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V. Gesheva et al.

generation of autoimmune symptoms and disease progression in the host animals. In this study, the donor and recipient mice differ in major histocompatibility complex haplotypes. Therefore, SCID cells cannot present MRL-derived ‘‘autoantigens’’, leaving this role to the transferred autoreactive B cells. The specific elimination of the latter restricts not only anti-dsDNA IgG production but also the activation and proliferation of autoreactive T cells. We propose that suppression of pathological dsDNA-specific B cells by the chimeric molecule results in a decreased presentation of autoantigens to T (CD4+) cells. This results in a limited activation and proliferation of T cells specific for nucleosomes, the primary self-antigen that induces SLE [44]. The successful cell transfer was demonstrated by flow cytometry analyses of SCID spleens and lymph nodes using anti-CD4 and anti-CD19-PE antibodies (data not shown). After the transfer, typical autoimmune syndromes were observed in the recipient SCID mice such as anti-dsDNA IgG antibodies, proteinuria and glomerulonephritis, confirming other authors’ data [26,29]. The obtained results suggested that a combined transfer of purified B and T cells from MRL/ lpr donor mice was sufficient to induce autoimmunity in the host SCID animals. The intravenous administration of chimeric molecules in MRL/lpr (B + T cells) transferred SCID model combined or not with IVIg pretreatment resulted in a low level of IgG antiDNA antibodies and proteinuria after the first two weeks of cell transfer. In contrast, an increase of urine protein concentration, appearance of anti-DNA antibodies and deposition of IgG-containing immune complexes in the glomeruli were observed in the PBS-injected control SCID mice during the same period. No histology data of kidney pathology except for insignificant immune-complex depositions were detected in DNA-like chimera injected animals. Reciprocal regulation mechanisms exist in the healthy immune system that provides a balance between Th1 and Th2 cytokines in case of pathological stimulation [48]. These mechanisms do not seem to function in SLE patients and lupus-prone mice, leading to significantly increased IFN-g levels, generation of pathogenic autoantibodies and self-specific T cells. IFN-g is the main pathogenetic factor for development of lupus symptoms, as the administration of IFN-g accelerates disease development in both mice and humans [34]. Several lines of evidence suggest that IL-10 plays also a critical role in the immunopathogenesis of SLE. SLE patients and untreated MRL/lpr mice produce large amounts of IL-10 and PBMCs from patients with SLE exhibit increased IL-10 mRNA expression and increased spontaneous IL-10 production. In addition, the peripheral blood of SLE patients contains a significantly increased number of IL-10 secreting cells. Finally, the treatment of patients with active disease with anti-IL-10 antibodies suppresses lupus symptoms [34]. In our study, abnormally high IFN-g and IL-10 levels were observed in PBS-treated SCID mice transferred with MRL/lpr cells. IFN-g expression was suppressed after immunization with DNA-chimeric molecules, while IL-10 levels remained high. At the same time, IL-4 levels increased after treatment suggesting that in our model, IL-4 could be involved in the

Autoimmunity, Early Online: 1–11

restoration of the cytokine balance and down-modulation of autoimmune response in MRL/lpr mice. The specificity of the chimera’s action was already proven in our studies [1,21], but long-living plasma cells are very resistant to therapeutic intervention [9]. The positive effect of treatment in the presented model could be explained by the limited number of transferred B cells that are efficiently inhibited by the administration of the DNA-like chimeric molecules. Treatment of transferred SCID mice with IVIg or with the DNA-like chimera alone limited the disease symptoms, but subsequent combination of both agents resulted in an additive beneficial effect. The potential targets (in the presented model) are dsDNA-specific B-lymphocytes, which are eliminated specifically at an earlier stage of their differentiation. Some anti-dsDNA IgG antibody producing plasma cells may be targeted only by their FcgRIIb. Unfortunately, plasmacytes in lupus-prone mice generally do not express FcgRIIb and are protected from apoptosis [16]. Even after 10 weeks of treatment, some transferred plasma cells producing antibodies with anti-dsDNA specificity still survive and cannot be affected by the chimeric molecules. However, the continuous administration of a DNA-like chimera would block the appearance of newly differentiated diseaseassociated plasma cells. Long-lasting treatment is not possible in intact MRL/lpr mice with pathological genetic background, which provides newly generated self-reactive B cells. Administration of the chimera to lupus-prone animals prevented the appearance of disease-associated autoantibodies for a limited period, followed by development of anti-rat immunoglobulin antibodies between the sixth and the eighth weeks of the treatment. Even more, all mice, including the PBS-treated ones, developed antibodies against each of the individual components of the chimera after the age of 15 weeks (data not shown). Our transferred SCID model of mouse lupus permits the unlimited administration of any B cell depletion agent in order to study the post-elimination disease progression and autoreactive T cell behavior.

Declaration of interest None of the authors has any potential financial conflict of interest related to this manuscript. This work was supported by the Bulgarian National Science Fund [grants DRNF 02/11 and TK-317/07].

Author contributions A. T., N. M. and V. G. designed the approach of experiments. V. G., N. K., K. N., T. T. and N. M. performed the experiments. A. T., V. G. and M. N. analyzed the data. N. M., M. N. and A. T. wrote and edited the paper.

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