Isolation and Characterization of Two Human Monoclonal Anti-Phospholipid IgG from Patients with Autoimmune Disease

Article No. jaut.1999.0316, available online at http://www.idealibrary.com on

Journal of Autoimmunity (1999) 13, 215–223

Isolation and Characterization of Two Human Monoclonal Anti-Phospholipid IgG from Patients with Autoimmune Disease Christine von Landenberg1, Karl J. Lackner1, Philipp von Landenberg2, Bernhard Lang2 and Gerd Schmitz1 1 Institute for Clinical Chemistry and Laboratory Medicine and 2Department of Internal Medicine I, University Hospital of Regensburg, Germany

Received 22 December 1998 Accepted 20 May 1999 Key words: anti-phospholipid antibody, human, monoclonal

The antigenic specificity of anti-phospholipid antibodies (APA) is a matter of intensive investigation. To further characterize these antibodies, we attempted to isolate human monoclonal APA. B-cells of patients with at least one positive test for antibodies against cardiolipin, phosphatidylserine, â2-glycoprotein I (â2-GPI) or the lupus anti-coagulant were immortalized by transformation with Epstein-Barr virus and screened for production of specific IgG. Positive pools were fused with a heteromyeloma cell line and APA-secreting clones were isolated by standard procedures. Two monoclonal APA, HL-5B from a 51-year-old man with primary anti-phospholipid syndrome and recurrent cerebral microinfarctions, and RR-7F from a 48-year-old women with systemic lupus erythematosus but no evidence for thrombotic events were obtained. HL-5B is of the IgG2 subtype with ë light chains, while RR-7F is IgG2 with ê light chains. Both monoclonals show reactivity against cardiolipin and phosphatidylserine but lack reactivity against â2-GPI or lupus anti-coagulant activity. To yield the same OD in the cardiolipin and phosphatidylserine ELISAs RR-7F must be used in an approximately 10-fold higher concentration than HL-5B, indicating a lower affinity towards these antigens. Interestingly, both mAPA can bind to cardiolipin in the absence of â2-GPI. They do not cross-react with dsDNA but show reactivity against oxidized low-density lipoproteins. Analysis of the heavy chain mRNA of HL-5B and RR-7F showed that both are members of the VH3 family. While HL-5B shows extensive somatic mutations in the CDR1 and 2 regions, indicating that it was derived by a T cell-dependent antigen driven process, RR-7F is apparently germline encoded. The two monoclonal APA can be used as tools in further structural and functional analyses. © 1999 Academic Press

Introduction

associated with them. Initially, it was thought that APA can be distinguished by their phospholipid specificity alone. The typical antigens were cardiolipin and phosphatidylserine, both anionic phospholipids. In addition, some antibodies bound neutral phospholipids such as phosphatidylethanolamine [5]. The discovery that most APA require a protein cofactor to bind their phospholipid antigen was an important step forward in the understanding of their binding specificity. The first cofactor discovered was â2-glycoprotein I (â2-GPI), an apolipoprotein with a postulated anti-coagulatory function [6–8]. Further cofactors have since been discovered, including prothrombin [9] and annexin V [10, 11], both involved in hemostasis. There is evidence that cofactor specificity may be related to disease association and perhaps the pathogenic potential of APA [12, 13]. In fact, some researchers claim that the protein cofactors are the actual antigen [14, 15]. Other data suggest that

Anti-phospholipid antibodies (APA) have long been known. Besides their well known appearance in certain infectious diseases, such as syphilis, they have been associated with a clinical entity first described by Hughes and called the anti-phospholipid syndrome [1, 2]. Typical manifestations of the anti-phospholipid syndrome are recurrent arterial or venous thrombosis, recurrent abortions, and low platelet counts. However, numerous other clinical manifestations have been described in association with APA [3, 4]. Accordingly, the binding specificities of APA appear to be as heterogeneous as the clinical manifestations Correspondence to: Dr Karl J. Lackner, Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universita¨t Regensburg, D-93042 Regensburg, Germany. Fax: +49 941 944 6202. E-mail: [email protected] 215 0896–8411/99/060215+09 $30.00/0

© 1999 Academic Press

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oxidized phospholipids may be the relevant antigens for APA [16, 17]. Just recently another anionic phospholipid, bislysophosphatidic acid, a constituent of late endosomes was added to the list of potential antigens [18]. Besides the difficulties defining the relevant antigens of APA, little is known about their development. Currently, no anti-phospholipid-specific T cells have been described. Thus, it is unknown whether APA are natural antibodies or are derived from a T celldependent, antigen-driven process, even though the association of APA with the HLA system suggests such a mechanism [19]. The pathophysiological role of APA is also still not clear. Even though there is ample evidence that APA may play a causal role in the anti-phospholipid syndrome, the mechanisms by which these antibodies exert their effects are still poorly understood. For example, it has been shown that APA interfere with the protein C pathway [20], bind other potentially anti-coagulant proteins [8, 11], and can bind to and activate endothelial cells [21] and platelets [22]. Moreover, APA are able to induce an experimental anti-phospholipid syndrome with the typical manifestations of thrombosis, thrombocytopenia and fetal loss in various mouse models [23]. Most of these studies used mouse monoclonal antibodies, affinity purified human IgG fraction or human monoclonal IgM. Several reports describing human monoclonal IgM APA have provided especially relevant data [24–31]. Comparably little information regarding IgG anti-phospholipid antibodies is available [32– 36]. We have therefore started to clone human B cells synthesizing anti-phospholipid antibodies from patients with autoimmune disease. The monoclonal anti-phospholipid antibodies shall serve as tools for further studies regarding their antigen specificity and their effects on a cellular and molecular level. Analysis of the immunoglobulin genes in B cells will provide information on the processes that lead to the appearance of anti-phospholipid antibodies.

Materials and Methods Production of monoclonal antibodies Peripheral blood mononuclear cells were obtained from patients with at least one positive test for antibodies against cardiolipin, phosphatidylserine, â2-GPI or the lupus anticoagulant. They were isolated from 30 ml samples of heparinized blood by FicollHypaque centrifugation. Mononuclear cells were washed twice with PBS and counted using the trypan blue exclusion method. Cells were resuspended at a density of 3×106/ml in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 U/ml each of penicillin and streptomycin and 2 mM L-glutamine. Epstein-Barr virus (EBV) infection was performed by adding 2 ml EBV-containing media from the B95-8 cell line to 1 ml of the cell suspension followed by incubation for 12 h at 37°C in a humidified atmosphere containing 5% CO2. Thereafter,

cells were pelleted by centrifugation and resuspended in fresh RPMI medium supplemented as above containing 2 ìg/ml phytohemagglutinine and 1 ìg/ml cyclosporin A and seeded in 96-well plates at 105 cells/well. After 2–3 weeks, supernatants from wells with actively growing clones were screened for IgG-production. IgG-positive supernatants were tested for reactivity against cardiolipin, phosphatidylserine and â2-GPI. Cell populations with antiphospholipid-activity were expanded up to at least 3×107 cells. PEG fusion with the heteromyeloma cell line CB-F7 (a HAT-sensitive, ouabain resistant, non-secreting cell line) was performed in a 3:1 ratio of B cells to CB-F7 cells as described [37]. After fusion, cells were seeded at 1×106 cells/well in a 96-well tissue culture plate in RPMI supplemented with 10% heat-inactivated FCS, 100 U/ml each of penicillin and streptomycin and 2 mM L-glutamine. After 24 h, medium was replaced by HAT-ouabain-medium (RPMI supplemented as above with 5×10 −3 M hypoxanthin, 2×10 −5 M aminopterin, 8×10 −4 M thymidin and 1×10 −5 M ouabain). Wells showing positive growth were tested in the screening assays for IgG production and antiphospholipid activity as described above. Positive clones were further expanded in 24-well tissue culture plates and recloned successively at 10, 1 and 0.3 cells/well in 96-well plates until monoclonal. As feeder cells the irradiated mouse macrophage cell line P388 was used at 2×104 cells/well.

ELISA procedures To determine IgG in culture supernatants, ELISA plates (Maxisorp; NUNC) were coated with 100 ìl of Fc-specific mouse anti-human IgG (2 ìg/ml) (Dianova, Hamburg, Germany) in PBS for 12 h at 4°C and blocked with PBS/1% Tween 20 at 37°C for 1 h. Samples were diluted in PBS/0.1% Tween 20 and incubated at 37°C for 1 h. Purified human IgG (Dianova) in concentrations from 100 ng/ml to 6 ng/ ml was used to calibrate the ELISA. Bound IgG was detected with a peroxidase-coupled F(ab)specific mouse anti-human IgG (Sigma, Deisenhofen, Germany). ABTS was used as a peroxidase substrate. For determination of APA and other autoantibodies the following ELISA tests were used: Anti-cardiolipin and anti-â2-GPI ELISA from ELIAS (Freiburg, Germany), anti-phosphatidylserine ELISA from IMTEC (Zepernick, Germany), anti-dsDNA ELISA from Progen (Heidelberg, Germany) and anti-ox-LDL ELISA from IBL (Hamburg, Germany). All assays were performed according to the manufacturer’s instructions. The cut-off values for the ELISA tests are 18 GPL-U/ml and 10 MPL-U/ml for cardiolipin IgG and IgM [38], 15 U/ml for both PS-IgG and IgM, 15 U/ml for both â2-GPI-IgG and IgM and 400 mU/ ml for anti-oxLDL antibodies. Antibody sources were patient sera, cell culture supernatants or purified monoclonal antibodies. For IgG light chain and isotype determination ELISA plates (Maxisorp; NUNC) were coated with

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1 ìg/ml of the respective purified monoclonal APA in PBS for 12 h at 4°C and blocked with PBS/1% Tween 20 at 37°C for 1 h. Commercially available IgG subclass and light chain standards (Sigma) were used as controls. IgG subclass and light chain were detected by biotinylated mouse monoclonal antihuman IgG 1, 2, 3, 4 and anti-ê, ë antibodies (Sigma) (1 h, 37°C). Biotinylated antibodies were detected with a strepavidin–horseradish peroxidase conjugate (Amersham, Bruckinghamshire, UK) 1:1000 for 1 h at 37°C. ABTS was used as a peroxidase substrate.

Table 1. Autoantibody titers of the patients HL and RR, determined using commercially available ELISA tests

CL IgG [GPL-U/ml] CL IgM [MPL-U/ml] PS IgG [U/ml] PS IgM [U/ml] â2-GPI IgG [U/ml] â2-GPI IgM [U/ml] ox-LDL [mU/ml]

Patient 1 (HL)

Patient 2 (RR)

19 5 99 18 2 1 >1200

40 3 46 8 1 2 >1200

Detection of lupus anti-coagulant activity The lupus anti-coagulant activity of purified monoclonal APA was determined by a modified activated partial thromboplastin time (aPTT) test. One hundred microlitres of purified antibody at 1 ìg/ml and 0.1 ìg/ml in PBS were mixed with 100 ìl normal plasma and 10 ìl Pathromtin SL (Behring, Marburg, Germany). Clotting was initiated by adding 100 ìl 25 mM CaCl2 and the clotting time was recorded.

Determination of cDNA sequence encoding the VH region of the IgG heavy chain Total RNA was isolated from 106 hybridoma cells using the RNeasy kit (Quiagen, Hilden, Germany). RNA was eluted with 40 ìl H2O. To remove any residual DNA, RNA was subjected to a DNase digestion using RNase-free DNase I for 20 min at 37°C, followed by heat-inactivation at 65°C for 15 min. After cDNA-synthesis using oligo(dT) primer and AMV reverse transcriptase the cDNA was amplified using the following VH family-based primers located at 5′ and 3′ end of the rearranged V gene as described previously [39]: HuVH1/5 back 5′ CAG GTG CAG CTG CAG CAG TCT GG HuVH2 back 5′ CAG GTC AAC CTG CAG GAG TCT GG HuVH3 back 5′ GAG GTG CAG CTG CAG GAG TCT GG HuVH4 back 5′ CAG GTG CAG CTG CAG GAG TCG GG HuVH6 back 5′ CAG GTA CAG CTG CAG CAG TCA GG HuVH forward 5′ CTT GGT GGA GGC TGA GGA GAC GGT GAC C

3′; 3′; 3′; 3′; 3′; 3′.

PCR conditions were 30 s at 95°C, 45 s at 65°C for annealing, and 1 min at 68°C, repeated for 35 cycles. The Mg2+ concentration in the reaction was 1.5 mM. PCR products were directly sequenced using an ABI Prism Genetic Analyzer 310 sequencer (PE Applied Biosystems, Weiterstadt, Germany). The primers described above were used for sense and anti-sense sequencing. V region sequences were analysed and

aligned to homologous germline genes by reference to the V Base database (Center for Protein Engineering, Cambridge, UK).

Purification of monoclonal antibodies Culture supernatants were harvested and pooled. Monoclonal antibodies were precipitated from culture supernatants by addition of 50% saturated ammonium sulfate (w/v). The precipitate was further purified by protein G affinity chromatography according to the manufacturer’s (Pharmacia, Freiburg, Germany) instructions. Eluted antibody was dialysed against PBS and IgG concentration was determined as described above.

Results Mononuclear cells from 10 patients with antiphospholipid antibodies were isolated and immortalized as described above. Cells were screened for the production of IgG antibodies against cardiolipin, phosphatidylserine and â2-GPI. Two monoclonal antiphospholipid antibodies of the lgG-type, designated HL-5B and RR-7F, were successfully generated from two different patients. Patient 1 (HL) is a 51-yearold man, who presented at the age of 49 with impaired memory and intellectual function to the neurology department. He had a stroke with a temporary left-sided hemiparesis 7 years earlier. On nuclear magnetic resonance imaging there were multiple small vascular lesions, compatible with recurrent cerebral emboli. An angiography of the cranial arteries did not reveal any significant stenosis. An echocardiogram showed an open foramen ovale with no other abnormalities. There were no other symptoms indicative of thromboembolic disease. Laboratory analysis showed an increased titer for antiphosphatidylserine antibodies of the IgG type and a borderline IgM titer. Anticardiolipin titers were borderline, anti-â2-GPI was negative. No lupus anti-coagulant could be detected. Other autoantibodies except for antibodies against oxidized low density lipoproteins (ox-LDL) were negative (Table 1) Screening for known genetic risk factors for thrombosis, i.e. deficiencies of protein C, protein S and anti-thrombin III, and the presence of

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A

B 1.0 Anti-phopshatidylserine (OD405)

Anti-cardiolpin (OD405)

1.0

0.8

0.6

0.4

0.2

0.0 100 µg

10 µg 1 µg 100 ng Antibody concentration/ml

10 ng

0.8

0.6

0.4

0.2

0.0 100 µg

10 µg 1 µg 100 ng Antibody concentration/ml

10 ng

Figure 1. Binding curves of purified monoclonal antibodies HL-5B ( ) and RR-7F ( ) and a monoclonal IgG2 control (.) without phospholipid binding properties in a cardiolipin (A) and phosphatidylserine (B) ELISA. Each value represents the mean of two determinations.

1.5 Anti-ox-LDL (OD450)

the factor V-Leiden mutation were negative. A diagnosis of primary anti-phospholipid syndrome was made, and the patient was treated with vitamin K antagonists. Patient 2 (RR) is a 48-year-old female with systemic lupus erythematosus (SLE), first diagnosed 7 years ago. She had no evidence of thromboembolic disease. The major manifestation of her SLE was membranoproliferative glomerulonephritis with decreased glomerular filtration rate (40 ml/min) and proteinuria. She had central nervous system vasculitis 4 years ago which subsided under therapy with azathioprine and steroids and anti-phospholipid antibodies were detected 4 years ago. She had increased IgG titers against cardiolipin and phosphatidylserine with normal IgM-values. Similar to patient 1 she had neither antibodies against â2-GPI nor a lupus anti-coagulant (Table 1). She was only treated for her SLE without any anti-coagulant drugs or platelet inhibitors. For an initial characterization of the antibodies, HL-5B and RR-7F were probed with isotype-specific and light chain-specific antibodies. This analysis revealed that both belong to the IgG2 subclass. HL5B has ë light chains whereas RR-7F has ê light chains. Binding characteristics of the two antibodies were analysed in several different ELISAs. Both were used in different concentrations ranging from 10 ng/ml to 1.000 ìg/ml, depending on the assay. Results are presented in optical density (OD) units. As a negative control a monoclonal IgG antibody, IgG2 with ë light chains, from patient 2 was used. HL-5B binds to cardiolipin, phosphatidylserine and ox-LDL (Figures 1 & 2) but not to â2-GPI coated to irradiated microtiter plates (data not shown). RR-7F shows the same binding specificities as HL-5B. While the binding pattern in the anti-phospholipid assays and anti-ox-LDL assay of RR-7F are quite similar to the serum of patient 2 (RR), it should be noted that HL-5B shows the strongest reactivity against cardiolipin, which is discordant from the serum reactivity of patient 1 (HL) (Table 1)

1.0

0.5

0.0 1 mg

100 µg 10 µg Antibody concentration/ml

1 µg

Figure 2. Binding curves of purified monoclonal antibodies HL-5B ( ) and RR-7F ( ) and a monoclonal IgG2 control (.) without phospholipid binding properties in an ox-LDL ELISA. Each value represents the mean of two determinations. As HL-5B hybridomas produce about 0.1 of antibody compared to RR-7F, a maximum antibody concentration of only 100 ìg/ml of HL-5B could be achieved after purification.

Furthermore, if HL-5B is tested against cardiolipin or phosphatidylserine it already reaches the same OD as RR-7F at a concentration of approximately 0.1. On the other hand the reactivity against ox-LDL is similar for both monoclonals (Figures 1 & 2). Reactivity against nuclear antigens was excluded for both antibodies by indirect immunofluorescence and ELISA against dsDNA (data not shown). The monoclonal antibodies were also analysed for lupus anti-coagulant activity by adding them to a modified aPTT assay. Only monoclonal HL-5B caused minor prolongation of the aPTT at a concentration of 1 ìg/ml (Table 2) indicative of lupus anti-coagulant activity. To test potential cofactor requirements of the two monoclonal APA they were analysed against cardiolipin coated in the absence of serum proteins other than bovine serum albumin to microtiter plates. Since both

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Table 2. Analysis for lupus anti-coagulant activity of monoclonal APA aPTT (s) Antibody concentration HL-5B RR-7F IgG-control

0.1 ìg/ml

1 ìg/ml

73.9 75.2 70.4

100.1 83.6 79.7

The lupus anti-coagulant activity of monoclonal APA HL-5B, RR-7F and an IgG2 control was determined by a modified aPTT test. Normal plasma used in this test yielded an aPTT of 74.0 s. Each value represents the mean of two determinations.

antibodies were used after protein G purification, the preparations themselves did not contain bovine serum proteins from the cell culture media other than IgG. Thus, there was no known cofactor for antiphospholipid antibodies present in the ELISA. Both protein G purified antibodies, HL-5B and RR-7F, reacted in this assay against cardiolipin (Figure 3). The addition of human serum from individuals without anti-phospholipid antibodies did not increase binding, indicating that both monoclonals are cofactor-independent. When the VH genes of HL-5B and RR-7F were analysed after amplification of the appropriate coding sequence of the mRNA for IgG of the two clones, it was shown that both monoclonals belong to the VH3 gene family. HL-5B is most homologous to HSIGH353X [40]. RR-7F is most homologous to HSIGDP47 [41]. As expected, analysis of the D and J segments of both clones yielded less obvious results because of junctional diversity. In the J segments the VH gene of HL-5B shows the highest homology to JH6, wheras RR-7F showed highest homology to JH3. Comparison of the VH sequence of HL-5B with the germline sequence HSIGH353X showed extensive somatic mutation in CDR1 and 2 (Figure 4). HL-5B shares only 90% homology on the DNA level and 84% homology on the protein level, with a total of 14 amino acid exchanges. Four each of these exchanges are in CDR1 and CDR2, respectively. There is only one conservative substitution (Thr→Ser) in CDR2, whereas five substitions involve a change from an uncharged to a charged amino acid (Ser→Asp; Tyr→His; Ser→Lys; Ser→Arg; Tyr→Asp). Conversely four out of six amino acid exchanges in the framework regions are conservative. In contrast with HL-5B the VH sequence of RR-7F is highly homologous to the germline sequence of HSIGDP47, with 97% homology on the DNA level and 94% homology on the protein level (Figure 5). There are a total of five amino acid exchanges, one of them in CDR1 and two in CDR2. Of these five substitutions three are conservative exchanges, one in framework 1 and 2 respectively (Gly→Ala, Thr→Ser, Glu→Asp). If conservative exchanges are excluded, the homology to the germline encoded sequence is even greater (98%). Thus, the VH sequence of RR-7F is only slightly, if at all, different from the germline sequence.

Discussion Anti-phospholipid syndrome has many different facets and there are still many unanswered questions regarding its etiology and pathophysiology. Structural and functional analysis of APA and the B cells producing them are very likely to provide important insights into the mechanisms by which autoreactive APA are generated and their potential role in the pathophysiology of anti-phospholipid syndrome. It has been shown that APA of the IgG isotype [42] and especially of the IgG2 subclass [43] correlate more strongly with thrombosis in anti-phospholipid syndrome than IgM. However, most of the available information on monoclonal APA refers to IgM antibodies [24–31]. We therefore attempted to generate human monoclonal IgG APA rather than IgM and succeeded in the isolation of two human monoclonal APA. Both monoclonals are of the IgG2 subtype with one (HL-5B) having ë light chains and the other (RR-7F) having ê light chains. These were derived from two patients with quite different clinical manifestations. While patient 1 (HL) has evidence for recurrent cerebrovascular thrombotic or embolic events in the setting of primary antiphospholipid syndrome, the second patient has antiphospholipid antibodies associated with SLE. Until now patient 2 has no clinical manifestations suggestive of anti-phospholipid syndrome, and in particular she has no thrombosis. Both monoclonals show a broad specificity in the available clinical assays. They react with phosphatidylserine, cardiolipin and ox-LDL. A weak lupus anti-coagulant activity has only been found for antibody HL-5B. Neither reacts with â2-GPI. In fact, they do not even need â2-GPI or any of the other known proteins associated with anti-phospholipid antibodies as cofactor in the respective antiphospholipid ELISAs. This could be shown in an anticardiolipin ELISA performed in the absence of â2-GPI or any other serum protein that could function as a cofactor. This observation is of interest, since it has been suggested that the lack of cofactor requirement is typical for APA associated with infectious diseases [44]. Although both monoclonal antibodies show similar binding specificities, they apparently have different binding affinities to specific antigens. If used in ELISAs against cardiolipin and phosphatidylserine HL-5B generates a particular OD at a concentration of 0.1 approximately of RR-7F. Interestingly, this different binding affinity does not apply to ox-LDL. The apparent difference in affinity towards their antigen coincides with a quite different pattern of the V genes in the two B cell clones. HL-5B shows extensive mutations in its expressed VH gene compared to the respective germline gene. More than half of the amino acid substitutions involve a change from a neutral to a charged residue. Thus, HL-5B appears to have emerged from an antigen-driven affinity maturation process. On the other hand, RR-7F shows an almost germline configuration with only a few mutations in its expressed VH gene. It is likely that RR-7F did not undergo an affinity maturation and one would expect

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0.8

A

1.0

B

0.7 0.8

0.6 OD405

OD405

0.5 0.4 0.3 0.2

0.6

0.4

0.2

0.1 0.0 100 µg

10 µg 1 µg Antibody concentration/ml

100 ng

0.0 10 µg

1 µg 100 ng Antibody concentration/ml

10 ng

Figure 3. Binding of purified monoclonal antibodies RR-7F (A) and HL-5B (B) in a cardiolipin ELISA in the presence ( ) and absence ( ) of â2-glycoprotein I. Each value represents the mean of two determinations.

Figure 4. V region of HL-5B aligned to germline gene HSIGH353X.

a lower affinity to its antigen. In fact, it cannot be ruled out that RR-7F is a natural autoantibody. Comparing our own data with the human monoclonal anti-cardiolipin IgG-antibodies described until now [32–34], there are some unexpected findings. Similar to the two antibodies described here, none of these monoclonal APA need the presence of â2-GPI for binding to cardiolipin. This has been thought to be characteristic of non-thrombogenic APA associated

with infectious diseases [44]. Moreover, one of these antibodies is regarded as pathogenic as it is able to induce thrombosis in an in vivo thrombosis model [32]; two others cause placental necrosis and fetal loss in mice [34]. Interestingly, the latter APA causing fetal loss in mice are derived from a patient with primary anti-phospholipid syndrome and from a healthy donor. Nevertheless, they cannot be distinguished either by their pathogenic potential or by their

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Figure 5. V region of RR-7F aligned to germline gene HSIGDP47.

immunoglobulin gene usage, as both antibodies are germline encoded. On the other hand, there are APA of the IgG type isolated from SLE patients which show somatic mutations in their VH and VL genes similar to HL-5B, indicating an affinity maturation process [33]. An intriguing observation is the fact that the affinity-matured antibody HL-5B was isolated from a patient with the clinical manifestations of thromboembolic events typical for primary anti-phospholipid syndrome, while RR-7F, which is germline or close to germline and has an apparently lower affinity to its antigens, was isolated from a patient with SLE without thromboembolic complications. Even though it cannot be ruled out that HL-5B is not involved in the clinical manifestations of patient HL and may not be representative for the spectrum of APA present in this patient, the results obtained with monoclonal APA by others and us suggest that â2-GPI-dependent binding to phospholipids may not be generally required for APA to be pathogenic. Furthermore, our data also support the presence of specific T cells providing help to the antiphospholipid antibody-producing B cells. This could be anticipated, since there are data indicating that the anti-phospholipid syndrome is HLA-associated. Shoenfeld et al. have shown that anti-CD4 treatment can prevent experimental anti-phospholipid syndrome and that transfer of experimental antiphospholipid syndrome by bone marrow transplantation depends on the presence of T cells [45, 46].

Clearly, further work is necessary to define the relevance of different immunocompetent cells in the etiology and pathophysiology of the antiphospholipid syndrome in man and their potential role in determining the pathogenicity of APA found in different conditions. In addition, the pathogenic potential of cofactor-independent anti-phospholipid antibodies needs further consideration.

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