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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Laboratoire d'Immunopathologie and
Laboratoire d'Hématologie, Institut d'Hématologie et
d'Immunologie, Hôpital Civil, Faculté de Médecine de
Strasbourg, Strasbourg, France; Unité 143 INSERM, Hôpital
de Bicêtre, France; and Laboratoire de Pathologie Comparée,
INRA/CNRS URA 2209, Saint-Christol Lez-Alès, France.
The mechanism underlying the prothrombotic state that characterizes
the primary antiphospholipid syndrome proves to be difficult to define
mainly because of the variety of the phospholipid and protein targets
of antiphospholipid antibodies that have been described. Much of the
debate is related to the use of polyclonal antibodies during the
different antiphospholipid assays. To better describe the
antiphospholipid antibodies, a strategy was designed to analyze the
reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal
level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were
cloned in order to express human recombinant antibodies in insect cells
by a baculovirus expression system. The molecular analysis, the fine
specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis
reveals the extreme heterogeneity of these antibodies, which could
account for the difficulties in the previous attempts to define the
pathogenic antiphospholipid response. This approach should help to
unravel the complex antiphospholipid immune response and the
mechanism of the prothrombotic state associated with these
antibodies, but it could also shed some light on their possible origins.
(Blood. 2001;97:3820-3828) Antiphospholipid autoantibodies
(aPLs)1 are associated with thrombosis, recurrent fetal
loss, and thrombocytopenia in patients with antiphospholipid syndrome
(APS).2-4 The mechanism of the characteristic
prothrombotic state related to aPLs during APS is still under
debate.5 Much of it is related to the definition of the
precise nature of aPL specificity, which remains unclear because of the
number of possible antigenic targets discovered over the last few
years. It is now accepted that plasma proteins play an important role
in aPL reactivity.6,7 The most common protein cofactors
are The major methods currently in use for the production of human
monoclonal antibodies (mAbs) are Epstein-Barr virus
transformation,34 mouse-human hybridomas,35
or human-human hybridomas36 and fusion of Epstein-Barr
virus-transformed B lymphocytes with a malignant cell
line.37 These methods have their drawbacks38: In particular, Epstein-Barr virus infection leads to the
immortalization of selected B cells, which could engender problems of
biased sampling. The culture of human hybridomas is not only laborious,
but the hybridomas are difficult to obtain in a stable manner and the yield is very poor due to the limited numbers of sensitized B cells in
peripheral blood. To overcome this problem, antigen preselection methods are used, the most common being the isolation of
antigen-specific subpopulations by cell sorting. This approach is
followed by the generation of variable (V) gene complementary DNA
libraries39 or phage-display combinatorial
libraries.40 However, these libraries in fact
constitute a pool of Ig sequences, where the heavy (H) and light (L)
chain are assembled randomly. The probability of selecting Abs formed
by a few H and L chain genes occurring in vivo is probably low. To get
around this problem, alternative approaches have been proposed:
Original VH/VL combinations can be preserved by
the technique of "in-cell PCR"41 or by a single-cell culture system of B cells involving clonal expansion.42
To circumvent all these problems, we propose a method based on a
combination of both the antigen-specific selection of single cells
sorted by flow cytometry using structurally bilayered anionic PL (CL)
and, also, a cloning of VH and VL genes
originating from individual anti-CL-specific B cells by a single-cell
polymerase chain reaction (PCR) technique. For the generation of mAbs,
the corresponding Ig genes are cloned in transfer vectors in order to
express human recombinant Abs in insect cells using a baculovirus expression system. In this paper we describe our method for isolating monoclonal anticardiolipin (aCL) IgG Abs. We also report the
specificity and the cofactor dependency of a series of monoclonal IgGs
allowing breakdown of the polyclonal aCL response in a patient
suffering from primary APS. This clonal analysis revealed the extreme
heterogeneity of aCL Abs in terms of molecular structure, fine
specificity, and cofactor dependency.
The patient
Cell labeling for flow cytometry analysis
Cells were defrosted, washed in PBS, resuspended in PBS 2% FCS, and
seeded into microtiter plates at a final concentration of
3.105 cells per well. Cells were incubated at 4°C for 1 hour in the presence of the lipid vesicle suspension, washed twice with
150 mL PBS 2% FCS, and stained with R-phycoerythrin-conjugated
F(ab')2 antihuman IgG Fc Single-cell sorting Cells were sorted using an Epics Altra flow cytometer (Beckman Coulter, Brea, CA) equipped with an automatic cell deposition unit. Single cells were directly sorted into thin-wall polycarbonate 96-well plates (Costar, Corning, NY) containing 6 mL water and 3 mL 5 × concentrated reverse transcriptase (RT)-PCR buffer (Gibco, Life Technologies, Gaithersburg, MD). Plates were immediately frozen by putting them on dry ice and were stored at 80°C until use for DNA amplification.
Preparation of complementary DNA The RT reaction was performed using random hexamer pd(N)6 (Amersham Pharmacia Biotech, Germany) and Superscript RT (Gibco). To prepare complementary DNA, single cells were directly heated for 1 minute at 65°C, put on ice, and 6 mL RT mixture (dithiothreitol 1 mM, deoxyribonucleoside triphosphate 0.1 mM, ribonuclease inhibitor 40 U, pd(N)6, and Superscript RT [25 U]) immediately added. Samples were incubated at 37°C for 1.5 hours. The enzyme was inactivated for 3 minutes at 95°C. A -actin PCR amplification was used as a control for the RT step.
PCR amplification DNA amplification was carried out in 2 rounds of PCR (Figure 1A,C) using a DNA Thermal Cycler 9600 (PerkinElmer, Foster City, CA). For PCR amplification of rearranged VH and V genes, a seminested PCR
approach was chosen. The sequences of the sets of oligonucleotides used
as primers are from a previous publication.44
The first round of amplification was carried out in the same sample
tube as the RT reaction and in a final volume of 90 mL containing 50 mM
KCl; 10 mM Tris-HCl, pH 8.4; 2.5 mM MgCl2; 6 nM of each
dATP, dCTP, dGTP, and dTTP; 7.5 pM of each VH,
V The second round of amplification was performed in separate reactions
of 20 mL for each of the 6 VH and 6 V PCR product sequencing PCR products were purified by extraction of the DNA from 2% low-melt agarose gels. (Euromedex, France). They were then sequenced using the Big Dye Terminator cycle DNA sequencing kit (Applied Biosystems, Foster City, CA) and an automated DNA sequencer (310 Genetic Analyzer, PerkinElmer).VH and V products were sequenced with the primers used in the
second round of amplification. The V genes were analyzed using the
international ImmunoGeneTics (IMGT) database45,46 (http://
imgt.cines.fr).61
Cloning of PCR products The resulting amplified VH and V
genes originating from single-cell PCR were cloned in the transfer
vectors p119Cg1 and pBHuCk47, respectively, for expression in
recombinant baculovirus-infected insect cells.
Preparation of fragments to be inserted Because the 5' end of the amplified products did not contain the original extremity of the rearranged V regions, we used overlapping oligonucleotides and PCR to reconstitute the 15 lacking 5' codons (Figure 1B). The primers were designed according to the identified germline gene (VH or V) and are
accessible through our website (http://aloes.u-strasbg.fr/). The PCR
was performed with a Taq High Fidelity DNA polymerase (Roche). The
VH and V extended fragments were cleaved
with AvrII/ApaI and
SacI/XhoI, respectively. The fragments were then
cloned in a forced orientation into the cassette transfer vectors
(Figure 1B,D). Integrity of the cloned fragment sequences was verified
by sequencing of the plasmids (automated DNA sequencer).
Construction of recombinant baculovirus-producing human mAbs To obtain double-recombinant virus expressing the H and L chains, plasmidic DNA, p119Cg1, and pBHuCk containing the VH and VL gene fragments from the original cell were cotransfected with the baculovirus DNA. The cotransfections of plasmidic DNA, virus propagations, and plaque assays were performed using published methods.47,48 The efficiency of the cotransfection ranged from 20% to 50%. Assembled and secreted Abs were detected in the insect cell supernatant by a conventional antihuman IgG enzyme-linked immunosorbent assay (ELISA). Finally, mAbs were produced by Sf9 insect cell infection in culture medium without FCS.Purification of IgG Supernatants containing IgG were dialyzed, first against Tris ethylenediaminetetraacetic acid buffer (10 mM Tris HCl, 1 mM ethylenediaminetetraacetic acid) to increase the pH to 7.8 and, finally, against PBS, pH 7.8. IgG was purified with a protein A column (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. The eluate was dialyzed in PBS or in Tris buffer saline (TBS; 50 mM Tris buffer, 120 mM NaCl, 2.7 mM KCl, pH 7.5) and 0.1 mm filtered. The purity of human IgG was checked by gel migration under undenatured conditions showing no isolated H or L chains (data not shown), and the quantity of purified IgG was measured by a direct ELISA.Immunologic assays for IgG antiphospholipid antibodies Anti-PLs were measured by means of a modified ELISA. Polystyrene microtitration plates (Polysorp, Nunc, Roskilde, Denmark) were coated with CL, phosphatidylserine (PS; Sigma), or PC in 95% ethanol at 50 mg/mL. Uncovered plates were left to evaporate overnight at room temperature and saturated at 4°C for 2 hours with PBS containing 10% FCS. After washing with PBS, samples diluted in PBS 10% FCS were added to each well (in duplicate) and incubated at room temperature for 3 hours. After 3 washing steps in PBS 0.01% Tween 20, Abs were revealed by peroxidase-conjugated goat antihuman IgG (Jackson ImmunoResearch) diluted in PBS 0.01% Tween 20 for 50 minutes at 37°C, followed by O-phenylenediamine dihydrochloride peroxidase substrate (Sigma) after 3 washing steps. Plates were read at 492 nm using a Titertek Multiskan (Labsystems, Helsinki, Finland). The background resulting from binding to wells treated with ethanol alone was subtracted from that of the PL-coated wells.Purified aCLs were tested for their serum independency. Briefly, plates were coated with anionic PLs in ethanol and blocked with 1% bovine serum albumin (BSA) (Euromedex) in PBS. Test samples were diluted in PBS containing 1% BSA. Monoclonal aCLs were tested for their cofactor dependency. Their
reactivity with CL and PS was tested in the presence of purified human
cofactors: The patient serum and purified monoclonal IgGs were also tested
directly with each of the previous human cofactors, Natural An V was purified from human placenta according to a
previous report.43 Human Inhibition experiments For the inhibition studies, CL vesicles (1 mM) were incubated 30 minutes at 4°C with the purified cofactors before they were mixed at various dilutions (1:10 000, 1:50 000, 1:100 000, 1:200 000) with the mAbs at a fixed concentration (10 mg/mL) for 30 minutes at 37°C. A total of 50 mL of the mixture was transferred to the wells of plates coated as above with CL and purified cofactors. The assays were performed as described above. Data were expressed as optical density (OD) values of mAb binding to solid-phase CL/cofactor complexes.Lupus anticoagulant test The lupus anticoagulant (LA) activity of the mAbs was determined by a modified dRVVT (dilute Russell's viper venom test (LAC Screen Instrumentation Laboratory, Milano, Italy) according to the manufacturer's instructions. LA activity is considered as positive when the clotting time ratio of LAC Screen/LAC Confirm is higher than 1.2.Prothrombinase functional assay The prothrombinase assay used here was adapted from a previous publication.49The inhibition of the prothrombinase assay was measured using irrelevant human polyclonal IgG representative of 100% activity. Microtiter plates (Maxisorp, Nunc) were coated overnight at room temperature with 5 mg/mL human serum albumin in TBS 1 mM CaCl2. PL liposomes (3.8 mM) composed of 33% PS and 67% PC were diluted at 1:200 000 in TBS CaCl2, 3 mg/mL human serum albumin. A total of 50 mL IgG Abs was mixed with 250 mL PL liposome suspension and incubated for 15 minutes at room temperature. After 3 washing steps of the precoated plates with TBS CaCl2, 100 mL of the mixture was added to the well (in duplicate). Then, each well was supplied with factor V, factor Xa, factor II, and CaCl2 at a final concentration of 30 pM, 5 pM, 1 mM, and 2 mM, respectively. Plates were incubated for 15 minutes at 37°C. The conversion of PT to thrombin was revealed by adding 50 mL per well of chromozyme TH (Roche), a chromogenic substrate for thrombin, at a final concentration of 0.75 mM. Linear absorbance changes were recorded at 405 nm using a microtitration plate reader equipped with kinetics software.
Flow cytometry analysis and sorting PBMCs originating from patient CIC were analyzed in their ability to react with CL vesicles. Figure 2A shows that fluorochrome-labeled lipid vesicles reacted with a low proportion of IgG-bearing cells (8%). This reactivity was specific for CL-expressing vesicles because (1) PC vesicles were unreactive with IgG-bearing cells (Figure 2C), (2) unlabeled CL vesicles were able to inhibit up to 70% the CL staining of IgG+ cells (Figure 2D) and, finally, (3) F(ab')2 antihuman Fab Abs were able to block up to 80% the binding of CL vesicles to IgG-bearing B cells (Figure 2F). The cell sorting was performed on the double-positive cells (labeled CL vesicles or IgG) as described in "Patient, materials, and methods" (Figure 2A). Notably, cells from a healthy control donor contained only 0.3% of such double-positive cells (Figure 2B).
Single-cell variable region gene amplifications Sorted single cells were subjected to RT-PCR for IgG V region genes. Ninety-eight CL+/IgG+ single cells were analyzed for their VH and V gene
rearrangements. Our method was able to amplify the VH
region in 58% of the cells and the V region in 61% of
the cells. Both V regions were amplified in 48 single cells
representing 49% of the analyzed cells.
The PCR products were directly sequenced, and the first 5 couples
of VH and V Monoclonal anticardiolipin antibody production and specificities As described in Figure 1, we first extended the 5' and 3' regions of the amplified V products to clone the entire VH/D/JH and V/J
regions into transfer vectors for the baculovirus insect cell
expression system. The plasmids were sequenced to ensure the integrity
of the cloned fragments. The first 5 pairs of VH and
VL regions gave rise to 5 different monoclonal IgG Abs (CIC
01, CIC 03, CIC 11, CIC 14, CIC 19) whose specificity was determined
after protein A purification. Figure 3
shows the reactivity of the 5 mAbs with anionic PL (CL and PS) and with
a neutral PL (PC). All of them reacted to various degrees in a
dose-dependent manner with the anionic PL in the presence of FCS. None
of them reacted with the neutral PL. In addition, none of them
recognized PL alone (without FCS). For comparison, we also purified by
the same procedure (protein A column) the serum patient IgG fraction and showed that the aCL activity was detectable at an IgG concentration of 20 mg/mL (Figure 3). These results clearly demonstrate the efficiency of the sorting and amplification strategies that were employed to break up the aCL polyclonal Ab response in this
single patient.
Cofactor dependency The 5 aCL monoclonal IgGs were tested for their cofactor requirement in anti-PL binding and compared with the patient's serum reactivity. Purified human2GP1, PT, Prot S, Prot C, and An V were
added during the ELISA procedure of anti-PL (CL or PS) binding (Figure
4A), and mAb cofactor dependencies were
considered significant when they increased the IgG binding to PL by at
least 30%. The cofactor dependency of the mAbs was extremely
heterogeneous: 2GP1 enhances the binding of CIC 03 to anionic
CL-coated plates, PT enhanced the binding of CIC 19 to CL, and both
cofactors enhance CIC 14 anti-CL activity. CIC 01 was not influenced by
any one of the purified proteins. Interestingly, the cofactor
dependency was identical with that of PS (Figure 4B).
The apparent multireactivity of 3 mAbs (CIC 03, CIC 14, CIC 19) was
further tested by performing binding inhibition experiments where the
binding of the Abs to solid-phase CL-purified cofactor complexes was
measured after incubating the Abs with liquid-phase CL vesicles
previously coated either with the same cofactor (homologous inhibition)
or a different cofactor (heterologous inhibition). The results are
described in Figure 5. The inhibition
curves of CIC 14 binding on solid-phase CL-
In vitro functional tests of aCL antibodies LA activity was performed using a dRVVT. One (CIC 01) of the 5 mAbs was considered positive, prolonging the clotting time. This prolongation was reversed by adding platelet PL (Table 1). We also tested the ability of the mAbs to inhibit the generation of thrombin in a prothrombinase activity test. The mechanism of this assay involves the Ab binding to PL and is mainly dependent on PS. The results are shown in Table 1. CIC 01 and CIC 03 markedly inhibit in a dose-dependent manner the generation of thrombin; CIC 11 is unable to do it, and CIC 14 as well as CIC 19 are intermediate. Notably, CIC 01 has both the capacity to prolong the clotting time during the dRVVT and to inhibit the thrombin generation during the prothrombinase activity test, which was not the case for CIC 03 effective only in the second test.
Molecular analysis of the aCL IgG antibodies The analysis of the V region sequences is summarized in Table 2, leading to a few remarks. First, there is no apparent bias in the gene family usage: 3 of the autoantibodies use a VH gene originating from the main human VH family (VH3, CIC 14, CIC 11, CIC 19), one uses a VH4 gene (CIC 03), and the last one uses a VH5 gene. Second, the sizes of the third heavy chain complementary determining regions (CDRs) are heterogeneous, ranging from 11 to 18 amino acids. Third, the ratios of replacement-to-silent changes (R/S) in the CDRs compared with the frameworks show that the CIC 01 and CIC 11 VH regions have significant R/S ratios in the CDRs, as well as the CIC 14 V region, possibly
indicating an antigen-driven process in the selection of the Ab
production B cells. All the sequences are available through GenBank
(AF301481, AF301482, AF301483, AF301484, AF301485, AF301486, AF301487, AF301488, AF301489, AF301490). Their detailed analysis with
the others originating from the same patient is beyond the scope of
this paper and will be described elsewhere (manuscript in
preparation). Notably, however, the activity of the most
mutated Ab (CIC 01) was not influenced in vitro by one of the tested
cofactors but still had LA activity and inhibited the generation
of thrombin.
The precise characterization of aPL associated with an increased thrombotic state is an important goal in order to understand the mechanism that links such autoantibodies to thrombosis and to better stratify the risk of thrombotic event in subjects with APS. The analysis of polyclonal purified aPL or serum aPL already suggests the complexity of the immune response against PL or PL complexed to proteins.6-8,10 Such a complexity at the polyclonal level certainly explains the difficulties in the mechanistic approach of aPL associated with thrombosis. The description of a few mAbs directed against PL, originating from different patients, also suggests that these Abs are indeed heterogeneous.32-33 Our approach combining a step of specific B-cell selection, a step of amplification of the IgG V region genes, and a step of in vitro Ab production confirms and extends previous observations, showing, in a single patient suffering from APS, that aCL Abs are extremely heterogeneous at the clonal level. It is clear that this situation is not peculiar to patient CIC because the molecular analysis of the VH gene usage by aCL Abs obtained following the same approach in a second patient suffering from APS gives the same picture of extreme molecular heterogeneity of aCL (to date, from the first 20 analyzed single cells, 4 different VH families were amplified and the first 7 sequences obtained were distinct; data not shown). The methodology described here could be applied to the general analysis
of any specific B-cell response, providing that the antigen is
purified. The specificity of the sorting of aCL B cells is already
suggested by the binding inhibition experiments (Figure 2) and
definitely demonstrated by the binding of the first 5 produced Abs to
CL. As described in "Patient, materials, and methods," the process
of the V region cloning implicated a preliminary extension, mainly on
the 5' side, of the products of amplification. This extension was
performed according to the previously determined germline genes, making
it possible that a few mutations located in the first framework were
missing in the final entire V region. However, aCL specificity was
preserved even though we cannot totally rule out that rare missing
mutations could affect Ab affinity. This method appears extremely
powerful in breaking down the polyclonal Ab response revealing, in this
case, at an unexpected rate the functional and molecular diversities of
aCL Abs. The diversities could be even more important, given that we
did not try to analyze The CIC serum reactivity is in fact very difficult to interpret (Figure 4): How many different Abs are involved; how frequent is the multireactivity of the different aCLs; which of these Abs are potentially pathogenic? The analysis of the first 5 aCLs shows that each one has a different reactivity profile with CL, PS in the presence or absence of cofactors. No Abs react with cofactors alone. Finally, only one Ab (CIC 01) was found positive in the in vitro functional tests: even though these tests could not be sufficient to identify the prothrombotic activity of an Ab, CIC 01 could be a candidate for further analysis of thrombogenic potential. This last Ab could also be responsible for the weak and transient LA activity detected in patient's plasma. The molecular analysis also gives a picture of high heterogeneity:
different VH and V The observed diversity of aCL Abs could explain some of the known difficulties in defining in vivo pathogenic Abs. Thus, it will be important to test our monoclonal aCL IgGs in their ability to bind to endothelial cells,56-58 to induce tissue factor expression on monocytes,59 to interfere with the function of activated Prot C24,60 and, finally, to help in elaborating a murine in vivo model of thrombosis.
We thank Professor P. De Groot for help and assistance and for welcoming P.L. in his laboratory.
Submitted September 5, 2000; accepted February 14, 2001.
Supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM CRI 9702). P.L. was supported by fellowships from the Fondation pour la Recherche Médicale, France, and the Association Alsacienne pour la Recherche et la Formation en Médecine Interne, France.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Thierry Martin, Laboratoire d'Immunopathologie, Institut d'Hématologie et d'Immunologie, Hôpital Civil, 1, place de l'Hôpital, 67091 Strasbourg Cedex, France; e-mail: thierry.martin{at}hemato-ulp.u-strasbg.fr.
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© 2001 by The American Society of Hematology.
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  X. Shen, G.-b. Hu, S.-j. Jiang, F.-r. He, W. Xing, L. Li, J. Yang, H.-f. Zhu, P. Lei, and G.-x. Shen Engineering and characterization of a baculovirus-expressed mouse/human chimeric antibody against transferrin receptor Protein Eng. Des. Sel., December 1, 2009; 22(12): 723 - 731. [Abstract] [Full Text] [PDF]
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P. Lieby, A. Soley, A.-M. Knapp, M. Cerutti, J.-M. Freyssinet, J.-L. Pasquali, and T. Martin Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals Blood, October 1, 2003; 102(7): 2459 - 2465. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(Jackson Immunoresearch, West
Grove, PA) for 15 minutes at 4°C. Finally, cells were washed twice
with PBS 2% FCS and resuspended in 100 mL PBS 2% FCS containing
propidium iodide at a final concentration of 5 mg/mL. Inhibition
experiments were performed with unlabeled vesicles and an antihuman Fab
fragment Ab (Bio-Yeda, Rehovat, Israel). For these inhibition
experiments, cells were preincubated either with unlabeled CL or PC
vesicles at different concentrations, 1:10 000, 1:1000, or 1:100, for
1 hour or with the antihuman Fab Ab (1, 10, or 20 mg/mL) for 15 minutes. The cells were analyzed by flow cytometry using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). Data acquisition and
analysis were conducted with Cell-quest software.
), PT
(
), Prot S (*), Prot C (
) coated
vesicles or vesicles alone (
). The vesicles were used at a
dilution of 1:200 000 (a), 1:100 000 (b), 1:50 000 (c), and
1:10 000 (d). Inhibition curves of the CIC 14 binding on
CL/
-bearing B cells.
the producing B cells being just
reactivated by the autoimmune state