Clin. exp. Immunol. (1991) 85, 379-385
ADONIS
000991049100246E
IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus T. H. WINKLER, S. JAHN* & J. R. KALDEN Institute for Rheumatology and Clinical Immunology, Department of Medicine III, University Erlangen-Nuernberg, Erlangen, and *Institute for Medical Immunology, Department of Medicine, Charite, Humbold-University, Berlin, Germany
(Acceptedfor publication 15 April 1991)
SUMMARY We describe the production of six mouse-human heterohybridomas secreting human IgG antidsDNA antibodies derived from patients with systemic lupus erythematosus (SLE). Peripheral blood cells used for fusion experiments were from patients who were shown to have high numbers of antiDNA secreting B cells in the peripheral blood. All monoclonal antibodies bind to dsDNA in ELISA systems, five are reactive with Crithidia lucilae kinetoplasts and three precipitate dsDNA in the Farr assay. Inhibition studies revealed a remarkable specificity for certain polynucleotide structures. To our knowledge these are the first hybridomas described in the human system that secrete anti-dsDNA antibodies of the IgG class. Keywords anti-DNA antibodies human monoclonal antibodies IgG autoantibodies systemic lupus erythematosus
INTRODUCTION Serum autoantibodies against dsDNA are characteristic for patients with systemic lupus erythematosus (SLE) (Tan, 1982) and SLE-prone mice strains (Theofilopoulos & Dixon, 1985). Their presence correlates with disease activity (Swaak et al., 1979), and a deposition of DNA/anti-DNA immune complexes in affected tissues has been demonstrated (Lambert & Dixon, 1968). However, a paradoxical finding seems to be that antiDNA-secreting B cells can readily be obtained from the normal human B cell repertoire after polyclonal activation and immortalization with Epstein-Barr virus (EBV) (Seigneurin et al., 1988; Casali & Notkins, 1989). These so-called natural autoantibodies are generally of the IgM isotype and react with rather low affinities with DNA and various other antigens. In contrast, the characteristic anti-dsDNA antibodies from SLE patients are of IgG isotype, and high avidity antibodies of cationic charge are associated with lupus nephritis (Rothfield & Stoller, 1967; Ebling & Hahn, 1980). It is still unclear whether the IgG antidsDNA antibodies of high avidity characteristic of active SLE are the class-switched and somatically mutated products of the natural autoantibody repertoire. Monoclonal antibodies have been important tools to study the specificity of anti-DNA antibodies, and facilitate insights into molecular mechanisms underlying the development of
pathogenic autoantibodies (Shoenfeld et al., 1983; Kofler et ai., 1987; Shlomchick et al., 1990). To study the activated autoimmune B cell repertoire in the peripheral blood of individual SLE patients we chose the hybridoma technology to immortalize B cells, because it has been shown that activated B cells form hybridomas at least 100 times more efficiently than resting B cells (Anderson & Melchers, 1978). Here we describe, to our knowledge for the first time, the generation of stable human monoclonal IgG anti-DNA antibodies using a mouse-human heteromyeloma cell line and in vitro unstimulated lymphocytes from SLE patients with a high disease activity and significantly increased number of specific antibody secreting B cells.
MATERIALS AND METHODS Patients and control subjects For the generation of human monoclonal antibodies we studied three patients with SLE who fulfilled the diagnostic criteria of the American Rheumatism Association (Tan et al., 1982) in an active disease state with high levels of anti-dsDNA antibodies, low levels of serum C4, increased blood sedimentation rate (BSR), decreased CH50 and severe nephritis with proteinuria (patients no. 1 and 2) or severe skin involvement without involvement of visceral organs (patient no. 3). For spot-ELISA we also tested two SLE patients in remission, one patient with rheumatoid arthritis and one healthy blood donor. Human hybridomas Peripheral blood mononuclear cells (PBMC) were obtained by leukapheresis from patients 1-3 and further purified by a
Correspondence: T. H. Winkler, Institut fur Rheumatologie und klinische Immunologie, Krankenhausstrasse 12, D-852 Erlangen, Germany.
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Lymphoprep (Nycomed) gradient centrifugation. Mononuclear cells were frozen in RPMI 1640, 20% fetal calf serum (FCS), 8% dimethylsulfoxide in aliquots of 5 x 107 mononuclear cells and stored in liquid nitrogen until fusion. For fusion experiments, the mouse-human heteromyeloma cell line CB-F7, a HATsensitive, ouabain-resistant cell line established for the development of human monoclonal antibodies (Grunow et al., 1988) was used. Mononuclear cells were fused with the CB-F7 cell line by PEG 1500 (Boehringer, Mannheim, Germany) immediately after thawing. The fusion protocol was performed according to Grunow et al. (1988). Cells were seeded in flat-bottomed microtitre plates (Becton Dickinson) with 105 cells/well, 100 ul/ well. After 24 h, 100 Ml/well double-concentrated selection medium containing 0-2 mm hypoxanthine, 0-8 gM aminopterin and 32 HM thymidine were pipetted to the wells. In most of the experiments culture wells with growing hybridomas were tested for antibody production usually after 21 days. Anti-DNA secreting hybridoma cells were cloned and recloned up to five times by limiting dilution using spleen cells (5 x 104/well) from BALB/c mice as feeders. The monoclonal IgM antibody CB15 was derived from a fusion of spleen cells from a patient with idiopathic thrombocytopenia (ITP) with the cell line P3X63 (Kiessig et al., 1990). The monoclonal IgM antibodies 20.G7, 2B.C5 and 39.F8 were derived from fusions of PBMC from patients with SLE and were reactive only with denatured DNA.
Anti-DNA detection systems Culture supernatants were tested for anti-DNA antibodies in ELISA as recently described (Winkler et al., 1988). Briefly, heatdenatured DNA (ssDNA) from calf thymus (Sigma) or native dsDNA from calf thymus (Sigma) were coated to ELISA microtitre plates (Nunc) after precoating with poly-L-lysin (Sigma). After washing with PBS, 0-05% Tween (PBS-T) and post-coating with PBS-T containing 10% FCS, the culture supernatants were incubated for 2 h at 37°C. Human immunoglobulin was detected by means of phosphatase-conjugated anti-human IgM or IgG antisera (Medac, Hamburg, Germany), diluted 1/1000 in PBS-T and after three final washes by addition of p-nitrophenylphosphate (2-5 mg/ml) in Tris-HCI buffer, pH 9-8. Determination of IgG subclass was performed by an IgG subclass specific anti-DNA ELISA as described (Winkler et al., 1988). Light chain type was determined by using light-chainspecific phosphatase-conjugated second antibodies (Medac) in the above-mentioned ELISA system. Indirect immunofluorescence on fixed Crithidia luciliae cells was performed as previously published (Aarden, de Groot & Feltkamp, 1979). Commercially available slides were used for the assay (Central Laboratory for Blood Transfusion, Amsterdam, The Netherlands). The Farr assay was performed essentially as described by Aarden, Lakmaker & Feltkamp (1976b). The hybridoma supernants were diluted in human gammaglobulin (Sandoz) as a carrier for precipitation. As a source for dsDNA, an EcoRI cut plasmid DNA pWH802 was used (Unger, Klock & Hillen, 1984); this was kindly provided by W. Hillen and labelled with 32P-dATP by filling up the protruding ends with klenow polymerase (Pharmacia) in the presence of 32P-dATP and dTTP. Precipitated DNA was determined in a scintillation counter (LKB Wallac) using the 3H channel (Czerenkov radiation).
ELISAs for analysis of polyreactivity The reactivity of the hybridoma supernatants to various antigens was further tested by ELISA. One-hundred microlitres/well of a 10 pg/ml solution of cardiolipin (Sigma) in ethanol were evaporated overnight in microtitre plates (Flow Labs) at 4°C. The wells were postcoated with 1% bovine serum albumin (BSA) in PBS for 2 h and the hybridoma supernatants were incubated in the wells. Detection of bound IgG was as for the anti-dsDNA ELISA, except that 1% BSA was present in the PBS for washing and incubation of second antibody. Other antigen-specific enzyme immunoassays were performed as described (Kiessig et al., 1990). Chondroitin sulphate (ChS), tetanus toxoid (TT), myosin, actin, poly-ribose-phosphate from Haemophilus influenzae coupled to diphtheria toxoid (PRP-D), phosphatidylcholine coupled to BSA (PC) and vimentin (all from Sigma, except for PRP-D from Roehm Pharma, Weiterstadt, Germany, and tetanus toxoid from Behring, Marburg, Germany) were coated to the ELISA plates (5 pg/ml in carbonate buffer, pH 9 6). The plates were post-coated with 1% BSA in PBS-T and the supernatants of the hybridomas were incubated as for the anti-dsDNA ELISA. All monoclonal antibodies were titrated on all antigens. Calculation ofdissociation constants (Kd) ofanti-DNA antibodies For the measurement of the Kd in solution, the method of Friguet et al. (1985) was used. The monoclonal antibodies were incubated with increasing amounts of DNA (heat-denatured and native calf thymus DNA, linearized plasmid DNA) in PBST containing 5 mm EDTA for 24 h at room temperature. The concentrations of the antibodies were 35 ng/ml for 33.C9, 80 ng/ ml for 32.B9, 400 ng/ml for 35.21, 1 pg/ml for 33.H I 1, 100 ng/ml for 33.F 12, 20 ng/ml for 19.E7, 500 ng/ml for CB 15, 1 ,g/ml for 39.F8, 500 ng/ml for 20.G7 and 250 ng/ml for 2B.C5. The mixtures were transfered into ssDNA or dsDNA coated ELISA plates. After 1 h of incubation the plates were washed and a biotinylated sheep anti-human immunoglobulin antibody and streptavidin-horseradish peroxidase complexes were used to detect IgG or IgM antibodies. The biotin-streptavidin system further enhances sensitivity of the ELISA (Winkler et al., 1988) and makes it possible to use the antibodies in concentrations below the dissociation constant. The antigen binding in presence of soluble ligand was expressed as percentage of binding activity in the absence of ligand. The Kd were calculated by Scatchard analysis as described by Friguet et al. (1985). Inhibition studies For the inhibition studies diluted culture supernatants were preincubated in PBS-T containing varying amounts of the following synthetic or natural polynucleotides: poly (A), poly (C), poly (G), poly (G) - poly (C), poly (dG) -poly (dC), poly (dA) - poly (dT), poly (dG-dC), poly (dA-dT), poly (dG-dme5C), phage lambda DNA, dsDNA from calf thymus and heat-denatured DNA (ssDNA) from calf thymus. All polynucleotides were purchased from Sigma, except for poly (dG-dme5C) which was obtained from Pharmacia. After 12 h of incubation, the supernatants with the polynucleotides were transferred to dsDNA-coated ELISA plates under conditions as described above. A dilution curve of the culture supernatant without competitors in PBS-T was run in parallel on the same plate. Data were expressed as residual binding in percent of the IgG binding to the solid-phase DNA in the absence of inhibitor.
Monoclonal IgG anti-DNA antibodies Spot-ELISA Enumeration of total immunoglobulin and anti-DNA-specific antibody-secreting cells was done by a spot ELISA essentially as described by Sedgwick & Holt (1983). Patients PBMC were isolated, washed five times with PBS, counted and definite cell numbers were pipetted into DNA or anti-immunoglobulin precoated 96-well microtitre plates. As control antigens tetanus toxoid (Behring) and a tetracyclin repressor protein tetB (kindly provided by W. Hillen) were coupled to the microtitre plates. The cells were incubated without vibration in culture medium for 3 h at 37°C. After incubation, cells were washed out with five washes of PBS and a second alkaline-phosphatase-conjugated anti-IgM or anti-IgG antibody (1/1000) was incubated for 2 h. After three final washing steps the spots were developed with BCIP (1 mg/ml) in 1% agarose. RESULTS Production of human monoclonal autoantibodies of the IgG isotype Several fusions with cryoconserved mononuclear cells from three SLE patients in active state of disease with significantly elevated numbers of IgG anti-DNA-secreting B cells (see below) were performed. Fusion frequencies were between one to three growing hybridoma cells per 105 PBMC. From several hundred Table 1. Characterization of human anti-dsDNA hybridomas
Anti-dsDNA Patient no.
Hybridoma
Isotype
ELISA
Crithidia
Farr
K
+
+
+
IgG3, A
+ + + +
+ + +
+ -
+
+
+
19.E7
3
IgGl,
32.B9
IgGl, K IgG1,A
33.C9
2 2 2 2
35.21
1
33.F12 33.HI1
IgG2, K IgG2, A
-
381
growing hybridomas, 17 primary cultures secreted IgG antiDNA antibodies. Eleven of the initial IgG anti-DNA secreting cultures lost their immunoglobulin production soon after the initial screening (after 4-6 weeks). However, six stable IgG secreting human anti-DNA hybridomas were established after two to five recloning steps. The amount of IgG secreted in culture by the hybridoma cells ranged from 3 to 35 pg/ml. Table I displays the isotypes and the binding to DNA in the three commonly used anti-dsDNA antibody assay systems. All IgG anti-DNA antibodies bind to dsDNA in the ELISA system. In contrast, only one out of 12 hybridoma supernatants with IgM antibodies binding to denatured DNA also reacted with dsDNA (not shown). Five of the IgG anti-DNA hybridomas exhibited the typical immunofluorescence staining of the kinetoplast of Crithidia luciliae. Antibody 33.H 1 I showed a staining of the cytoplasm of the Crithidia cells (not shown). Three of the monoclonal IgG anti-DNA antibodies precipitated dsDNA in the Farr assay (Table 1). Only the antibody 33.F12 showed cross-reactivity with cardiolipiwrin the direct ELISA (Table 2). No significant reactivity of the IgG anti-dsDNA antibodies with several antigens could be demonstrated (Table 2). In comparison, the IgM monoclonal antibody CB 15 showed typical polyreactivity and three other IgM antiDNA antibodies derived from SLE patients showed significant binding to cardiolipin or vimentin.
Spot ELISA When the number of anti-DNA antibody and total immunoglobulin secreting cells in PBMC were calculated, surprisingly high numbers of anti-DNA-secreting cells were present in the three SLE patients with high disease activity. About 10% of IgG-secreting cells produced dsDNA specific antibodies. In addition, a dramatic activation of the B cell compartment was obvious in these patients and in the patient with rheumatoid arthritis as compared with the healthy control and with two SLE patients under remission (Table 3). The sensitivity and specificity of the assay were confirmed by following experimental data: (i) hybridoma cells secreting antidsDNA antibodies (from the clone 32.B9) could be individually counted on plates coated with DNA or anti-IgG but not with other antigens (tetanus toxoid or tetracyclin repressor protein)
Table 2. Antigen-binding activity of human monoclonal antibodies
Clone
Isotype
dsDNA
19.E7
IgG IgG IgG IgG IgG IgG
++ ++ ++ ++ ++ ++
IgM IgM IgM IgM
-
32.B9 33.F12 33.HII 33.C9 35.21 CBI5 20.G7 2B.C5 39.F8
-
ssDNA
Cardiolipin
+
-
++ ++ ++ ++ + + ++ ++ ++
++
Chondroitin sulphate
Tetanus toxoid
Myosin
-
-
-
_ -
-
-
++
+ ++ +
-
-
-
-
-
-
Actin
PRP-D
+ -
+ -
-
Phosphatidylcholine Vimentin
_ + -
++ ++
-
PRP-D, poly-ribose-phosphate (from H. influenzae) coupled to diphtheria toxoid. Each antibody was titrated to every antigen starting with I pg/ml. Scale: + +, OD > 1 000 at 250 ng/ml; +, OD > 0 250 at I ,ug/ml; and -for OD < (blank + 3 s.d.).
T. H. Winkler, S. Jahn & J. R. Kalden
382
Table 3. Immunoglobulin-secreting cells in patients with SLE with different disease activity, a patient with rheumatoid arthritis (RA) and a healthy blood donor
Antibody-secreting cells/ 106 PMNCt Patient no.
IgM
IgG
Serum anti-dsDNA (U/ml)*
Total
dsDNA
Total
dsDNA
340 850 135 146 35 7 6
3380+1205$ 4400+980 320 + 56 260+97 234+ 102 1345+623 110 +65
745+ 127 245 + 56 20+ 6 16+ 11 < 10 < 10 < 10
4460+1400 5156 + 1090 986 + 265 300+65 156+43 2356+487 68 + 34
410+67 1080 + 234 110+ 38 < 10$ < 10 < 10 < 10
SLE I SLE 2 SLE 3 SLE 4 SLE 5 RA Healthy donor
Serial dilutions of suspensions of PBMC starting with 105/well were incubated on DNA- or anti-immunoglobulin-precoated ELISA plates in RPMI 1640, 100/o FCS for 3 h. Patients 1-3 were in an active disease state with either glomerulonephritis (patients 1 and 2) or dramatic skin lesions (patient 3); patients 4 and 5 were with no evidence for disease activity. * Determined with the Farr assay (Amersham, Braunschweig, Germany), calibrated with the international anti-dsDNA standard preparation (Wo/80). t Mean + s.d. of triplicate wells. + No spots visible in highest cell concentration (105/well). Table 4. Kd (mol//) of human monoclonal anti-DNA antibodies
Ligand Clone
Isotype
Source
dsDNA
ssDNA
33.C9 32.B9 35.21
33.Hll 33.F12 19.E7
IgG IgG IgG IgG IgG IgG
SLE SLE SLE SLE SLE SLE
9 1 x 10-9 9-6lx10-9 19x 10o-, 6-6x 10-'
1 6x 10-8 1 x lo-8 2-2 x 10-7 2-7x 10-7 7-4x 10- 7 7-9x 10-6
CB15 39.F8 20.G7 2B.C5
IgM IgM IgM IgM
ITP SLE SLE SLE
Nil* Nil Nil Nil
*
1-8x 10-6 3-9x 10-6
Kd value too high to be calculated (> 5 x
6-9x 7-2x 2-1 x 5-8 x
10-7 10-6 10-5 10-5
10-4).
or anti-IgM; (ii) no spots were found with PBMC from healthy donors on dsDNA coated plates; and (iii) no spot or very weak spots developed after incubation of PBMC at 4°C.
K,, values of monoclonal anti-DNA antibodies The Kd values of the monoclonal antibodies were calculated for dsDNA and ssDNA as soluble ligands. The values were calculated by assuming a molecular weight of 660 or 330 for the average molecular weight of the monomeric unit of dsDNA or ssDNA respectively. This assumption was made for the following reasons: the size of the DNA used for the experiments is heterogeneous (> 10 kbp); due to the size of the DNA many antibody molecules can be bound to one DNA molecule (Aarden, de Groot & Lakmaker, 1976a); and the size of the
recognized epitope is not known. As shown in Table 4, the Kd values of most of the IgG monoclonal antibodies for ssDNA were two to three magnitudes lower than those of the IgM antibodies. For dsDNA the difference was obvious, as none of the tested IgM antibodies showed significant binding to dsDNA from calf thymus or plasmid DNA. The size of the DNA had no influence on our calculation with the assumption described above, as the Kd values of the IgG anti-dsDNA binding antibodies were the same for the heterogeneous sized calf thymus DNA and the homogeneous, linearized 3-7-kbp plasmid pWH802 as ligand (not shown). Inhibition studies In order to analyse further the specificity of the IgG hybridoma antibodies, various polynucleotides were used as competitors for the binding of the monoclonal antibodies to dsDNA. As shown in Fig. 1, each of the monoclonal antibodies exhibited an individual binding pattern to different nucleic acid structures. The binding of all antibodies was inefficiently inhibited by different ssRNA and dsRNA structures. Only p(G) * p(C) competed significantly for the binding to dsDNA of the antibodies 33.HI I and 33.C9. The antibodies 32.B9, 35.21 and 19.E7 showed a stronger reactivity with native lambda DNA than with denatured DNA. The antibody 32.B9 was inhibited to an equal extent by native lambda DNA, poly(dA-dT) and poly(dG-d~meC) but very inefficiently by poly(dG-dC). Poly (dG) poly(dC) was recognized by this antibody, whereas poly (dA) poly(dT) was not (Fig. Id). Similar complex binding patterns were observed for all the monoclonal anti-DNA antibodies of IgG isotype. The antibodies 33.F 12, 33.HI I and 33.C9 showed similar reactivity against the different polynucleotides, and denatured DNA, dsDNA and p(dG) - p(dC) were the best competitors for all three antibodies. Interestingly, these hybridomas were derived from a single patient. Identity however was ruled out by the differential usage of light and heavy chains (Table 1).
383
Monoclonal IgG anti-DNA antibodies (a )
(c)
(b)
;0~.
100
-
0
80 60
1
40
1
0-
z 0
a-
20
Q)
(e )
(d)
.r_
in
10
0-1
0- 11 10
~0
. _
_
w
-~~
(f)
100
0
c
80 _
0
v-
801
m
60
40
20
a
0
40
iii
11
I
-,----Io1, ,,,,,
O
f
20 0
10
10
0.l
Soluble antigen (yg/mI)
Fig. 1. Dose-dependent inhibition of the binding of six different human monoclonal anti-DNA IgG antibodies (a, 33.F12; b, 33.H 1l; c, 33.C9; d, 32.B9; e, 35.21; f, 19.E7) to solid-phase dsDNA from calf thymus by various polynucleotides. Identical amounts of each monoclonal antibody (0 5 pg/ml) were incubated 12 h with increasing amounts of soluble dsDNA from lambda phage (0), heatdenatured DNA from calf thymus (0), p(dG-dC) (-), p(dA-dT) (El), p(dG) p(dC) (v), p(dA) * p(dT) (v), p(G) p(C) (* only for a-c) or p(dG-dme5C) (A* only for d-f). The mixtures were transferred into ELISA plates coated with dsDNA and the binding of each antibody in the presence ofeach different soluble competitor was measured and expressed as percentage of the dsDNA binding activity in absence of free ligand. -
DISCUSSION In the past, several attempts have been made to establish monoclonal anti-DNA antibodies from SLE patients (Schoenfeld et al., 1983; Rauch, Massicote & Tannenbaum, 1985; Hoch & Schwaber, 1986). As far as we are aware, all published antibodies were of the IgM isotype and thus may reflect the socalled 'natural' antibody repertoire with polyreactive binding to several auto- and xenoantigens (Ternynck & Avrameas, 1986; Casali & Notkins, 1989). In the present communication we demonstrated for the first time stable human-mouse heteromyelomas secreting IgG antibodies against dsDNA. For the fusion experiments, in vitro unstimulated lymphocytes from patients with an active disease and high numbers of anti-DNA secreting B cells in the circulation were used. We were able to generate IgG anti-dsDNA antibody-secreting hybridomas only from patients with highly active disease. This might be taken as circumstantial evidence that IgG anti-dsDNA antibodies are indeed involved in pathogenic mechanisms of SLE. The data presented show a high frequency of B cells secreting anti-dsDNA antibodies in the peripheral blood of three patients in active disease state. As up to nearly 20YO, of the total IgG producing B cells secrete specific antibody, a polyclonal activation of B cells as a single trigger mechanism seems to be unlikely. lshigatsubo et al. (1989) recently also analysed the
-
numbers of anti-DNA-secreting B cells in the blood of SLE patients, applying a similar assay system. In their communication seven out of 29 patients with SLE exhibited similar high numbers of anti-DNA-secreting B cells, but no differentiation of the isotypes was made. It may be of interest that in our rather small study of five SLE patients, no correlation with the antidsDNA serum levels could be found (not shown). It therefore appears worthwhile to analyse the number and frequency of anti-DNA-producing B cells among total antibody forming cells in longitudinal studies, to see whether the occurrence of the high numbers of antigen-specific, activated B cells correlates with the disease activity. All of the established monoclonal antibodies of the IgG isotype reacted with dsDNA in the ELISA, five were also reactive with dsDNA in the Crithidia luciliae assay and three precipitated dsDNA in the Farr assay under 2 molar ammonium sulphate and were therefore of high avidity. Similar proportions in the reactivity in the three assays for anti-dsDNA antibodies were shown in a study with hybridomas from lupusprone mice (Smeenk et al., 1988). This reactivity pattern as well as the isotype distribution of the hybridomas reflect the reactivity and isotypes of anti-dsDNA antibodies found in SLE patients (Winkler et al., 1988). These monoclonal antibodies should be useful for reference purposes in the different anti-DNA assays and for the otherwise
384
T. H. Winkler, S. Jahn & J. R. Kalden
difficult quantification of IgG subclasses of anti-DNA antibodies (since all the three IgG subclasses increased in patients' sera (Winkler et al., 1988) were obtained). Analysis of the fine specificity of the IgG monoclonal antibodies revealed interesting binding characteristics. In contrast to many of the monoclonal IgM anti-DNA antibodies (Lafer et al., 1981; and own data, not shown) only one monoclonal IgG antibody cross-reacted with cardiolipin. This result confirms data published by Smeenk, Lucassen & Swaak (1987), indicating that only low avidity anti-DNA antibodies cross-react with cardiolipin. Highly specific reaction patterns of the IgG monoclonal anti-dsDNA antibodies with certain nucleic acids were demonstrated in inhibition studies. The binding to dsDNA was generally inhibited very inefficiently by ssRNA or dsRNA structures. With poly-deoxyribonucleotides the antibodies showed a striking selective binding, for example a clear preference for poly(dA-dT) over poly(dG-dC). This suggests the involvement of specific hydrogen bonds between the antibody and bases in DNA grooves (Stollar, Zon & Pastor, 1986). Three of the antibodies bound more efficiently to native than to denatured DNA, suggesting a recognition of the native B DNA structure. However, the base composition of the DNA structure seems not to be directly responsible for the binding of the antibodies, as, for example, antibody 32.B9 reacted with poly(dA-dT), poly(dG-dme5C) and poly(dG) * poly(dC) but only very weakly with poly(dG-dC) and poly(dA) poly(dT). The antibody-binding epitope may depend on additional conformational factors such as helix structure, number of nucleotides per helix turn, depth of the grooves, and the potential to form higher organized structures like supercoils and stem-loops. The data argue for an important difference between polyreactive IgM moncolonal antibodies derived from patients with SLE and other autoimmune and infectious diseases and even healthy subjects, and monoreactive IgG anti-dsDNA monoclonal antibodies specific for SLE. Conclusions that have been drawn from the analysis of the polyreactive repertoire may have limited significance for human SLE. Studies are now in progress in our laboratory to characterize the genetic elements used by the IgG anti-DNA antibodies and the expression of common idiotypes to gain further insights into the molecular mechanisms of autoantibody production in human SLE.
ACKNOWLEDGMENTS We wish to thank S. Kiessig for assistance with the determination of dissociation constants, A. Beck for expert technical assistance in cell culture and R. Salinger for performance of leukapheresis. This work was supported by the Deutsche Forschungsgemeinschaft, grant Ka 325/11 1.
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ANDERSON, J. & MELCHERS, F. (1978) The antibody repertoire of hybrid cell lines obtained by fusion of X63-AG8 myeloma cells with mitogenactivated B-cell blasts. Curr. Top. Microbiol. Immunol. 81, 130. CASALI, P. & NOTKINS, A.L. (1 989) Probing the human B cell repertoire with EBV: polyreactive antibodies and CD5 B lymphocytes. Annu. Ret. Immunol. 7, 513. EBLING, F. & HAHN, B.H. (1980) Restricted subpopulations of DNA antibodies in kidneys of mice with systemic lupus: comparison of antibodies in serum and renal eluates. Arthritis Rheum. 23, 392. FRIGUET, B., CHAFFOTTE, A.F., DJAVADI-OHANIANCE, L. & GOLDBERG, M.E. (1985) Measurement of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods, 77, 305. GRUNOW, R., JAHN, S., PORSTMANN, T., KIESSIG, S.S., STEINKELLER, H., STEINDL, F., MATTANOWITCH, L., GORTLER, L., DEINHARDT, F., KATTINGER, H. & VON BAEHR, R. (1988) The higher efficiency, human B cell immortalizing heteromyeloma CB-F7. J. Immunol. Methods, 106, 257. HOCH, S. & SCHWABER, J. (1986) Specificity analysis of human antiDNA antibodies. J. Immunol. 136, 892. ISHIGATSUBO, Y., SAKAMOTO, H., HAGIWARA, E., AOKI, A., SHIRAI, A., TANI, K., OKUBO, T. & KLINMAN, D.M. (1989) Quantitation of autoantibody-secreting B cells in systemic lupus erythematosus. Autoimmunity, 5, 71. KIESSIG, S.T., JAHN, S., HIEPE, F., VOLK, H.D., FIETZE, E., ZUGEHOR, M., PORSTMANN, T. & VON BAEHR, R. (1990) Multireactive human monoclonal antibodies. Allerg. Immunol. 36, 163. KOFLER, R., NOONAN, D.J., STROHAL, R., BALDERAS, R.S., MOLLER, N.P., DIXON, F.J. & THEOFILOPOULOS, A.N. (1987) Molecular analysis of the murine lupus-associated anti-self response: involvement of a large number of heavy and light chain variable region genes. Eur. J. Immunol. 17, 91. LAFER, E.M., RAUCH, J., ANDRZEJEWSKI, C., MUDD, D., FURIE, B., SCHWARTZ, R.S. & STOLLAR, B.D. (1981) Polyspecific monoclonal
lupus autoantibodies reactive with both polynucleotides and phospholipids. J. exp. Med. 153, 897. LAMBERT, P.H. & DIXON, F.J. (1968) Pathogenesis of the glomerulonephritis in NZB/W mice. J. exp. Med. 127, 507. RAUCH, J., MASSICOTE, H. & TANNENBAUM, H.T. (1985) Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics. J. Immunol. 134, 180. ROTHFIELD, N.F. & TOLLAR, B.D. (1967) The relation of immunoglobulin class, pattern of anti-nuclear antibody and complement fixing antibodies to DNA in sera from patients with systemic lupus erythematosus. J. clin. Invest. 46, 1785. SEDGWICK, J.D. & HOLT, P.G. (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods, 57, 301. SEGNEURIN, J.M., GUILBERT, B., BOURGEAT, M.J. & AVRAMEAS, S. (1988) Polyspecific natural antibodies and autoantibodies secreted by human lymphocytes immortalized with Epstein-Barr virus. Blood, 71, 581.
SHLOMCHIK, M., MASCELLI, M., SHAN, H., RADIC, M.Z., PISETSKY, D., MARSHAK-ROTHSTEIN, A. & WEIGERT, M. (1990) Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. J. exp. Med. 171, 265. SHOENFELD, Y., RAUCH, J., MASSICOTE, H., DATTA, S.K., ANDRESCHWARTZ, J., STOLLAR, B.D. & SCHWARTZ, R.S. (1983) Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas. N. Engl. J. Med. 308, 414. SMEENK, R.J., LUCASSEN, W.A. & SWAAK, T.J. (1987) Is anticardiolipin activity a cross-reaction of anti-DNA or a separate entity? Arthritis Rheum. 30, 607. SMEENK, R.J.T., BRINKMAN, K., VAN DEN BRINK, H.G. & WESTGEEST, A.A.A. (1988) Reaction pattern ofmonoclonal antibodies to DNA. J. Immunol. 140, 3786.
Monoclonal IgG anti-DNA antibodies STOLLAR, B.D., ZON, G. & PASTOR, R.W. (1986) A recognition site on synthetic helical oligonucleotides for monoclonal anti-native DNA autoantibody. Proc. ntl A ca(l. Sci. USA, 83, 4469. SWAAK, A.J.G., AARDEN, L.A., STATIUS VAN EPs, L.W. & FELTKAMP, T.E.W. (1979) Anti-dsDNA and complement profiles as prognostic
guides in SLE. Arthritis Rheuniat. 22, 226. TAN, E.M. (1982) Antibodies to nuclear antigens: their immunology and medicine. Adr. Inimunol. 33, 167. TAN. E.M., COHEN, A.S., FRIES, J.F., MASI, A.T., MCSHANE, D.J., ROTHFIELD, N.F., SCHALLER, J.G., TALAL, N. & WINCHESTER, R.J. ( 1982) The 1982 revised criteria for the classification of systemic lupus eryThematosus. Arthritis Rheumn. 25, 1271. TERNYNCK, T. & AVRAMEAS, A. (1986) Murine natural monoclonal
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autoantibodies: a study of their polyspecificities and their affinities. ImmunIol. Rev. 94, 99.
THEOFILOPOULOS, A.N. & DIXON, F.J. (1985) Murine models of systemic lupus erythematosus. Adi'. Immunol. 37, 269. UNGER, B., KLOCK. G. & HILLEN, W. (1984) Nucleotide sequence of the repressor gene of the RAI tetracycline resistance determinant: structural and functional comparison with three related Tet repressor genes. Nucleic Acids Res. 12, 7693. WINKLER, T.H., HENSCHEL, T.A.. KALIES, I., BAENKLER, H.W., SKVARIL, F. & KALDEN, J.R. (1988) Constant isotype pattern of anti-dsDNA antibodies in patients with systemic lupus erythematosus. C/in. exp. Iflnillninol. 72, 434.
ADONIS
000991049100246E
IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus T. H. WINKLER, S. JAHN* & J. R. KALDEN Institute for Rheumatology and Clinical Immunology, Department of Medicine III, University Erlangen-Nuernberg, Erlangen, and *Institute for Medical Immunology, Department of Medicine, Charite, Humbold-University, Berlin, Germany
(Acceptedfor publication 15 April 1991)
SUMMARY We describe the production of six mouse-human heterohybridomas secreting human IgG antidsDNA antibodies derived from patients with systemic lupus erythematosus (SLE). Peripheral blood cells used for fusion experiments were from patients who were shown to have high numbers of antiDNA secreting B cells in the peripheral blood. All monoclonal antibodies bind to dsDNA in ELISA systems, five are reactive with Crithidia lucilae kinetoplasts and three precipitate dsDNA in the Farr assay. Inhibition studies revealed a remarkable specificity for certain polynucleotide structures. To our knowledge these are the first hybridomas described in the human system that secrete anti-dsDNA antibodies of the IgG class. Keywords anti-DNA antibodies human monoclonal antibodies IgG autoantibodies systemic lupus erythematosus
INTRODUCTION Serum autoantibodies against dsDNA are characteristic for patients with systemic lupus erythematosus (SLE) (Tan, 1982) and SLE-prone mice strains (Theofilopoulos & Dixon, 1985). Their presence correlates with disease activity (Swaak et al., 1979), and a deposition of DNA/anti-DNA immune complexes in affected tissues has been demonstrated (Lambert & Dixon, 1968). However, a paradoxical finding seems to be that antiDNA-secreting B cells can readily be obtained from the normal human B cell repertoire after polyclonal activation and immortalization with Epstein-Barr virus (EBV) (Seigneurin et al., 1988; Casali & Notkins, 1989). These so-called natural autoantibodies are generally of the IgM isotype and react with rather low affinities with DNA and various other antigens. In contrast, the characteristic anti-dsDNA antibodies from SLE patients are of IgG isotype, and high avidity antibodies of cationic charge are associated with lupus nephritis (Rothfield & Stoller, 1967; Ebling & Hahn, 1980). It is still unclear whether the IgG antidsDNA antibodies of high avidity characteristic of active SLE are the class-switched and somatically mutated products of the natural autoantibody repertoire. Monoclonal antibodies have been important tools to study the specificity of anti-DNA antibodies, and facilitate insights into molecular mechanisms underlying the development of
pathogenic autoantibodies (Shoenfeld et al., 1983; Kofler et ai., 1987; Shlomchick et al., 1990). To study the activated autoimmune B cell repertoire in the peripheral blood of individual SLE patients we chose the hybridoma technology to immortalize B cells, because it has been shown that activated B cells form hybridomas at least 100 times more efficiently than resting B cells (Anderson & Melchers, 1978). Here we describe, to our knowledge for the first time, the generation of stable human monoclonal IgG anti-DNA antibodies using a mouse-human heteromyeloma cell line and in vitro unstimulated lymphocytes from SLE patients with a high disease activity and significantly increased number of specific antibody secreting B cells.
MATERIALS AND METHODS Patients and control subjects For the generation of human monoclonal antibodies we studied three patients with SLE who fulfilled the diagnostic criteria of the American Rheumatism Association (Tan et al., 1982) in an active disease state with high levels of anti-dsDNA antibodies, low levels of serum C4, increased blood sedimentation rate (BSR), decreased CH50 and severe nephritis with proteinuria (patients no. 1 and 2) or severe skin involvement without involvement of visceral organs (patient no. 3). For spot-ELISA we also tested two SLE patients in remission, one patient with rheumatoid arthritis and one healthy blood donor. Human hybridomas Peripheral blood mononuclear cells (PBMC) were obtained by leukapheresis from patients 1-3 and further purified by a
Correspondence: T. H. Winkler, Institut fur Rheumatologie und klinische Immunologie, Krankenhausstrasse 12, D-852 Erlangen, Germany.
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T. H. Winkler, S. Jahn & J. R. Kalden
Lymphoprep (Nycomed) gradient centrifugation. Mononuclear cells were frozen in RPMI 1640, 20% fetal calf serum (FCS), 8% dimethylsulfoxide in aliquots of 5 x 107 mononuclear cells and stored in liquid nitrogen until fusion. For fusion experiments, the mouse-human heteromyeloma cell line CB-F7, a HATsensitive, ouabain-resistant cell line established for the development of human monoclonal antibodies (Grunow et al., 1988) was used. Mononuclear cells were fused with the CB-F7 cell line by PEG 1500 (Boehringer, Mannheim, Germany) immediately after thawing. The fusion protocol was performed according to Grunow et al. (1988). Cells were seeded in flat-bottomed microtitre plates (Becton Dickinson) with 105 cells/well, 100 ul/ well. After 24 h, 100 Ml/well double-concentrated selection medium containing 0-2 mm hypoxanthine, 0-8 gM aminopterin and 32 HM thymidine were pipetted to the wells. In most of the experiments culture wells with growing hybridomas were tested for antibody production usually after 21 days. Anti-DNA secreting hybridoma cells were cloned and recloned up to five times by limiting dilution using spleen cells (5 x 104/well) from BALB/c mice as feeders. The monoclonal IgM antibody CB15 was derived from a fusion of spleen cells from a patient with idiopathic thrombocytopenia (ITP) with the cell line P3X63 (Kiessig et al., 1990). The monoclonal IgM antibodies 20.G7, 2B.C5 and 39.F8 were derived from fusions of PBMC from patients with SLE and were reactive only with denatured DNA.
Anti-DNA detection systems Culture supernatants were tested for anti-DNA antibodies in ELISA as recently described (Winkler et al., 1988). Briefly, heatdenatured DNA (ssDNA) from calf thymus (Sigma) or native dsDNA from calf thymus (Sigma) were coated to ELISA microtitre plates (Nunc) after precoating with poly-L-lysin (Sigma). After washing with PBS, 0-05% Tween (PBS-T) and post-coating with PBS-T containing 10% FCS, the culture supernatants were incubated for 2 h at 37°C. Human immunoglobulin was detected by means of phosphatase-conjugated anti-human IgM or IgG antisera (Medac, Hamburg, Germany), diluted 1/1000 in PBS-T and after three final washes by addition of p-nitrophenylphosphate (2-5 mg/ml) in Tris-HCI buffer, pH 9-8. Determination of IgG subclass was performed by an IgG subclass specific anti-DNA ELISA as described (Winkler et al., 1988). Light chain type was determined by using light-chainspecific phosphatase-conjugated second antibodies (Medac) in the above-mentioned ELISA system. Indirect immunofluorescence on fixed Crithidia luciliae cells was performed as previously published (Aarden, de Groot & Feltkamp, 1979). Commercially available slides were used for the assay (Central Laboratory for Blood Transfusion, Amsterdam, The Netherlands). The Farr assay was performed essentially as described by Aarden, Lakmaker & Feltkamp (1976b). The hybridoma supernants were diluted in human gammaglobulin (Sandoz) as a carrier for precipitation. As a source for dsDNA, an EcoRI cut plasmid DNA pWH802 was used (Unger, Klock & Hillen, 1984); this was kindly provided by W. Hillen and labelled with 32P-dATP by filling up the protruding ends with klenow polymerase (Pharmacia) in the presence of 32P-dATP and dTTP. Precipitated DNA was determined in a scintillation counter (LKB Wallac) using the 3H channel (Czerenkov radiation).
ELISAs for analysis of polyreactivity The reactivity of the hybridoma supernatants to various antigens was further tested by ELISA. One-hundred microlitres/well of a 10 pg/ml solution of cardiolipin (Sigma) in ethanol were evaporated overnight in microtitre plates (Flow Labs) at 4°C. The wells were postcoated with 1% bovine serum albumin (BSA) in PBS for 2 h and the hybridoma supernatants were incubated in the wells. Detection of bound IgG was as for the anti-dsDNA ELISA, except that 1% BSA was present in the PBS for washing and incubation of second antibody. Other antigen-specific enzyme immunoassays were performed as described (Kiessig et al., 1990). Chondroitin sulphate (ChS), tetanus toxoid (TT), myosin, actin, poly-ribose-phosphate from Haemophilus influenzae coupled to diphtheria toxoid (PRP-D), phosphatidylcholine coupled to BSA (PC) and vimentin (all from Sigma, except for PRP-D from Roehm Pharma, Weiterstadt, Germany, and tetanus toxoid from Behring, Marburg, Germany) were coated to the ELISA plates (5 pg/ml in carbonate buffer, pH 9 6). The plates were post-coated with 1% BSA in PBS-T and the supernatants of the hybridomas were incubated as for the anti-dsDNA ELISA. All monoclonal antibodies were titrated on all antigens. Calculation ofdissociation constants (Kd) ofanti-DNA antibodies For the measurement of the Kd in solution, the method of Friguet et al. (1985) was used. The monoclonal antibodies were incubated with increasing amounts of DNA (heat-denatured and native calf thymus DNA, linearized plasmid DNA) in PBST containing 5 mm EDTA for 24 h at room temperature. The concentrations of the antibodies were 35 ng/ml for 33.C9, 80 ng/ ml for 32.B9, 400 ng/ml for 35.21, 1 pg/ml for 33.H I 1, 100 ng/ml for 33.F 12, 20 ng/ml for 19.E7, 500 ng/ml for CB 15, 1 ,g/ml for 39.F8, 500 ng/ml for 20.G7 and 250 ng/ml for 2B.C5. The mixtures were transfered into ssDNA or dsDNA coated ELISA plates. After 1 h of incubation the plates were washed and a biotinylated sheep anti-human immunoglobulin antibody and streptavidin-horseradish peroxidase complexes were used to detect IgG or IgM antibodies. The biotin-streptavidin system further enhances sensitivity of the ELISA (Winkler et al., 1988) and makes it possible to use the antibodies in concentrations below the dissociation constant. The antigen binding in presence of soluble ligand was expressed as percentage of binding activity in the absence of ligand. The Kd were calculated by Scatchard analysis as described by Friguet et al. (1985). Inhibition studies For the inhibition studies diluted culture supernatants were preincubated in PBS-T containing varying amounts of the following synthetic or natural polynucleotides: poly (A), poly (C), poly (G), poly (G) - poly (C), poly (dG) -poly (dC), poly (dA) - poly (dT), poly (dG-dC), poly (dA-dT), poly (dG-dme5C), phage lambda DNA, dsDNA from calf thymus and heat-denatured DNA (ssDNA) from calf thymus. All polynucleotides were purchased from Sigma, except for poly (dG-dme5C) which was obtained from Pharmacia. After 12 h of incubation, the supernatants with the polynucleotides were transferred to dsDNA-coated ELISA plates under conditions as described above. A dilution curve of the culture supernatant without competitors in PBS-T was run in parallel on the same plate. Data were expressed as residual binding in percent of the IgG binding to the solid-phase DNA in the absence of inhibitor.
Monoclonal IgG anti-DNA antibodies Spot-ELISA Enumeration of total immunoglobulin and anti-DNA-specific antibody-secreting cells was done by a spot ELISA essentially as described by Sedgwick & Holt (1983). Patients PBMC were isolated, washed five times with PBS, counted and definite cell numbers were pipetted into DNA or anti-immunoglobulin precoated 96-well microtitre plates. As control antigens tetanus toxoid (Behring) and a tetracyclin repressor protein tetB (kindly provided by W. Hillen) were coupled to the microtitre plates. The cells were incubated without vibration in culture medium for 3 h at 37°C. After incubation, cells were washed out with five washes of PBS and a second alkaline-phosphatase-conjugated anti-IgM or anti-IgG antibody (1/1000) was incubated for 2 h. After three final washing steps the spots were developed with BCIP (1 mg/ml) in 1% agarose. RESULTS Production of human monoclonal autoantibodies of the IgG isotype Several fusions with cryoconserved mononuclear cells from three SLE patients in active state of disease with significantly elevated numbers of IgG anti-DNA-secreting B cells (see below) were performed. Fusion frequencies were between one to three growing hybridoma cells per 105 PBMC. From several hundred Table 1. Characterization of human anti-dsDNA hybridomas
Anti-dsDNA Patient no.
Hybridoma
Isotype
ELISA
Crithidia
Farr
K
+
+
+
IgG3, A
+ + + +
+ + +
+ -
+
+
+
19.E7
3
IgGl,
32.B9
IgGl, K IgG1,A
33.C9
2 2 2 2
35.21
1
33.F12 33.HI1
IgG2, K IgG2, A
-
381
growing hybridomas, 17 primary cultures secreted IgG antiDNA antibodies. Eleven of the initial IgG anti-DNA secreting cultures lost their immunoglobulin production soon after the initial screening (after 4-6 weeks). However, six stable IgG secreting human anti-DNA hybridomas were established after two to five recloning steps. The amount of IgG secreted in culture by the hybridoma cells ranged from 3 to 35 pg/ml. Table I displays the isotypes and the binding to DNA in the three commonly used anti-dsDNA antibody assay systems. All IgG anti-DNA antibodies bind to dsDNA in the ELISA system. In contrast, only one out of 12 hybridoma supernatants with IgM antibodies binding to denatured DNA also reacted with dsDNA (not shown). Five of the IgG anti-DNA hybridomas exhibited the typical immunofluorescence staining of the kinetoplast of Crithidia luciliae. Antibody 33.H 1 I showed a staining of the cytoplasm of the Crithidia cells (not shown). Three of the monoclonal IgG anti-DNA antibodies precipitated dsDNA in the Farr assay (Table 1). Only the antibody 33.F12 showed cross-reactivity with cardiolipiwrin the direct ELISA (Table 2). No significant reactivity of the IgG anti-dsDNA antibodies with several antigens could be demonstrated (Table 2). In comparison, the IgM monoclonal antibody CB 15 showed typical polyreactivity and three other IgM antiDNA antibodies derived from SLE patients showed significant binding to cardiolipin or vimentin.
Spot ELISA When the number of anti-DNA antibody and total immunoglobulin secreting cells in PBMC were calculated, surprisingly high numbers of anti-DNA-secreting cells were present in the three SLE patients with high disease activity. About 10% of IgG-secreting cells produced dsDNA specific antibodies. In addition, a dramatic activation of the B cell compartment was obvious in these patients and in the patient with rheumatoid arthritis as compared with the healthy control and with two SLE patients under remission (Table 3). The sensitivity and specificity of the assay were confirmed by following experimental data: (i) hybridoma cells secreting antidsDNA antibodies (from the clone 32.B9) could be individually counted on plates coated with DNA or anti-IgG but not with other antigens (tetanus toxoid or tetracyclin repressor protein)
Table 2. Antigen-binding activity of human monoclonal antibodies
Clone
Isotype
dsDNA
19.E7
IgG IgG IgG IgG IgG IgG
++ ++ ++ ++ ++ ++
IgM IgM IgM IgM
-
32.B9 33.F12 33.HII 33.C9 35.21 CBI5 20.G7 2B.C5 39.F8
-
ssDNA
Cardiolipin
+
-
++ ++ ++ ++ + + ++ ++ ++
++
Chondroitin sulphate
Tetanus toxoid
Myosin
-
-
-
_ -
-
-
++
+ ++ +
-
-
-
-
-
-
Actin
PRP-D
+ -
+ -
-
Phosphatidylcholine Vimentin
_ + -
++ ++
-
PRP-D, poly-ribose-phosphate (from H. influenzae) coupled to diphtheria toxoid. Each antibody was titrated to every antigen starting with I pg/ml. Scale: + +, OD > 1 000 at 250 ng/ml; +, OD > 0 250 at I ,ug/ml; and -for OD < (blank + 3 s.d.).
T. H. Winkler, S. Jahn & J. R. Kalden
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Table 3. Immunoglobulin-secreting cells in patients with SLE with different disease activity, a patient with rheumatoid arthritis (RA) and a healthy blood donor
Antibody-secreting cells/ 106 PMNCt Patient no.
IgM
IgG
Serum anti-dsDNA (U/ml)*
Total
dsDNA
Total
dsDNA
340 850 135 146 35 7 6
3380+1205$ 4400+980 320 + 56 260+97 234+ 102 1345+623 110 +65
745+ 127 245 + 56 20+ 6 16+ 11 < 10 < 10 < 10
4460+1400 5156 + 1090 986 + 265 300+65 156+43 2356+487 68 + 34
410+67 1080 + 234 110+ 38 < 10$ < 10 < 10 < 10
SLE I SLE 2 SLE 3 SLE 4 SLE 5 RA Healthy donor
Serial dilutions of suspensions of PBMC starting with 105/well were incubated on DNA- or anti-immunoglobulin-precoated ELISA plates in RPMI 1640, 100/o FCS for 3 h. Patients 1-3 were in an active disease state with either glomerulonephritis (patients 1 and 2) or dramatic skin lesions (patient 3); patients 4 and 5 were with no evidence for disease activity. * Determined with the Farr assay (Amersham, Braunschweig, Germany), calibrated with the international anti-dsDNA standard preparation (Wo/80). t Mean + s.d. of triplicate wells. + No spots visible in highest cell concentration (105/well). Table 4. Kd (mol//) of human monoclonal anti-DNA antibodies
Ligand Clone
Isotype
Source
dsDNA
ssDNA
33.C9 32.B9 35.21
33.Hll 33.F12 19.E7
IgG IgG IgG IgG IgG IgG
SLE SLE SLE SLE SLE SLE
9 1 x 10-9 9-6lx10-9 19x 10o-, 6-6x 10-'
1 6x 10-8 1 x lo-8 2-2 x 10-7 2-7x 10-7 7-4x 10- 7 7-9x 10-6
CB15 39.F8 20.G7 2B.C5
IgM IgM IgM IgM
ITP SLE SLE SLE
Nil* Nil Nil Nil
*
1-8x 10-6 3-9x 10-6
Kd value too high to be calculated (> 5 x
6-9x 7-2x 2-1 x 5-8 x
10-7 10-6 10-5 10-5
10-4).
or anti-IgM; (ii) no spots were found with PBMC from healthy donors on dsDNA coated plates; and (iii) no spot or very weak spots developed after incubation of PBMC at 4°C.
K,, values of monoclonal anti-DNA antibodies The Kd values of the monoclonal antibodies were calculated for dsDNA and ssDNA as soluble ligands. The values were calculated by assuming a molecular weight of 660 or 330 for the average molecular weight of the monomeric unit of dsDNA or ssDNA respectively. This assumption was made for the following reasons: the size of the DNA used for the experiments is heterogeneous (> 10 kbp); due to the size of the DNA many antibody molecules can be bound to one DNA molecule (Aarden, de Groot & Lakmaker, 1976a); and the size of the
recognized epitope is not known. As shown in Table 4, the Kd values of most of the IgG monoclonal antibodies for ssDNA were two to three magnitudes lower than those of the IgM antibodies. For dsDNA the difference was obvious, as none of the tested IgM antibodies showed significant binding to dsDNA from calf thymus or plasmid DNA. The size of the DNA had no influence on our calculation with the assumption described above, as the Kd values of the IgG anti-dsDNA binding antibodies were the same for the heterogeneous sized calf thymus DNA and the homogeneous, linearized 3-7-kbp plasmid pWH802 as ligand (not shown). Inhibition studies In order to analyse further the specificity of the IgG hybridoma antibodies, various polynucleotides were used as competitors for the binding of the monoclonal antibodies to dsDNA. As shown in Fig. 1, each of the monoclonal antibodies exhibited an individual binding pattern to different nucleic acid structures. The binding of all antibodies was inefficiently inhibited by different ssRNA and dsRNA structures. Only p(G) * p(C) competed significantly for the binding to dsDNA of the antibodies 33.HI I and 33.C9. The antibodies 32.B9, 35.21 and 19.E7 showed a stronger reactivity with native lambda DNA than with denatured DNA. The antibody 32.B9 was inhibited to an equal extent by native lambda DNA, poly(dA-dT) and poly(dG-d~meC) but very inefficiently by poly(dG-dC). Poly (dG) poly(dC) was recognized by this antibody, whereas poly (dA) poly(dT) was not (Fig. Id). Similar complex binding patterns were observed for all the monoclonal anti-DNA antibodies of IgG isotype. The antibodies 33.F 12, 33.HI I and 33.C9 showed similar reactivity against the different polynucleotides, and denatured DNA, dsDNA and p(dG) - p(dC) were the best competitors for all three antibodies. Interestingly, these hybridomas were derived from a single patient. Identity however was ruled out by the differential usage of light and heavy chains (Table 1).
383
Monoclonal IgG anti-DNA antibodies (a )
(c)
(b)
;0~.
100
-
0
80 60
1
40
1
0-
z 0
a-
20
Q)
(e )
(d)
.r_
in
10
0-1
0- 11 10
~0
. _
_
w
-~~
(f)
100
0
c
80 _
0
v-
801
m
60
40
20
a
0
40
iii
11
I
-,----Io1, ,,,,,
O
f
20 0
10
10
0.l
Soluble antigen (yg/mI)
Fig. 1. Dose-dependent inhibition of the binding of six different human monoclonal anti-DNA IgG antibodies (a, 33.F12; b, 33.H 1l; c, 33.C9; d, 32.B9; e, 35.21; f, 19.E7) to solid-phase dsDNA from calf thymus by various polynucleotides. Identical amounts of each monoclonal antibody (0 5 pg/ml) were incubated 12 h with increasing amounts of soluble dsDNA from lambda phage (0), heatdenatured DNA from calf thymus (0), p(dG-dC) (-), p(dA-dT) (El), p(dG) p(dC) (v), p(dA) * p(dT) (v), p(G) p(C) (* only for a-c) or p(dG-dme5C) (A* only for d-f). The mixtures were transferred into ELISA plates coated with dsDNA and the binding of each antibody in the presence ofeach different soluble competitor was measured and expressed as percentage of the dsDNA binding activity in absence of free ligand. -
DISCUSSION In the past, several attempts have been made to establish monoclonal anti-DNA antibodies from SLE patients (Schoenfeld et al., 1983; Rauch, Massicote & Tannenbaum, 1985; Hoch & Schwaber, 1986). As far as we are aware, all published antibodies were of the IgM isotype and thus may reflect the socalled 'natural' antibody repertoire with polyreactive binding to several auto- and xenoantigens (Ternynck & Avrameas, 1986; Casali & Notkins, 1989). In the present communication we demonstrated for the first time stable human-mouse heteromyelomas secreting IgG antibodies against dsDNA. For the fusion experiments, in vitro unstimulated lymphocytes from patients with an active disease and high numbers of anti-DNA secreting B cells in the circulation were used. We were able to generate IgG anti-dsDNA antibody-secreting hybridomas only from patients with highly active disease. This might be taken as circumstantial evidence that IgG anti-dsDNA antibodies are indeed involved in pathogenic mechanisms of SLE. The data presented show a high frequency of B cells secreting anti-dsDNA antibodies in the peripheral blood of three patients in active disease state. As up to nearly 20YO, of the total IgG producing B cells secrete specific antibody, a polyclonal activation of B cells as a single trigger mechanism seems to be unlikely. lshigatsubo et al. (1989) recently also analysed the
-
numbers of anti-DNA-secreting B cells in the blood of SLE patients, applying a similar assay system. In their communication seven out of 29 patients with SLE exhibited similar high numbers of anti-DNA-secreting B cells, but no differentiation of the isotypes was made. It may be of interest that in our rather small study of five SLE patients, no correlation with the antidsDNA serum levels could be found (not shown). It therefore appears worthwhile to analyse the number and frequency of anti-DNA-producing B cells among total antibody forming cells in longitudinal studies, to see whether the occurrence of the high numbers of antigen-specific, activated B cells correlates with the disease activity. All of the established monoclonal antibodies of the IgG isotype reacted with dsDNA in the ELISA, five were also reactive with dsDNA in the Crithidia luciliae assay and three precipitated dsDNA in the Farr assay under 2 molar ammonium sulphate and were therefore of high avidity. Similar proportions in the reactivity in the three assays for anti-dsDNA antibodies were shown in a study with hybridomas from lupusprone mice (Smeenk et al., 1988). This reactivity pattern as well as the isotype distribution of the hybridomas reflect the reactivity and isotypes of anti-dsDNA antibodies found in SLE patients (Winkler et al., 1988). These monoclonal antibodies should be useful for reference purposes in the different anti-DNA assays and for the otherwise
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T. H. Winkler, S. Jahn & J. R. Kalden
difficult quantification of IgG subclasses of anti-DNA antibodies (since all the three IgG subclasses increased in patients' sera (Winkler et al., 1988) were obtained). Analysis of the fine specificity of the IgG monoclonal antibodies revealed interesting binding characteristics. In contrast to many of the monoclonal IgM anti-DNA antibodies (Lafer et al., 1981; and own data, not shown) only one monoclonal IgG antibody cross-reacted with cardiolipin. This result confirms data published by Smeenk, Lucassen & Swaak (1987), indicating that only low avidity anti-DNA antibodies cross-react with cardiolipin. Highly specific reaction patterns of the IgG monoclonal anti-dsDNA antibodies with certain nucleic acids were demonstrated in inhibition studies. The binding to dsDNA was generally inhibited very inefficiently by ssRNA or dsRNA structures. With poly-deoxyribonucleotides the antibodies showed a striking selective binding, for example a clear preference for poly(dA-dT) over poly(dG-dC). This suggests the involvement of specific hydrogen bonds between the antibody and bases in DNA grooves (Stollar, Zon & Pastor, 1986). Three of the antibodies bound more efficiently to native than to denatured DNA, suggesting a recognition of the native B DNA structure. However, the base composition of the DNA structure seems not to be directly responsible for the binding of the antibodies, as, for example, antibody 32.B9 reacted with poly(dA-dT), poly(dG-dme5C) and poly(dG) * poly(dC) but only very weakly with poly(dG-dC) and poly(dA) poly(dT). The antibody-binding epitope may depend on additional conformational factors such as helix structure, number of nucleotides per helix turn, depth of the grooves, and the potential to form higher organized structures like supercoils and stem-loops. The data argue for an important difference between polyreactive IgM moncolonal antibodies derived from patients with SLE and other autoimmune and infectious diseases and even healthy subjects, and monoreactive IgG anti-dsDNA monoclonal antibodies specific for SLE. Conclusions that have been drawn from the analysis of the polyreactive repertoire may have limited significance for human SLE. Studies are now in progress in our laboratory to characterize the genetic elements used by the IgG anti-DNA antibodies and the expression of common idiotypes to gain further insights into the molecular mechanisms of autoantibody production in human SLE.
ACKNOWLEDGMENTS We wish to thank S. Kiessig for assistance with the determination of dissociation constants, A. Beck for expert technical assistance in cell culture and R. Salinger for performance of leukapheresis. This work was supported by the Deutsche Forschungsgemeinschaft, grant Ka 325/11 1.
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