CELLULAR IMMUNOLOGY W,44-52 (1986)

Use of Anti-idiotypic Antibodies to Explore Genetic Mechanisms of Production of Anti-DNA Antibodies’

ANNE DAVIDSON, NADINE CHIEN, LUCILLE FRANK, ROBERTA HALPERN, SCOTT SNAPPER, KARIN ZUPKO, AND BETTY DIAMOND

Departments of Microbiology and Immunology and Medicine, Albert Einstein College ofMedicine, and Irvington House Institute for Medical Research, Bronx, New York 10461

Received November 15. 1985; accepted November 17, 1985

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies with a broad range of antigenic specificities, including specificity for double-stranded DNA. Analysis of the idiotypic profile of anti-DNA antibodies both in humans and mice has demonstrated presence of cross-reactive idiotypes, suggesting that they arise from a restricted number of germ- line genes. Our laboratory has previously reported the generation of 31, a monoclonal anti-idiotypic antibody which recognizes a cross-reactive idiotype on anti-DNA antibodies in a majority of unrelated humans with SLE. We have recently studied the expression of 31 in sera of three human kindreds with familial SLE. We found 6 of 8 SLE patients and 15 of 19 unaffected family members had elevated 31 reactivity. Eleven of these family members had no anti-DNA activity despite elevated 31 reactivity, suggesting that expression of this idiotype in certain indivduals is part of the normal immune response. In another set of experiments using an in vitro culture system we examined somatic mutants of the S107 mouse myeloma cell line. This line makes an antibody which bears the T 15 idiotype, a common idiotype on antibodies to the bacterial antigen phosphoryl choline (PC). U4, a mutant, makes an immunoglobulin which varies by one amino acid from the parent protein, retains the T 15 idiotype, but loses reactivity with PC and acquires reactivity with DNA. We have found that some anti-DNA antibodies in mice with spontaneous lupus and in mice immunologically induced to make anti-DNA antibodies bear the Tl5 idiotype and may represent somatic mutants arising in vivo. 0 1986 Academic PISS, Inc.

INTRODUCTION

Systemic lupus erythematosus (SLE) is a disease characterized by the production of autoantibodies, and in particular antibodies to double-stranded DNA (dsDNA). While much has been learned in recent years of the fine specificity and cross reactivity of anti-DNA antibodies in human and murine lupus (reviewed in (l)), neither the regulation of production of anti-DNA antibodies nor the molecular genetics of their formation is understood. In recent years, idiotypic analysis of antibody molecules has become a tool for understanding the genetic basis of antibody formation and the cellular interactions that regulate specific antibody production. We have therefore been pursuing an idiotypic analysis of anti-DNA antibodies in both human lupus and murine models in order to better understand the factors that lead to expression of anti-DNA antibodies.

’ Presented at the Boehringer Ingelheim Centennial Symposium, “Frontiers in Molecular Immunology: Impact on Human Disease,” held in Danbury, Connecticut, August 14-16, 1985.

44

0008-8749186 $3.00 Copyright Q 1986 by Academic Press, Inc. All rigbu of reproduction in any form reserved

ORIGINS OF ANTI-DNA ANTIBODIES 45

The idiotype of an immunoglobulin molecule is formed, in composite, of a series of antigenic determinants, or idiotopes, on the variable region of the molecule and is defined serologically by anti-idiotypic antibodies. The structural basis of an idiotype is complex and may differ from one antibody molecule to another (2). It is clear, however, that the idiotypic repertoire is largely determined by variable region genes in the germ line and that antibodies which share idiotypic determinants are, in general, encoded by the same variable region gene or gene family. Diversification of these antibodies by combinatorial mechanisms and somatic mutation results in a large family of idiotype-bearing antibodies, not all of which are directed to the same antigen (3). Furthermore, because idiotypic determinants are found throughout the immunoglob- ulin variable region, changes in idiotype expression may occur without affecting antigen binding (4). Differences in idiotype expression from one individual to another may therefore reflect polymorphisms of the variable region genes, somatic mutation of variable region genes, or as yet incompletely understood regulatory events.

Idiotypes form a complex communication network between B and T lymphocytes as both B cells and T cells bear idiotypic determinants. This interacting cellular network appears to play a part in regulation of the immune response. Additionally, responses of T cells to administration of exogenous idiotype or anti-idiotype can result in long lasting modification of the functional idiotypic repertoire (5, 6).

ORIGIN AND SPECIFICITY OF ANTI-DNA ANTIBODIES

Antibodies to nuclear antigens are characteristically found in patients with systemic lupus erythematosus. Antibodies to double-stranded DNA have been studied in detail because they are relatively specific for SLE, they tend to fluctuate with disease activity, and because the presence of deposits of DNA-anti-DNA complexes in injured tissues of SLE patients, especially kidneys, implies a role for these antibodies in disease patho- genesis (7).

Study of the fine specificity of anti-dsDNA antibodies has until recently been ham- pered by the heterogeneity of circulating anti-DNA antibodies. Hybridoma technology has made it possible to study monoclonal anti-DNA antibodies and has led to huther understanding of their antigenic specificity (1). Using monoclonal anti-DNA antibodies, Schwartz, Stollar, and their colleagues demonstrated that the antigenic specificity of these antibodies is less restricted than previously thought. A single monoclonal anti- DNA antibody can bind to a variety of antigens including synthetic polynucleotides, single-stranded DNA, double-stranded DNA, cardiolipin, and other phospholipids (8). Furthermore, monoclonal anti-DNA antibodies can also bind to cytoskeletal pro- teins such as vimentin (9). Thus the same antibody can account for many of the serologic abnormalities found in patients with SLE.

Many questions still remain to be answered about anti-DNA antibodies. First, the nature of the antigen that elicits anti-DNA antibodies is unknown. Experimental im- munization of mice with DNA or oligonucleotides results in antibodies reactive against nucleotides but fails to elicit the polyreactive antibodies typical of SLE (10). Immu- nization of mice with cardiolipin, however, leads to formation of antibodies that cross- react with DNA and show the pattern of antigenic cross-reactivity of autoimmune anti-DNA antibodies (11). Stollar and Schwartz have therefore proposed that the anti- DNA antibodies in SLE are formed against a phosphodiester epitope common to DNA, cardiolipin, and other phospholipids and that the anti-DNA antibodies found

46 DAVIDSON ET AL.

in lupus may actually be elicited by a cross-reacting antigen, perhaps a bacterial phos- pholipid (1).

Second, it is not clear what genes encode anti-DNA antibodies. There may be specific immunoglobulin variable region genes, perhaps present only in autoimmune individ- uals, the encode anti-DNA antibodies. Alternatively anti-DNA antibodies may arise from somatic mutation of variable region genes that encode antibodies used in the normal immune response. Recently several different approaches have been used to examine this question. Studies by our laboratory and by others have indicated that anti-DNA antibodies in human SLE are restricted with respect to idiotype expression ( 12- 14). We generated monoclonal anti-idiotypes to anti-DNA antibodies from a pa- tient with SLE. One of these, 31, has been extensively studied. 31-reactive antibodies are expressed in low titer in normal individuals. Elevated titers of 31 reactivity are found in over 85% of SLE patients with anti-DNA antibodies. Results have shown that 31 is a dominant idiotype on these anti-DNA antibodies while 31-reactive antibodies in normal individuals do not bind DNA (15). The presence of high titers of a cross- reactive idiotype on anti-DNA antibodies in genetically unrelated patients with SLE suggests that the same germ-line gene or gene family encodes these autoantibodies. The presence of this idiotype on nonautoreactive antibodies in SLE patients and normal individuals suggests the germ-line gene or gene family encoding anti-DNA antibodies also encodes antibodies in the normal immune response. Mutation of the gene or gene family encoding 31-reactive antibodies may result in anti-DNA antibodies.

Another approach has been to look for production of anti-DNA antibodies by normal individuals. Cairns and colleagues have generated hybridoma antibodies specific for anti-dsDNA from normal human tonsils (16), and Rauch et al. have generated such hybridomas from peripheral blood lymphocytes of patients with rheumatoid arthritis ( 17). These studies indicate that B cells capable of forming autoantibodies are present in individuals without SLE. Furthermore in the latter study, it was found that some of the anti-DNA antibodies generated from patients with rheumatoid arthritis share a cross-reactive idiotype with anti-DNA antibodies from SLE patients, suggesting that the germ-line genes used to form anti-DNA antibodies in lupus patients exist in in- dividuals without SLE and without anti-DNA activity.

A final approach has been to examine directly the genes encoding hybridoma anti- DNA antibodies. Eilat and colleagues sequenced the heavy and light chain variable region of a monoclonal anti-DNA antibody derived from an autoimmune NZB/W mouse ( 18). The heavy chain variable region is homologous to the T 15 heavy chain gene, a gene used to encode antibodies to bacterial polysaccharides in many strains of mice. Kofler et al. showed that a monoclonal IgM anti-DNA antibody derived from a MRL/lpr mouse uses variable genes which are also used to encode antibodies to exogenous antigens in nonautoimmune mice (19). That similarly, genes encoding anti-DNA antibodies also encode antibodies to exogenous antigen in humans is sug- gested by the observation of Naparstek et al. that some human hybridoma anti-DNA antibodies share amino acid sequence homology with a human IgM myeloma protein that binds a Klebsiella cell wall component (20).

Third, it is still not known what regulates the production of autoantibodies in normal and autoimmune individuals. Some workers have demonstrated auto-anti-idiotypic antibodies in serum of patients with SLE during clinical remission and in serum of family members and suggest that these play a regulatory role (21, 22). Studies of T cells in patients with SLE have suggested that there is a defect in T-suppressor cells

ORIGINS OF ANTI-DNA ANTIBODIES 47

in patients with SLE but it it still not clear whether this is a primary or secondary defect (23). Autoimmune T-helper cells have been isolated from patients with other autoimmune diseases (24, 25) but similar studies remain to be undertaken for SLE.

An idiotypic analysis of anti-DNA antibodies may reveal the nature of the genes encoding the antibodies, whether they exist in normal individuals, or whether they represent germ-line sequences or an accumulation of somatic mutations. It might teach us whether there are antigens other than DNA that elicit production of these or related antibodies in normal and autoimmune individuals and whether some sub- populations of anti-dsDNA antibodies are more pathogenic than others. Finally, an idiotypic analysis may lead us to an understanding of the role of T cells in regulating expression of anti-DNA antibodies, whether there are T-helper cells supporting the response and whether there is a defect in autoimmune individuals in T-suppressor cells that might normally inhibit autoantibody formation.

USE OF ANTI-IDIOTYPIC ANTIBODIES TO DETECT ANTI-DNA ANTIBODIES

The anti-idiotype 31 recognizes an idiotypic determinant present on anti-DNA an- tibodies in a majority of patients with SLE (15). 31 does not compete with DNA for binding to anti-DNA antibodies; therefore, it is not directed to the antigen-binding site (12). Because of this 31 is able to detect anti-DNA antibodies which are bound in immune complexes and not detected by conventional DNA-binding assays. In previous studies we have shown that some SLE patients with high titer 31 reactivity who are in serologic remission have anti-DNA antibodies that are “unmasked” when sera are analyzed on isoelectric focusing gels under conditions that dissociate immune com- plexes. In these patients the presence of 31-bearing antibodies with alkaline isoelectric points correlates with anti-DNA activity (26).

Because 31 is able to detect anti-DNA antibodies that are not detected by conven- tional anti-DNA assay, we asked whether titers of idiotype in serum of patients with SLE might be a better marker of disease activity than anti-DNA activity. We analyzed sequential serum samples from 2 1 patients with SLE for expression of three idiotypes defined by three mouse monoclonal anti-idiotypic antibodies, 31, 8.12 and 1.12, all of which recognize both free and bound anti-DNA antibodies. 8.12 and 1.12 recognize anti-DNA antibodies in about 50% of patients with SLE. Periods of disease flare and remission were identified by chart review and levels of anti-DNA and complement were available for each time point. Forty-one periods of clinically increased disease activity were observed and correlated with serologic activity. The following findings are summarized in Table 1: In 73% of the episodes, at least one idiotype was elevated above the patient’s own baseline levels. In 68%, total hemolytic complement decreased. In less than half DNA increased. The presence of high titers of idiotype in conjunction with low levels of complement in 14 of 2 1 patients may indicate presence of immune complexes. These complexes may possibly have pathogenic potential even when anti- DNA antibodies are not detectable by conventional assays. This study suggests that using a panel of monoclonal anti-idiotypic antibodies may supply a better index of disease activity than the currently used indices.

Similar findings have been reported by Isenberg et al. who used one monoclonal and two polyclonal anti-idiotypic antisera to analyze sequential serum samples from

48 DAVIDSON ET AL.

TABLE 1

Levels of Anti-DNA Antibodies, CH50, and Idiotype during Disease Flare and Remission in 2 1 Patients with SLE

% Abnormal % Changing with during remission disease flares

Anti-DNA antibodies 29 (612 1) 44 (18/41) CH50 76 (16/21) 68 (28/41) Any idiotype 86 (18/21) 73 (30/41)

12 patients with SLE. They also found cases where fluctuations in idiotype titer reflected clinical disease activity more accurately than titer of complement or anti-dsDNA activity (27).

USE OF ANTI-IDIOTYPIC ANTIBODIES TO EXPLORE MECHANISMS OF PRODUCTION OF ANTI-DNA ANTIBODIES IN HUMAN SLE

In SLE it is clear from epidemiologic analysis of families and studies of twins with SLE that there is a genetic tendency toward development of disease (28-30). The molecular basis for this genetic predisposition has yet to be elucidated. In order to determine whether inheritance of expression of a particular idiotype might contribute to the genetic predisposition to SLE, we examined expression of 31 reactivity in the sera of 27 patients and unaffected relatives from three human kindreds with SLE. High titers of 31 reactivity were found in 6 of 8 SLE patients and 15 of 19 family members. When sera were analyzed on isoelectric focusing gels under dissociating conditions, cationic 31-reactive bands were found in 5 of 8 SLE patients and 4 of 19 family members.

Sera were then examined for anti-DNA activity by a Millipore filter assay. Four of eight SLE patients and two unaffected relatives had high titer anti-DNA activity by this assay. All these individuals had high titer 31 reactivity. In order to determine whether the other relatives with high titer 31 reactivity in their serum had “masked” anti-DNA antibodies, we analyzed sera on isoelectric focusing gels under dissociating conditions, transferred the gels to nitrocellulose, and probed the blots with radiolabeled DNA. Two additional SLE patients and two additiona family members had anti- DNA antibodies revealed by this technique. Furthermore all patients and family members with cationic 31-reactive bands had anti-DNA antibodies present either free or in masked form in their serum. These data suggest to us that the DNA-binding potential of 31-reactive antibodies is related not only to primary amino acid sequence but also to charge.

In 11 unaffected family members there was high titer 31 expression in the absence of DNA binding, suggesting to us that there is genetic regulation of 31 idiotype expres- sion and that this regulation appears to be independent of antigen-binding specificity. Furthermore these data show that idiotypes used for formation of anti-DNA antibodies are also used by normal individuals in the course of a normal immune response. This implies a structural similarity between normal antibodies and autoantibodies. The molecular basis for this may be somatic mutation (3 1).

ORIGINS OF ANTI-DNA ANTIBODIES 49

USE OF ANTI-IDIOTYPIC ANTIBODIES TO EXPLORE THE ROLE OF SOMATIC MUTATION IN THE GENERATION OF

ANTI-DNA ANTIBODIES

In collaboration with Dr. Matthew Scharff we have recently demonstrated in an in vitro murine tissue culture system that a single amino acid change in a myeloma protein results in the generation of an autoantibody from an antibody with antibacterial specificity (32). The mouse myeloma protein studied was S107 which is an anti-phos- phorylcholine antibody bearing the T 15 idiotype. Phosphorylcholine is the dominant antigen in the cell wall of the bacterium Streptococcus pneumonias and the T 15 idiotype is the dominant antibody made in several mouse strains in response to pneumococcal antigen (33,34). Dr. Scharff’s laboratory has generated in vitro derived somatic variants of the S107 protein that differ by only one or two amino acids from the parental protein (35). One of these, U4, differs from S107 by a single amino acid at residue 35 in the first hypervariable region of the heavy chain. U4 has lost reactivity with phos- phorylcholine but has acquired reactivity with double-stranded DNA and other phos- phorylated macromolecules, including cardiolipin and protamine (32). This pattern of antigenic reactivity is similar to that of the spontaneously arising anti-DNA anti- bodies found in murine and human SLE (8). This raises the possibility that autoan- tibodies may arise in vivo also by somatic mutation from antibodies used in the normal immune response to foreign antigen.

In order to further explore this question we have been studying anti-DNA activity and expression of the T15 idiotype in both autoimmune mice and immunologically manipulated nonautoimmune mice. In the first system we examined anti-DNA an- tibodies from MRL/lpr mice for the expression of T15 idiotype. MRL/lpr mice spon- taneously develop anti-DNA antibodies and a lupus-like syndrome (36). Anti-DNA antibodies were precipitated from serum with double-stranded DNA, dissolved in 8 it4 urea, and displayed on isoelectric focusing gels. The gels were transferred to nitrocellulose and then probed with rabbit anti-T 15 antibody followed by enzyme- conjugated anti-rabbit antibody and substrate. Figure 1 shows that the precipitated

PH

-1-uu MRVlPr NWNZW !m?BAm/c

FKL 1. Western blot analysis of the. Ih@ing of qqbbit anti-T 15 antisemm to DNA-anti-DNA precipitates t%om MRL/& mice and NZB/NZW mice. -S 107 protein was displayed as a contro1 for the anti-T1 5 antiserum. No immunoglobulin is precipitated by the addition of DNA .to BAL.B/c serum.

50 DAVIDSON ET AL.

anti-DNA antibody population includes T 15-bearing antibodies and that these anti- bodies are cationic.

In order to augment expression of T 15 in MRL mice we have neonatally primed the mice with T 15 idiotype to fix this idiotype in the repertoire and then immunized with phosphorylcholine. This protocol results in marked enhancement of the T 15 response to phosphorylcholine and of TlS-positive anti-DNA antibodies and has therefore provided the opportunity to generate T 15-positive anti-DNA hybridomas for molecular analysis (37). The study demonstrates that exposure to a bacterial antigen augments production of both idiotype-positive antibacterial antibodies and idiotype- positive anti-DNA antibodies (Table 2).

Recently we have used another murine model to analyze anti-DNA antibodies in a normal mouse strain. In this set of experiments we immunized BALB/c mice with a monoclonal antibody to I-J, a marker found on T-suppressor cells. Immunization with anti-I-J antibody has been shown to block T-suppressor cell activity in vivo (38). We therefore thought that anti-I-J antibody might inhibit T-suppressor cells that are presumed to be important in preventing autoimmunity in normal individuals. Ad- ministration of anti-I-Jd antibody to BALB/c mice results in hypergammaglobulinemia and in the generation of high titers of anti-DNA antibodies, some bearing the T 15 idiotype. We believe this occurs as a result of interference with T-suppressor-cell func- tion. Sera from these manipulated BALB/c mice were displayed on isoelectric focusing gels, transferred to nitrocellulose, and probed with anti-T 15 antibody. We found that the T 15 positive antibodies in these mice focused at alkaline pH in contrast to the T 15 antibodies seen in naive mice (Fig. 2).

These data indicate that autoantibodies formed in our in vivo model use idiotypes and hence germ-line genes used in normal immune response to bacterial antigens and that these DNA-binding T 15 antibodies are more cationic than T 15-positive antibodies seen in naive mice. Generation of hybridoma antibodies from these mice will enable us to determine whether the cationic DNA-binding antibodies arise by somatic mu- tation of the germ-line genes.

SUMMARY

Anti-idiotypic antibodies are important immunologic tools for obtaining both structural and genetic information about antibody molecules and for exploring the regulation of their production.

TABLE 2

TlS-Bearing Anti-DNA and Anti-PC Antibodies in MRL/lpr Mice Primed with S107 and Boosted with PC

Immunization No. of mice Titer of anti-PC T I5 +’

antibodies Titer of anti-DNA T15 +a

antibodies

s107 &PC 5 0.83 (0.66-0.93) 0.13 (0.07-0.30) PC 4 0.44 (0.25-0.78) 0. I 1 (0.03-0.25) s107 3 0.01 (O-.01) 0.04 (0.0 l-0.08) None 3 0.01 (O-.01) 0.01 (O-0.02)

’ Mean OD,, with range in parentheses.

ORIGINS OF ANTI-DNA ANTIBODIES 51

PH

-UV MRL BALE/c aI-Jd

FIG. 2. Western blot analysis of the binding of polyclonal rabbit anti-T I5 antiserum to serum from MRL/ Ipr mice, BALB/c mice, and BALB/c mice treated with anti- lJd antibody. Lanes l-2, MRL/lpr mice; lanes 3-9, unimmunized BALB/c mice; lanes 5-8, BALB/c mice treated with anti-I-Jd antiserum. Note the ap pearance of cationic T 15positive bands in the anti-I-Jd-treated BALB/c mice.

The anti-idiotype 31 defines a dominant idiotype present on anti-DNA antibodies in a majority of genetically unrelated patients with SLE. We have demonstrated in our families with SLE that family members preferentially express the 31 idiotype and that in most of these family members the idiotype is not on DNA-binding antibodies. We speculate that the genetic predisposition to SLE consists in part of the use of particular idiotypes in the normal immune response. Random somatic mutation may then convert these antibodies to autoantibodies. Mutations at important structural sites or those resulting in an acquisition of cationic charge may result in increased affinity for DNA. Some idiotypes such as 31 may be more likely than others to undergo “pathogenic” mutations and acquire autoreactivity.

We have demonstrated both in autoimmune and in immunologically manipulated nonautoimmune mice that some anti-DNA antibodies bear an idiotype commonly found on antibacterial antibodies and that these idiotype positive autoantibodies bear a cationic charge.

Examination of the differences between 31-positive DNA-binding and non-DNA- binding antibodies in our human patients with SLE and of the T 15bearing antibodies that bind DNA and those that do not in our mouse in vivo model will help to elucidate the molecular mechanisms responsible for the generation of autoantibodies, and their regulation.

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