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Idiotypic analysis constitutes a very important and primary approach in the understanding of a variety of antibody responses. Antibodies associated with autoimmune diseases are mostly defined according to their antigen binding specificities. They may also be classified according to the idiotypes which are antigenic determinants located in the variable regions of the antibodies. Idiotypes(id) are important firstly because they are a special category of autoantigens which are immunogenic and they are also targets of regulatory processes. As the immune system recognizes these idiotypes, anti-idiotype (anti- id) antibodies are produced thus leading to a regulatory network of interacting ids and anti-id antibodies. Disturbances in the idiotypic network may result in a selective polyclonal B cell expansion which may lead to the initiation of an autoimmune disease. SLE is characterized by polyclonal B cell activation

directed against a broad range of self antigens such as DNA. Both in patients with SLE and in mice with a lupus like disease, lupus associated ids on anti-DNA antibodies have been described (Shoenfeld et al 1991, Ravirajan et al 1991, Uner et al 1994, Knupp et al 1992, Ebling et al 1993, Greenspan et al 1993). Idiotypes may be beneficial in distinguishing between pathogenic and non-pathogenic autoantibodies. The 8.12 idiotype found on anti-DNA antibodies in sera of patients with SLE have been known to contribute to to pathogenicity as they can be found in glomerular deposits on renal biopsies (Diamond and Schwartz 1987). Manipulation of the idiotypic network may thus be important in generating reagents for down regulation of pathogenic subsets of autoantibodies. Also idiotypes may serve as phenotypic markers for protective immune responses. The 8.12 + antibodies have also been shown to display potentially protective anti­ microbial specificities and are routinely generated in non-autoimmune individuals in response to vaccination with pneumococcal polysaccharide (Livneh et al 1994). Also murine anti-PC antibodies expressing the T15 idiotype have been shown to be protective against pneumococcal infection (Briles et al 1981). Immunization with anti-idiotypes and idiotypes may change the autoantibody expression in autoimmune diseases. Also internal image bearing anti-idiotypes may provide an effective tool in the treatment of tumors (George et al 1987). Thus idiotypes may be of importance as V region markers in antibodies, as regulators of the immune response and as tools in tumor therapy.

Various groups have reported idiotypes associated with anti-DNA antibodies. Approximately thirty common idiotypes have been described on hybridoma derived or affinity purified anti-DNA antibodies (Watts et al 1990).

Shoenfeld et al (1983) and Isenberg et al (1984) have described the 16/6 idiotype expressed on a human monoclonal anti-DNA antibody that reacts with poly (dT), poly (I) and single stranded DNA. The 16/6 idiotype is found in about 50% of lupus patients , notably those with active disease. An idiotope designated as 9G4 is a marker for immunoglobulins which utilise a particular Vh gene, the V4-34. This idiotope which is found on anti-DNA antibodies has been identified in 45% of sera from patients with SLE. It has also been identified occasionally amongst patients with other autoimmune rheumatic diseases. The 9G4 levels are found to fluctuate with disease activity and this idiotope was detected in 3/11 SLE renal biopsies tested. Its presence was associated with the HLA markers AI and B8 (Potter et al 1993, Isenberg et al 1993, Isenberg et al 1998).

Blanco et al (1994) reported the WRI176 beta idiotope which was located on a IgM kappa anti-DNA human monoclonal antibody. The WRI176 beta idiotope is found on the heavy chain and outside the binding site of the antibody. This idiotope occurs in about 44% of SLE patients and is also frequently found in other rheumatic and infectious diseases and in some healthy first degree relatives of SLE patients. Kalsi et al (1995b) have identified and characterized three idiotypes on human monoclonal anti-DNA antibodies. RT-6 is a human monoclonal antibody which binds histones and Sm/RNP expresses a private idiotype (Id). Its expression is limited to a small number of human monoclonal antibodies and is also found to occur in sera of patients with infectious diseases. RT-72 Id and RT-82 Id are expressed on polyreactive monoclonal antibodies and have a public distribution. These two Ids are found on monoclonal antibodies from murine and human adult and foetal tissues and in sera from patients with

SLE, rheumatoid arthritis (RA) and other autoimmune diseases. No correlation with disease activity, IgM or IgG levels was observed with either RT-72 or RT- 84 Id. RT-6 Id and RT-72 Id are located on the framework regions of the jj, heavy chain whereas RT-84 Id is present on the kappa light chain, within the binding site.RT-72 Id and RT-84 Id have also been found in immunoglobulin deposits in lupus renal sections.

Ehrenstein et al (1994a) identified two idiotypes present on human IgG anti-DNA antibody, D5 derived from a patient with active SLE. Two idiotypes designated D-5RI Id and D5MI Id are defined by a polyclonal and monoclonal anti-Id respectively. The idiotypes are found only in the IgG fraction of human sera. About 30% of SLE patients express either of the two idiotypes. The idiotypes are present on both DNA binding and non DNA binding fractions of lupus sera. D5Id was found in 6/10 renal lupus biopsies as compared to 2/15 disease control renal biopsies in which immunoglobulin was deposited. D-5RI Id was not found in renal biopsies.

Ehrenstein et al (1994b) also characterized another idiotype, B3-Id in patients with active SLE. This idiotype is present on the lambda light chain and is at or near the binding site for double stranded DNA. The lambda chain, as characterized by nucleotide sequencing was found to be 90% homologous to the V Lambda 2.1 germline gene which is involved in coding for the nephritogenic anti-DNA antibodies carrying the 8.12 idiotype. This idiotype is associated with active disease and is the first to be derived from a human monoclonal anti-DNA antibody of the IgG class.

Ravirajan et al (1995) investigated phospholipid binding specificity of IgM monoclonal antibodies generated from the spleen of two patients (RT and RSP) with active SLE and also studied their idiotype expression. The idiotypes, RT-84Id and H3 are commonly expressed in sera of patients with SLE. It was observed that 75% of the phospholipid binding antibodies from the RT clones expressed RT-84 Id but none from the RSP clones did so and the H3 Id was expressed only by the RT-83 antibody which is also a monoclonal phospholipid binding antibody obtained from patient RT. Anti-phospholipid antibodies are associated with a clotting tendency and recurrent fetal loss in a subset of SLE patients (Feinstein et al 1985, Kalunian et al 1988, Meroni et al 1991, Bagger et al 1993, Gharavi et al 1996, Nojima et al 2001).

Three other idiotypic systems, F4, 31 and 8.12 have been well characterized. The F4 idiotype is a heavy chain determinant expressed on IgG immunoglobulins and is associated with specificity for double stranded DNA. The F4 idiotype is not present in the repertoire of nonautoimmune individuals or on anti-pneumococcal antibodies. It is present in about 50% of lupus patients with anti-dsDNA activity (Davidson et al 1989). The F4 determinant is present on the heavy chain variable region and is expressed almost exclusively on IgG antibodies. In studies by Manheimer-Lory et al (1997), most of the F4+ antibodies from EBV transformed B cell lines from lupus patients used the Vh3 gene family for their heavy chains. The 31 and 8.12 idiotypes are present in high titer in lupus sera and are also present on anti-pneumococcal antibodies from non autoimmune individuals (Grayzel et al 1991, Livneh et al 1994).

The 31 idiotype is present on the kappa light chains of anti-dsDNA antibodies. It occurs in elevated titers in about 80% of patients with SLE whose serum is dsDNA reactive and in clinically unaffected family members. The 31 idiotype is found in both the kidney and skin of the lupus patients. The 31 idiotype is encoded by the V J genefamily.

The 8.12 idiotype is present on lambda light chains (Livneh et al 1987, Paul et al 1992). About 50% of SLE patients show elevated titers of 8.12+ antibodies. 8.12 antibodies can be found in the glomeruli of the lupus patients. 8.12 reactive antibodies are not elevated in family members. 8.12+ antibodies are also part of the protective antibody repertoire since vaccination with pneumococcal polysaccharide results in production of 8.12+ anti-pneumococcal antibodies (Livneh et al 1994). Sharing of the 8.12 idiotype by the anti-DNA and anti-pneumococcal antibodies may indicate that anti-dsDNA antibodies may be activated in an anti-pneumococcal response. Anti-DNA antibodies are also known to crossreact with other bacterial antigens such as lipid A (Spellerberg et al 1995). Also Diamond and Scharff (1984) showed that a single amino acid substitution of alanine for glutamic acid at residue 35 on the CDRl of the heavy chain converts a protective T15+ anti-PC antibody to one with DNA specificity. In studies by Limpansithikul et al (1995) murine crossreactive anti-dsDNA and anti-PC antibodies not only protect against lethal pneumococcal infection but are also pathogenic and deposit in kidneys of SCID mice. On the basis of these results, studies on crossreactivity of human anti-dsDNA antibodies to phosphorylcholine were undertaken by me.

Paul et al (1993), characterized two anti-DNA antibodies bearing the 8.12

idiotype from EBV transformed B cell lines from one patient with SLE (KS3)

and another with no autoimmune disease (SD6). Both the IgG antibodies (KS3

and SD6) expressed the 8.12 idiotype and bound dsDNA. The lambda light

chains of these antibodies were encoded by the VÀII gene family. The KS3

heavy chain was found to be encoded by a Vh4-DM1-DQ52-Jh6B-Cy1 gsne rearrangement and the SD6 heavy chain is encoded by a VH3-D21/9-JH6b-Cyl rearrangement. Both the antibodies were found to be somatically mutated. The

KS3 monoclonal antibody displays mutation in the complementarity determining

regions (CDRs) and the SD6 antibody shows mutations in the framework regions. The 8.12 antibodies are exclusively encoded by the VxII genes and maps to the

vicinity of the light chain CDRl (Paul et al 1992).

Idiotypic analysis are of potential utility to the clinician as anti-idiotypes may distinguish between pathogenic and nonpathogenic subsets of autoantibodies. One study in SLE patients has suggested that those in remission have anti-idiotypic antibodies to anti-DNA antibodies whereas the same patients during flares lack anti-idiotypic antibody (Zouali et al 1983). Furthermore, passive administration of anti-idiotypic antibodies has been shown to decrease anti-DNA antibody production and attenuate disease in mice for a short period of time (Hahn et al 1983). Anti-idiotypes to pathogenic anti-DNA antibodies have also been used to suppress lupus nephritis (Silvestris et al 1996). Uner et al (1994) have shown that treatment with antibodies reactive with the nephritogenic idiotype IdLNFl suppressses its production and leads to prolonged survival of (NZBxSWR)Fl mice.

Hence, the idiotypic network could be exploited for therapeutic purposes. Although these studies are provocative, however, the role of the idiotype network in SLE with regard to therapy remains uncertain mainly because even though the literature suggests that anti-idiotypes to antidsDNA may be effective in animals, there is no experience in humans to inform us.