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Lupus-prone (NZB x NZW)F1 mice spontaneously develop elevated titers of anti-DNA Abs that contain T cell determinants in their V(H) regions. We have previously shown that tolerization with an artificial peptide based on these T cell determinants (pConsensus (pCons)) can block production of anti-DNA Abs and prolong survival of the mice. In this study, we show that this protection depends in part on the generation of peripheral TGFbeta- and Foxp3-expressing inhibitory CD8+ (Ti) cells. These CD8+ Ti cells suppress anti-DNA IgG production both in vitro and in vivo and require up-regulated expression of both Foxp3 and TGFbeta to exert their suppressive function, as indicated by microarray analyses, small interfering RNA inhibition studies, and blocking experiments. Additionally, CD8+ Ti cells from pCons-tolerized mice were longer-lived suppressors that up-regulated expression of Bcl-2 and were more resistant to apoptosis than similar cells from naive mice. These data indicate that clinical suppression of autoimmunity after administration of pCons depends in part on the generation of CD8+ Ti cells that suppress secretion of anti-DNA Ig using mechanisms that include Foxp3, TGFbeta, and resistance to apoptosis.

T cells that recognize nucleoproteins are required for the production of anti-dsDNA Abs involved in lupus development. SmD1 83-119 (a D1 protein of the Smith (Sm) proteins, part of small nuclear ribonucleoprotein) was recently shown to provide T cell help to anti-dsDNA Abs in the NZB/NZW model of lupus. Using this model in the present study, we showed that high dose tolerance to SmD1 (600-1000 microg i.v. of SmD1(83-119) peptide/mo) delays the production of autoantibodies, postpones the onset of lupus nephritis as confirmed by histology, and prolongs survival. Tolerance to SmD1 83-119 was adoptively transferred by CD90+ T cells, which also reduce T cell help for autoreactive B cells in vitro. One week after SmD1 83-119 tolerance induction in prenephritic mice, we detected cytokine changes in cultures of CD90+ T and B220+ B cells with decreased IFN-gamma and IL-4 expression and an increase in TGFbeta. Increased frequencies of regulatory IFN-gamma+ and IL10+ CD4+ T cells were later detected. Such regulatory IL-10+/IFN-gamma+ type 1 regulatory T cells prevented autoantibody generation and anti-CD3-induced proliferation of naive T cells. In conclusion, these results indicate that SmD1 83-119 peptide may play a dominant role in the activation of helper and regulatory T cells that influence autoantibody generation and murine lupus.

Immunization with portions of a murine antibody to DNA induced Ig peptide-reactive peripheral CD8+ inhibitory T (Ti) cells in non-autoimmune (BALB/c x NZW) F1 (CWF1) mice. Those Ti suppressed in vitro production of IgG anti-DNA by lymphocytes from MHC-matched, lupus-prone (NZB x NZW) F1 (BWF1) mice, primarily via secretion of transforming growth factor-beta (TGF-beta). However, splenic CD8+ cells from immunized BWF1 mice failed to suppress anti-DNA. Therefore, BWF1 mice were studied for defects in peripheral CD8+ T cells. The potential to suppress autoimmunity mediated by activated CD4+ helper T and B cells in BWF1 mice was assessed. As BWF1 mice aged, peripheral CD8+ T cells expanded little; fewer than 10% displayed surface markers of activation and memory. In contrast, quantities of splenic CD4+ T and B cells increased; high proportions displayed activation/memory markers. In old compared to young BWF1 mice, splenic cell secretion of two cytokines required for generation of CD8+ T effectors, IL-2 and TGF-beta, was decreased. Immunizing BWF1 mice activated peptide-reactive CD8+ T cells, but their number was decreased compared to young BWF1 or old normal mice. While peptide-reactive splenic CD8+ T cells from immunized BWF1 mice did not survive in short-term cultures, similar CD8+ T cell lines from immunized CWF1 mice expanded and on transfer into BWF1 mice delayed autoimmunity and prolonged survival. Therefore, CD8+ T cells in old BWF1 mice are impaired in expansion, acquisition of memory, secretion of cytokine, and suppression of autoimmunity. Understanding these defects might identify targets for therapy in systemic lupus erythematosus., (Copyright 2004 Wiley-VCH Verlag GmbH & Co.)

We have recently shown that tolerogenic administration of an artificial peptide (pConsensus) that is based on sequences within the V(H) regions of several murine anti-dsDNA Ig delays appearance of autoantibodies in female (New Zealand Black (NZB) x New Zealand White (NZW))F(1) (NZB/W F(1)) mice and significantly prolongs their survival. The aim of this study was to characterize the T cell population(s) involved in pConsensus-induced down-regulation of autoimmune responses in tolerized NZB/W F(1) mice. Using MHC class II dimers loaded with tolerogenic peptide, we found that pCons favored expansion of peptide-reactive CD4(+)CD25(+) regulatory T cells (T(R)) that inhibited in vitro production of anti-dsDNA Ab-forming cells. Suppression by T(R) was abrogated by the presence in culture of Ab to glucocorticoid-induced TNFR family member 18 or to TGFbeta latency-associated protein. These findings suggest possible relevance of Ag specificity in the mechanism of T(R)-mediated immune tolerance to Ig-derived peptides in NZB/W F(1) mice.

Peptides from VH regions of antibodies to DNA drive immune responses in systemic lupus erythematosus (SLE). We studied peptide-induced cytokine release by peripheral blood mononuclear cells (PBMC) of patients, the influence of peptide concentration, disease characteristics and HLA-D haplotypes. Cells secreting cytokines (IFNgamma, IL-2, IL-4 and IL-10) were measured by ELISPOT in PBMC from 31 patients with SLE and 20 matched healthy controls in response to seven peptides (A-G) from the CDR1/FR2 to CDR2/FR3 VH regions of human anti-DNA MAbs. Disease activity was assessed by SELENA-SLEDAI. HLA-DR and -DQ alleles were determined by molecular typing techniques. PBMC from significantly higher proportions of SLE patients than controls responded to VH peptides by generating IFNgamma and IL-10. Type of cytokines released in response to at least one peptide (D) depended on antigen concentration. Cytokine release was not associated with clinical features of SLE except for disease duration. A shift occurred from IFNgamma, IL-4 and IL-10 production in early disease to IL-4 and IL-10 in late disease (suggesting increasing TH2-like responses over time). Three peptides (B, D, G) were more stimulatory in the SLE patients than controls. Although none of the peptides was restricted by any particular MHC class II allele, among responders there was increased prevalence of HLA- DQB1*0201 and/or DRB1*0301, alleles known to predispose to SLE. Thus, responses to some VH peptides are more frequent in SLE and vary with disease duration. Increased responses in individuals with HLA class II genotypes that predispose to SLE suggest that peptide presentation by those molecules permits brisker peripheral blood cell responses to autoantibody peptides, thus increasing risk for disease.

Current understanding of the role of leptin has expanded from its narrow association with obesity to a variety of effects on different biological processes including immune function. More specifically, leptin links nutritional status and energy balance to regulation of pro-inflammatory T-helper 1 immune responses. This has prompted several studies of targeted intervention with leptin antagonists in rodents to suppress onset and/or progression of chronic inflammation and autoimmunity. This review presents current preclinical evidence and potential applications for leptin-based immune approaches aimed at improving therapy for chronic and autoimmune conditions.

Objective: The C-terminal peptide of amino acids 83-119 of the SmD1 protein is a target of the autoimmune response in human and murine lupus. This study was undertaken to test the hypothesis that SmD1(83-119)-reactive T cells play a crucial role in the generation of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies., Methods: Splenic or lymph node T cells derived from unmanipulated as well as SmD1(83-119)-immunized NZB/NZW mice were analyzed in vitro by enzyme-linked immunospot (ELISpot) assay to determine T cell help for anti-dsDNA generation induced by the SmD1(83-119) peptide. Cytokines expressed by these T cells were measured by ELISpot assay, enzyme-linked immunosorbent assay, and flow cytometry. SmD1(83-119)- and ovalbumin-specific T cell lines were generated and characterized., Results: The SmD1(83-119) peptide, but not the control peptides, significantly increased the in vitro generation of anti-dsDNA antibodies in cultures from unmanipulated NZB/NZW mice. Interferon-gamma (IFNgamma), interleukin-2 (IL-2), IL-4, transforming growth factor beta, and IL-10 production increased in response to the peptide in young mice; only IFNgamma and IL-2 were increased in older, diseased mice. Activation of SmD1(83-119)-reactive T cells by immunization of NZB/NZW mice resulted in elevated anti-dsDNA synthesis and, later, increased antibodies to SmD1(83-119). Most cells in SmD1(83-119)-specific CD4+ T cell lines helping both antibodies had increased intracellular expression of IFNgamma, and most expressed both IFNgamma and IL-4., Conclusion: The SmD1(83-119) peptide plays an important role in generating T cell help for autoantibodies, including anti-dsDNA, and activates different subsets of T cells as defined by distinct cytokine expression. This peptide is an interesting target structure for the modulation of autoreactive T cells, and its characterization may contribute to our understanding of the role of autoantigen-reactive T cells in the pathogenesis of SLE.

Many individuals develop a single or a few brief episodes of autoimmunity from which they recover. Mechanisms that quell pathologic autoimmunity following such a breakdown of self-tolerance are not clearly understood. In this study, we show that in nonautoimmune mice, dsDNA-specific autoreactive B cells exist but remain inactive. This state of inactivation in dsDNA-specific B cells could be disrupted by autoreactive Th cells; in this case T cells that react with peptides from the V(H) region of anti-DNA Abs (hereafter called anti-V(H) T cells). Immunization with anti-DNA mAb, its gamma-chain or peptides derived from its V(H) region induced anti-V(H) Th cells, IgG anti-dsDNA Ab, and proteinuria. The breakdown of B cell tolerance in nonautoimmune mice, however, was short-lived: anti-DNA Ab and nephritis subsided despite subsequent immunizations. The recovery from autoimmunity temporally correlated with the appearance of T cells that inhibited anti-DNA Ab production. Such inhibitory T cells secreted TGFbeta; the inhibition of anti-DNA Ab production by these cells was partly abolished by anti-TGFbeta Ab. Even without immunization, nonautoimmune mice possess T cells that can inhibit autoantibody production. Thus, inhibitory T cells in nonautoimmune mice may normally inhibit T-dependent activation of autoreactive B cells and/or reverse such activation following stimulation by Th cells. The induction of such inhibitory T cells may play a role in protecting nonautoimmune mice from developing chronic autoimmunity.

Objective: To test the hypothesis that an artificial peptide, based on an algorithm describing T cell stimulatory sequences from the VH regions of murine IgG antibodies to DNA, is an effective tolerogen in vivo in the (NZB/NZW)F1 (BWF1) mouse model of systemic lupus erythematosus., Methods: Using proliferative T cell responses to 439 Ig peptides, an algorithm was constructed that describes the amino acid sequences likely to stimulate BWF1 T cells. Stimulatory (pConsensus [pCONS]) or nonstimulatory (pNegative [pNEG]) peptides were synthesized. Groups of 10-week-old (healthy) or 20-week-old (diseased) BWF1 mice received monthly intravenous injections of 1,000 microg of peptide or saline. Ex vivo splenic T cell responses and in vivo clinical effects were measured., Results: Tolerance was induced by pCONS, but not by pNEG, with respect to ex vivo T cell proliferation and T cell help for antibodies to DNA. T cell help for IgG anti-DNA was impaired not only after T cell stimulation by pCONS but also after stimulation by some peptides from nucleosomal and Ro antigens. Treatment with pCONS significantly delayed the onset of nephritis and inhibited increases in the plasma levels of total IgG, IgG antibodies to DNA, nucleosome, cardiolipin, interferon-gamma, and interleukin-4. In contrast, antibody responses to an exogenous antigen were not impaired. Survival was significantly prolonged in both healthy and diseased mice treated with pCONS., Conclusion: Induction of immune tolerance in response to treatment with pCONS in autoreactive T cell helper populations is highly effective in delaying the appearance of multiple autoantibodies, cytokine increases, and nephritis in BWF1 mice, and dramatically prolongs survival. A striking effect is the ability of the peptide to tolerize T cell help for anti-DNA that is induced by multiple, structurally unrelated self antigens.

Individuals with systemic autoantibody-mediated diseases such as lupus have polyclonal T and B cell activation. Yet, autoantibody production is restricted to certain autoantigens. The mechanisms underlying this phenomenon remain unclear. We propose three potential mechanisms by which autoreactive helper T cell responses diversify to become polyclonal, yet are restricted to certain antigens. First, using a model where self-Ig peptides spontaneously activate T cells and modulate disease in lupus mice, we demonstrate that the numbers of autoantibody-augmenting T helper peptides increased across the Ig molecule as mice aged ("intramolecular determinant spreading"). Secondly, a single T cell hybridoma established from a (NZB x NZW)F1 mouse immunized with one self-Ig peptide recognized several Ig-derived determinants, which had little sequence homology with the immunizing peptide. Such determinant degeneracy can lead to polyclonality. To explore a mechanism for restriction to certain autoantigens, a protein database search was done for homologies with sequences of selected stimulatory Ig peptides. Identical sequences of such determinants were not found in murine proteins other than Ig. These occurred infrequently in nonautoantibody Ig, but quite commonly in lupus-related autoantibodies such as antibodies to DNA, cardiolipin, and erythrocytes. Thus, determinant spreading and degenerate recognition in T cells coupled with recurring use of T cell determinant sequences among autoantibodies result in polyclonality that is restricted to certain autoantigens.

Young NZB/NZW F1 (BWF1) mice develop T cell repertoires that are spontaneously stimulated by peptides derived from the VH regions of BWF1 J558-encoded autoantibodies (autoAb) to DNA, but not to VH region peptides of a J558-encoded antibody to an exogenous antigen. Immunization of young BWF1 mice with selected Ig-derived peptides accelerates anti-DNA production and nephritis, and immune tolerance induction to a combination of these determinants delays anti-DNA production and disease onset. To further characterize this immunoregulatory circuitry, we asked whether this phenomenon of spontaneous T cell activation by VH region peptides is restricted to anti-DNA Ab of the VH J558 family, and what are the charge and structural attributes of these T cell determinants? We studied spontaneous T cell proliferative responses to peptides derived from an autoAb to DNA constructed from VH 7183 and found that it contains several T cell determinants. Both charge and size of certain amino acids (AA) within each peptide seemed to be important. Peptides containing arginine (R) or glutamic acid (E) were more likely to be T cell determinants than peptides without those AA; replacement of charged AA with uncharged AA abolished T cell recognition of a peptide. We previously reported that some Abs to DNA are enriched in R in their VH; pathogenic BWF1 IgG anti-DNA are enriched in positively and negatively charged AA in VH regions. Therefore, we speculate that peptides from natural IgM autoAb may initially activate BWF1 T cells, and as somatic mutations of Ig occur, charged AA introduced into V regions increase the number of T cell determinants, thus favoring upregulation of pathogenic Ab subsets. Therefore, in predisposed individuals, the ability of T cells to recognize more charged T cell determinants in autoAb may be one mechanism promoting development of disease.

Mechanisms that initiate and maintain autoantibody (autoAb) production in individuals with autoimmune diseases like SLE are poorly understood. Inadequate suppression of autoreactive T cells and/or unusual activation of T and B cells may underlie the persistence of pathogenic autoAbs in lupus. Here, we examine the possibility that in mice with lupus, autoAb molecules may be upregulating their own production by activating self-reactive T cells via their own processed peptides; downregulation of this circuit may decrease autoAb production and delay the development of lupus. We found that before the onset of clinical disease, lupus-prone (NZB/NZW) F1 [BWF1] (but not MHC-matched nonautoimmune mice) developed spontaneous T cell autoimmunity to peptides from variable regions of heavy chains (VH) of syngeneic anti-DNA mAbs but not to peptides from the VH region of an mAb to an exogenous antigen. Tolerizing young BWF1 mice with intravenous injections of autoAb-derived determinants substantially delayed development of anti-DNA antibodies and nephritis and prolonged survival. Thus, in such an autoAb-mediated disease, the presence of autoreactive T cells against VH region determinants of autoAbs may represent an important mechanism involved in the regulation of autoimmunity. Our findings show that tolerizing such autoreactive T cells can postpone the development of an autoimmune disease like SLE.

(NZB x NZW) F1 (BWF1) mice develop spontaneous T cell autoimmunity to VH region determinants of syngeneic anti-DNA before the onset of clinical disease. In this study, we characterized the immunogenicity, MHC binding, and lymphokine secretion patterns induced by T cell determinants from the VH region of one such anti-DNA mAb (A6.1) and examined their role in the regulation of autoimmunity. Determinants were identified by proliferation of syngeneic splenic T cells from young, unprimed BWF1 mice in response to overlapping 12-mer peptides representing the entire VH region sequence. Immunization of young BWF1 mice with any of three determinants (A6H 34-45 [p34], A6H 58-69 [p58], and A6H 84-95 [p84]) elicited proliferative responses upon in vitro recall. Upon immunization with the whole A6.1 molecule, however, proliferative responses could be recalled only to the p58 peptide, defining this as immunodominant. The other two peptides (p34 and p84) elicited minimal or no proliferation and could be termed cryptic. Proliferative responses elicited by the cryptic determinants were restricted by a single class II (I-Ed for p34 and I-Au for p84), whereas the immunodominant p58 determinant was restricted by both I-Ed and I-Eu. The cryptic p34 and p84 bound strongly to I-Ed and I-Au, respectively, whereas the immunodominant p58 peptide bound poorly to I-Ed. A6H p84 elicited T cells that secreted lymphokines in a pattern consistent with a Th1-like phenotype, whereas p58 induced a Th2-like cytokine pattern. Immunization with p34 or p84, or adoptive transfer of a p84-reactive T cell line to young BWF1 mice significantly increased IgG anti-DNA levels, accelerated nephritis, and decreased survival. In conclusion, in BWF1 mice, autoreactive T cells recognizing both cryptic and dominant self-determinants on anti-DNA autoantibodies escape deletion or anergy induction. Furthermore, since these cells are spontaneously activated before the onset of clinical disease, they may be involved in the development of the autoimmune process.

Murine monoclonal and human affinity-purified Abs to native DNA (anti-nDNA) that cross-react with the A and D SnRNP polypeptides were analyzed for direct injurious effects against cultured pig kidney (PK15) cells under ordinary cell-culture conditions. Of the two murine nephritogenic Abs derived from NZB/NZW F1 mice (BWds1 and BWds3), BWds1 initially bound to the cell surface and subsequently penetrated into cells localizing in nuclei and cytoplasm. BWds3 was consistently and abundantly associated with the surface of live cells without penetration. In the presence of rabbit C, BWds3 caused massive cell lysis (85% dead cells) whereas BWds1 had only a modest lytic effect (24% dead cells). One of the nonpathogenic murine anti-nDNA Abs (5GD5) that did not cross-react with the A and D polypeptides showed no interaction with PK-15 cells and had no injurious effects. Affinity-purified autoantibodies to nDNA isolated from two SLE patients with high anti-nDNA titers and clinically active lupus nephritis showed properties similar to the murine mAbs. They both strongly cross-reacted with the A and D SnRNP polypeptides and interacted with live PK-15 cells. One of them (Cr) penetrated into live cells and localized within cytoplasm and nuclei whereas the other (Pe) bound mostly to the cell surface and caused significant cell lysis in the presence of C. Results of this study suggest that the nephritogenic murine anti-nDNA as well as subpopulations of human anti-nDNA Abs could exert their injurious influence through direct interactions with kidney cells using two different pathogenic mechanisms (i.e., C-mediated cytotoxicity and potential cell cycle dysfunctions. Interestingly, cross-reactivity of anti-nDNA Abs with the A and D SnRNP polypeptides appears to be a prerequisite for their direct pathogenicity.

Three pathogenic and two non-pathogenic NZB/NZW F1 mAbs to DNA were compared. Pathogenicity was defined as the ability to induce nephritis in BALB/c mice. All mAbs were IgG2a or 2b, had high avidity for double-stranded DNA and fixed complement well. All three pathogens expressed idiotype IdGN2. Mice receiving pathogenic mAbs (compared with non-pathogenic) had more glomerular IgG deposits. The unique properties of two of the pathogens were: strong homogeneous staining of Hep-2 nuclei and the ability to bind (i) nucleosomes, (ii) histone (after mAb complexed with DNA), (iii) heparan sulfate in renal basement membranes (after complexing with DNA/histone) and (iv) nuclei in vivo. Comparison of nucleotide and amino acid sequences of the V regions of heavy and light Ig chains showed use of multiple VHDJH and V kappa J kappa gene families, with representation of several anti-DNA 'families' described by others. Arginine (R) occurred in the CDR2 or CDR3 of VH chains in all pathogens; R was absent in the CDRs of VH chains of non-pathogens. Positively and negatively charged AA were more frequent in VH CDR of pathogens than of non-pathogens. We hypothesize that the tertiary structure of mAbs determined by VH CDR regions permits stronger binding to negatively charged antigens (DNA and heparan sulfate) and to positively charged molecules (histone) in pathogens compared with non-pathogens.

Antibodies to native DNA (nDNA) in sera from patients with systemic lupus erythematosus have been found to frequently correlate with antibodies to the A and D SnRNP proteins measured in Western blot assays. 40 of 54 SLE (74.1%) sera with anti-nDNA bound to A and D proteins, while 9 of 113 sera (8%) without anti-nDNA bound the A and D proteins, P < 10(-8) by Fisher's exact test. Antibodies to nDNA correlated closely with anti-A and anti-D in seven of eight patients followed sequentially, r = 0.7865. Nine human polyclonal anti-nDNA populations were isolated from DNA cellulose columns. Seven reacted equally with A and D, and two reacted predominantly with D. Two of three murine monoclonal anti-DNA antibodies isolated from NZB/NZW F1 hybrid mice bound A and D equally in Western blot with a titer > 1/40,000. These reactions were directed to the unfolded A and D proteins measurable in Western blot since these monoclonals (and several of the human anti-nDNA populations) failed to react with native U1RNP in ELISA or in RNA immunoprecipitation experiments. These newly recognized cross reactions of anti-nDNA may amplify the immune response to DNA and be part of the original immunogenic drive.

Objective: To assess the ability of peptides derived from anti-DNA to stimulate syngeneic T lymphocytes and influence lupus in (NZB x NZW)F1 (NZB/W) mice., Methods: We synthesized (by Geysen pin method) overlapping 12-mer peptides recapitulating the amino acid sequence of the VH region of a nephritogenic monoclonal IgG2a anti-DNA antibody (A6.1) from an NZB/W mouse. Splenic T cells were cultured with the peptides; multiple experiments assayed 12-mer and 16-mer peptides which contained a triple-base motif (KFKGK). We immunized 20-week-old NZB/W mice with the 12-mer and evaluated them for evidence of nephritis and for quantities of antibodies in plasma and glomeruli., Results: Three clusters of peptides caused proliferation of spleen cells from young, nonimmunized mice. Both the 12-mer FYNQKFKGKATL and the 16-mer GDTFYNQKFKGKATLT peptides stimulated purified T cells. The KXKXK motif occurs in 15% of murine Ig VH (NBRF protein database), compared with 100% (6 of 6) of NZB/W anti-DNA monoclonal antibody. Immunization with the 12-mer peptide increased plasma levels of IgG, anti-DNA, and immune complexes, and levels of anti-DNA in glomeruli; nephritis was accelerated., Conclusion: NZB/W anti-DNA contain peptide sequences in their VH regions that stimulate self-T cells. At least one motif is frequent in NZB/W anti-DNA. If some activated T cells provide help, this mechanism may contribute to sustained up-regulation of autoantibodies in murine lupus.

Serum levels of IdGN2 (an idiotype enriched in nephritogenic antibodies), IdX (an idiotype not enriched in nephritogenic antibodies), IgG, and anti-DNA were measured in 23 Caucasian patients with lupus nephritis, in age- and sex-matched lupus patients without nephritis, and in similarly matched healthy individuals. Serum levels of IdGN2 were significantly higher in the patients with lupus nephritis than in those without, and they were higher in all lupus patients compared with the healthy control subjects. However, the same observations were true for serum levels of IdX. There were significant positive correlations between the serum levels of IgG, IdGN2, IdX, and anti-DNA. HLA typing at the DR and DQ loci was performed in 105 lupus patients of different races (Caucasian, black, and Asian/Polynesian/Filipino). Serum levels of IdGN2 in 83 of these individuals did not correlate with any of the HLA class II haplotypes currently known to predispose to lupus nephritis. We conclude that the high serum levels of IdGN2, which are characteristic of some patients with lupus nephritis, may often result from polyclonal B cell activation rather than from idiotype-specific upregulation associated with one or more of the class II genes that predispose to nephritis in this disease.

We have studied several monoclonal anti-double-stranded (ds) DNA antibodies for their ability to accelerate lupus nephritis in young NZB X NZW F1 female mice and to induce it in BALB/c mice. Two identified as pathogens in both strains have characteristics previously associated with nephritogenicity: expression of IgG2a isotype and IdGN2 idiotype. Both pathogenic antibodies used the combination of genes from the VHJ558 and VK9 subfamilies. Two weak pathogens failed to accelerate nephritis in young BW mice, but induced lupus nephritis in BALB/c mice. They both express IdGN2; one is cationic and an IgG3, the other is an IgG2a. Additional MAbs (some IgG2a, one IdGN2-positive) did not accelerate or induce nephritis. We have cloned and sequenced the variable regions of the immunoglobulin genes of one pathogenic autoantibody. No unique V, D, or J gene segments and no evidence of unusual mechanisms in generating diversity were used to construct this antibody. These data argue against use of unique abnormal Ig genes by systemic lupus erythematosus individuals to construct pathogenic autoantibody subsets. Instead, the major abnormality may be immunoregulatory.

Antibodies to self-antigens are characteristic of several human and murine autoimmune diseases. Subsets of those autoantibodies cause organ damage in some instances, such as IgG antibodies to DNA in human and murine systemic lupus erythematosus (SLE). Our experiments in the NZB/NZW F1 (BW) female mouse model of SLE were designed to define idiotypic (Id) structures on antibodies to DNA in attempts to distinguish pathogens from nonpathogens within the anti-DNA population. Two important findings emerged. First, the number of public Id expressed became relatively restricted as the mice aged, with three such Id (IdX, IdGN1 and IdGN2) dominating and accounting for 30 to 95% of the total serum IgG in all individual nephritic mice studied, and 81 to 86% of the total IgG in serum pools from 30-wk-old nephritic mice. Second, IdGN1 and IdGN2 constituted approximately 50% of the IgG deposited in glomeruli of nephritic mice; IdX was present in negligible quantities in glomeruli, whereas it was usually the most frequent Id in BW serum. These latter findings suggested that pathogens and nonpathogens can be distinguished by their idiotypy in this animal model. The finding of relative Id restriction suggests the occurrence of an idiotypic "spreading" phenomenon, in which a regulatory process appears as BW mice age that results in repeated selection and expansion of this small number of Id, one group of which, the IdGN, is pathogenic. This process was further suggested in experiments in which IdX was suppressed by administration of anti-IdX; the "escape" antibodies to DNA appearing after suppression of IdX were composed largely of IdGN1 and IdGN2, without a major contribution from Id-negative mutants. Defining the basis of this Id spreading or restriction phenomenon may provide important information regarding the pathogenesis of this autoimmune disease.

We propose that a major immunoregulatory abnormality in murine and human autoantibody-mediated disease is idiotypic spreading. By this mechanism, B cells with the genetic information to produce immunoglobulin (Ig) bearing certain public idiotypes (Ids) are selectively upregulated, probably by Id-recognizing helper T cells. The model in which we are testing the hypothesis is systemic lupus erythematosus (SLE) in humans and NZB/NZW F1 (BW) female mice. Recent experiments have shown that the number of public Ids expressed on the Ig of nephritic BW mice is quite restricted. IdX is the dominant Id on serum Ig; IdGN1 and IdGN2 are also common. All three Ids were initially derived from spontaneous antibodies to DNA. Together the three are present on 85% of the total Ig repertoire. Such restriction suggests idiotypic spreading. In glomerular Ig deposits from nephritic BW mice, IdGN1 and IdGN2 are found on 45% of the total Ig: IdX is present in minute amounts. Furthermore, suppression of IdGNs by administration of anti-IdGN1 to BW mice resulted in significant delay in the onset of nephritis, but the IdGNs escaped from control and eventually caused a fatal nephritis. Finally, studies of glomerular Ig deposits in renal biopsies of patients with SLE have shown that IdGN2 dominates such Ig, being present in 76% of renal biopsies from SLE patients and in 6% from patients with non-lupus immune nephritis. Therefore, we have concluded that IdGN1 and IdGN2 are markers of nephritogenic subsets of autoantibodies and are probably the products of idiotypic spreading most likely to cause disease. Finally, after a review of recent experiments suggesting the dominance of autoreactive, Mossman Type 2 T helper cells in nephritic BW mice, it is hypothesized that autoreactive, IdGN-recognizing helper T cells may be central to the sustained upregulation of pathogenic autoantibodies in murine and human SLE.

A monoclonal anti-idiotypic antibody (anti-Id) to a public idiotype (Id) present on spontaneous IgG antibodies to DNA from NZB/NZW F1 mice recognized similar determinants on polyclonal and monoclonal IgG anti-DNA antibodies from mice of the unrelated MRL/lpr and BXSB strains. Incubation of the anti-Id with four of five monoclonal Id in solid phase inhibited their ability to bind DNA; however, different Id+ antibodies recognized different epitopes within the DNA molecule. Therefore, the public Id was located close to the antigen-binding regions but did not comprise all of those regions. Analysis of multiple polyclonal and monoclonal antibodies to DNA showed the Id on all subclasses of IgG. However, antibodies bearing the Id carried a neutral or cationic charge (10 of 10 monoclonals with pI greater than 7 were Id+); the presence of the Id on anionic IgG (pI less than or equal to 7) was infrequent (one of 21 serums, one of eight monoclonal antibodies). Therefore, IgG autoantibodies to DNA are constructed from closely related public idiotypes in several mouse strains that spontaneously develop lupus, and that Id is restricted to antibodies with a pI of 7 or greater.

Among the autoantibodies that are known to play a role in the pathogenesis of autoimmune diseases, antibodies to DNA (anti-DNA) have been the subject of much study. Several interesting observations have resulted. The ability to make antibodies that bind DNA is not abnormal. Normal mice and humans can produce antibodies that bind DNA. On the other hand, large quantities of antibodies to DNA are found in the sera of patients with systemic lupus erythematosus (SLE), and complement-fixing antibodies to double-stranded (ds) DNA cause some of the tissue lesions, especially glomerulonephritis (GN). Why, then, do some individuals make anti-DNA that deposits in glomeruli, skin, and other tissue, resulting in organ damage? It is likely that disease results from a combination of several factors--ability to make pathogenic antibody subsets, inability to downregulate those subsets, and "tissue susceptibility" to injury from those antibodies and their immune complexes. This chapter will focus on the characteristics of pathogenic antibody subsets and their regulation.

There is good evidence that the idiotypic network of the immune system can be implicated in the synthesis of pathogenic subsets of autoantibodies. The individual with systemic lupus erythematosus must have the immunoglobulin gene information which permits synthesis of those idiotypes, helper T populations which drive or select for the B cells producing them, and inadequate mechanisms to suppress those activated effector cells.

A mechanism for sustained production of pathogenic autoantibody subsets in patients and mice with systemic lupus erythematosus may be centered on selective up-regulation of B cells bearing certain idiotypes. Public idiotypes are characteristic of some autoantibodies, including anti-DNA. Evidence is reviewed that suggests that immunoglobulins bearing certain public idiotypes, such as IdGN2, contain autoantibody subsets that are nephritogenic in human systemic lupus erythematosus and in New Zealand black/New Zealand white F1 mice. Up-regulation of such cells could promote development of nephritis. Work from several laboratories has shown that production of immunoglobulin G antibodies to DNA depends upon T cell help. In New Zealand black/New Zealand white F1 mice, cloned T cells are dominated by autoreactive cells that produce B cell growth factors. We suggest that this sustained release of B cell growth factors combined with selection by T helper cells for B cells bearing IdGN2 are a major mechanism for sustained up-regulation of nephritogenic subsets of autoantibodies.

A new method for measuring DNA antibody forming cells (DNA-AFC) using the enzyme-linked immunospot (ELISPOT) assay is described. This method uses enzyme-linked immunosorbent assay (ELISA) techniques applied to cells cultured on DNA-coated plates, which allows visual quantitation of spots representing imprints of specific antibodies from DNA-AFC. Specificity for DNA was confirmed by inhibition studies and lack of reactivity by anti-lysozyme hybridomas. Isotypes of IgG and IgM can be measured using the appropriate antisera in the assay. A study of 16 female (New Zealand black x New Zealand white)F1 ([NZB x NZW]F1) female mice showed significant correlation between age, rising blood urea nitrogen levels, and increasing proteinuria and increasing numbers of DNA-AFC. In contrast, the correlation between circulating antibodies to DNA (ELISA method) and clinical parameters of nephritis was not significant. Both the native DNA ELISPOT and the native DNA ELISA had similar significant linear correlations with age. This is the first report of use of the ELISPOT assay for measurement of DNA-AFC. The DNA-AFC measured by this method were specific and correlated with the presence of clinical nephritis in (NZB x NZW)F1 mice. This method should allow further study on the regulation of DNA-AFC in vitro and in vivo, and will be useful in the investigation of DNA-AFC and cellular mechanisms of autoimmunity.

Cloned hybrid cells, selected for their ability to secret an IgG 2a immunoglobulin specific for single-stranded (ss) nucleic acids, were obtained by fusion of spleen cells from an unimmunized autoimmune MRL/1 pr male mouse with nonsecreting myeloma cells (MOPC-21, line Sp2/0-Ag 14). Designated MRss-1, this monoclonal antibody was (i) propagated by intraperitoneal injection of hybrid cells to pristane-treated. Balb/c mice, (ii) purified from the bulk of other proteins in ascites extracts by chromatography with DEAE-Sephacel adsorbent, and (iii) radiochemically labeled via reductive methylation using NaB3H4 and formaldehyde. The binding of 3H-labeled antibody to immobilized ssDNA- agarose, calf thymus) or soluble (fd DNA) ssDNA was rapid and dependent upon ssDNA and ionic strength, but not hydrogen ion concentration. Optimal binding occurred in both low and intermediate salt concentration (0.1-0.25 M NaCl), yet was completely abolished above 0.30 M NaCl. The presence of guanine (Gua)-containing mono-, oligo-, and polynucleotides also abolished and/or decreased 3H-labeled antibody binding to ssDNA-agarose. In these competition assays, the amount of Gua-containing mono-and oligonucleotides required to inhibit antibody binding by 50% (0.2-1.0 mg/mL) exceeded those of poly(G), rRNA, and fd DNA (i.e., 0.03-0.1 microgram/mL) by 4 orders of magnitude. In contrast, (deoxy)ribose 5'-phosphate as well as other nucleic acid derivatives devoid of Gua failed to inhibit antibody binding. The above findings were substantiated by the observation that 3H-labeled antibody bound to guanosine (G)- and guanidylate (pG)-conjugated Sepharose, yet not to other nucleoside (A, C, and U)- or nucleotide (pA, pC, and pU)-conjugated adsorbents. Last, the introduction of the methyl group at the N-2, O-6, and N-7 positions in the Gua ring system completely abolished antibody binding. Collectively, these results demonstrate that the MRss-1 antibody recognized single-stranded nucleic acid substrates by virtue of their content of guanidylate residues and, more specificity, by the presence of the Gua base moiety.

The suppression of pathogenic antibodies to DNA in NZB/NZW f1 female mice was achieved by repeated inoculation of the mice with a monoclonal anti-idiotypic antibody (anti-Id). The anti-Id, an IgG1, kappa, was directed against a major cross-reactive idiotype (Id) on NZB/NZW IgG antibodies to DNA. One hundred micrograms of the anti-Id were inoculated i.p. every 2 wk, beginning at 6 wk of age (nondiseased mice--no circulating anti-DNA or proteinuria) or 20 wk of age (diseased mice--all with circulating anti-DNA, one-third with proteinuria). As controls, littermates received an IgG, kappa non-DNA-binding myeloma or no treatment. In the young mice, nephritis and anti-DNA antibodies appeared at the same time in all groups, and their circulating antibodies to DNA did not bear the target Id. In the older (20-wk-old) mice, survival was significantly prolonged because of delay in the onset of nephritis; the total quantities of antibodies to DNA were diminished, and the target Id, initially present on circulating IgG, was deleted. These benefits were transient; the suppression of antibodies was followed by the appearance of large quantities of anti-DNA that did not bear the major Id. Therefore, although administration of anti-Id was effective in reducing an undesirable antibody response after the target Id was present on circulating antibodies, the benefits were limited, probably by Id "switch" or by increased synthesis of pathogenic antibodies bearing a minor Id.

Suppression of circulating antibodies to double-stranded DNA was achieved in NZB/NZW f1 female mice by repeated administration of an IgG2a monoclonal antibody to DNA. Deaths from nephritis were delayed; glomerular deposition of IgG and of the cationic IgG DNA antibodies characteristic of murine lupus nephritis were diminished. Quantities of circulating antibodies to single-stranded DNA were not reduced compared with untreated or IgG myeloma-treated control mice. Antibodies directed against the monoclonal anti-DNA appeared in the circulation of treated mice after three inoculations of the idiotype. Those antibodies did not react with another monoclonal anti-DNA of the same allotype. One monoclonal anti-idiotypic antibody was obtained in hybridoma cultures derived from a spleen of a treated mouse. Cross-reactive or common idiotypes were found in 30-50% of NZB/NZW f1 sera and monoclonal DNA antibodies. Deletions of portions of the spectrotype of antibodies to DNA were found in sera containing anti-idiotypic antibodies, suggesting suppression of clones producing antibodies with isoelectric points similar to that of the immunizing idiotype. Deletions of some of the anti-idiotypic antibodies also occurred as the mice aged. Rheumatoid factors were not detectable in any sera. Therefore, administration of an antibody to DNA bearing an idiotype occurring with high frequency in NZB/NZW f1 females resulted in relatively specific suppression of the antibody response to double-stranded DNA, as well as suppression of nephritis. Reduction of anti-DNA synthesis by anti-idiotypic antibodies may have been an important suppressive mechanism. Experiments are in progress to test this hypothesis.

Indirect immunofluorescence with monoclonal antibodies to 6 different idiotypes was used to characterize immunoglobulins deposited in the glomeruli of renal biopsy samples from 32 patients with systemic lupus erythematosus (SLE) and 19 patients with nonlupus immune glomerulonephritis. IdGN2 was present in 75% of the biopsy specimens from SLE patients and in 6% of those from patients with non-lupus nephritis; IdGN1 occurred in 38% and 6%, respectively. The other idiotypes were not increased in biopsy samples from patients with SLE. Deposition of IdGN2 was associated with a subendothelial location of Ig and proliferative changes in the glomeruli. In studies of glomerular eluates from 4 immunosuppressed SLE patients, an average of 26% of total Ig and 37% of anti-DNA was composed of IdGN2. Compared with IdGN2- immunoglobulin, IdGN2+ immunoglobulin was enriched in IgG1 in all 4 eluates, and was enriched in high-avidity anti-DNA in 2 eluates. IdGN2 is a marker of antibody subsets that are characteristic of SLE and are associated with severe lupus nephritis.

A graft-vs-host (GVH) reaction of parental T cells in allogeneic F1 mice can lead to an autoimmune disease resembling human SLE. We analyzed the contribution of MHC genes to the development of IgG antinuclear antibody production and immune complex glomerulonephritis in MHC-congenic F1 recipients. DBA/2 T cells elicited IgG antibodies to histone, ssDNA, and dsDNA in all histoincompatible F1 recipients that were studied. The anti-DNA antibody responses were quantitatively similar among the F1 combinations and displayed comparable IgG2a subclass and cationic charge characteristics. In contrast, severe renal disease was manifested only in F1 mice that expressed H-2b encoded class II gene products. Disease susceptibility was associated with a decrease in circulating anti-DNA antibodies and a characteristic localization of immune complexes in the glomeruli. The data suggest that the production of potentially pathogenic IgG anti-nuclear antibodies is not sufficient for the development of renal disease in GVH-induced lupus. Thus, another event separate from autoantibody production is MHC dependent and appears to be critical for the formation and/or deposition of pathologic immune complexes.