Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.     

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.     

Antigen-specific therapy of murine lupus nephritis using nucleosomal peptides: tolerance spreading impairs pathogenic function of autoimmune T and B cells

In the (SWR x NZB)F1 mouse model of lupus, we previously localized the critical autoepitopes for nephritogenic autoantibody-inducing Th cells in the core histones of nucleosomes at aa positions 10-33 of H2B and 16-39 and 71-94 of H4. A brief therapy with the peptides administered i.v. to 3-mo-old prenephritic (SWR x NZB)F1 mice that were already producing pathogenic autoantibodies markedly delayed the onset of severe lupus nephritis. Strikingly, chronic therapy with the peptides injected into 18-mo-old (SWR x NZB)F1 mice with established glomerulonephritis prolonged survival and even halted the progression of renal disease. Remarkably, tolerization with any one of the nucleosomal peptides impaired autoimmune T cell help, inhibiting the production of multiple pathogenic autoantibodies. However, cytokine production or proliferative responses to the peptides were not grossly changed by the therapy. Moreover, suppressor T cells were not detected in the treated mice. Most interestingly, the best therapeutic effect was obtained with nucleosomal peptide H416-39, which had a tolerogenic effect not only on autoimmune Th cells, but autoimmune B cells as well, because this peptide contained both T and B cell autoepitopes. These studies show that the pathogenic T and B cells of lupus, despite intrinsic defects in activation thresholds, are still susceptible to autoantigen-specific tolerogens.     

Histone peptide-induced nasal tolerance: suppression of murine lupus

Induced mucosal tolerance has been shown to be beneficial in preventing or treating a number of murine and human autoimmune disorders. However, this particular form of therapy has not been thoroughly tested in systemic lupus erythematosus. In this study, we investigated the conditions for induction of nasal tolerance using a histone peptide named H471 expressing a dominant T cell epitope in the histone protein H4 of mononucleosome in lupus-prone SNF(1) female mice. We also tested the effect of chronic peptide nasal treatment on the development of autoimmune reactivities in these mice. Results demonstrated that a dose-dependent nasal tolerance to peptide H471 can be achieved before or after peptide sensitization in SNF(1) mice. In addition, tolerance to mononucleosomes was induced by nasal instillation of SNF(1) mice with H471. This was accompanied by an increase in IL-10 and suppression of IFN-gamma production by lymph node cells. Suppression of Th1-type cytokines was also observed in SNF(1) mice that were nasally administered with H471 before intradermal injection with the peptide. Finally, chronic nasal instillation of mice with the H471 peptide not only suppressed the development of autoantibodies, but also altered the severity of glomerulonephritis in lupus-prone SNF(1) mice.     

CD4+ T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3

We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2(d/d)) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4(+) T cells from unprimed (New Zealand Black (NZB) x New Zealand White (NZW))F(1) lupus mice (H-2(d/z)). None of the 11 H4 peptides was recognized by CD4(+) T cells from (NZB x NZW)F(1) mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-gamma upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-gamma and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF(1) (H-2(d/q)) and MRL/lpr (H-2(k/k)) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB x NZW)F(1) mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.     

Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets

We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 mug nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H4(71-94) was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+ CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN-gamma responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90-100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+ CD25+ and CD8+ Treg cells produced and required TGF-beta1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+ CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF-beta-producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.     

Evidence for multiple shared antigenic determinants within Ro60 and other lupus-related ribonucleoprotein autoantigens in human autoimmune responses

Ab responses directed against several ribonucleoprotein (RNP) Ags are a characteristic feature of systemic lupus erythematosus (SLE). Previous work in our laboratory using mouse model systems had revealed that both epitope spreading and inherent cross-reactivity between ribonucleoproteins contributes to the observed multiple specificities in autoimmune sera. We have now extended these studies to human autoimmune responses. Using purified polyclonal and mAbs derived from SLE patients, cross-reactivity between Ro60 and SmD was demonstrated. The cross-reactive epitope was mapped to nonhomologous regions on Ro60(481-505) and SmD(88-102). Five mAbs specifically recognized apoptotic cells, demonstrated variable levels of cross-reactivity toward other nonhomologous ribonucleoprotein targets and bound multiple, nonoverlapping and nonhomologous epitopes on Ro60. Our study demonstrates that cross-reactivity between frequently targeted autoantigens is an important aspect of human systemic autoimmune responses. The presence of multiple cross-reactive epitopes on Ro60 might be important for the generation of anti-Ro60 Ab in SLE patients and in normal individuals displaying no evidence of clinical disease.     

Localization of autoepitopes on the PCM-1 autoantigen using scleroderma sera with autoantibodies against the centrosome

Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portions of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434–1465, and 1661–1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434–1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.     

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.     

T cell reactivity to MHC class II-bound self peptides in systemic lupus erythematosus-prone MRL/lpr mice

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03-5.01) and C3H mice (stimulation index = 2.03-3.75). IL-2 and IFN-gamma production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2(k), mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, beta(2)-microglobulin, and MHC class II I-A(k)beta. The first three peptides were isolated from I-E(k) molecules and the last peptide was bound to I-A(k). T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity.     

Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H4(71-94), when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-beta. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H4(71-94) peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-beta, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H4(71-94) was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H4(71-94) peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.     

Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry

The origins of autoimmunity in systemic lupus erythematosus (SLE) are thought to involve both genetic and environmental factors. To identify environmental agents that could potentially incite autoimmunity, we have traced the autoantibody response in human SLE back in time, prior to clinical disease onset, and identified the initial autoantigenic epitope for some lupus patients positive for antibodies to 60 kDa Ro. This initial epitope directly cross-reacts with a peptide from the latent viral protein Epstein-Barr virus nuclear antigen-1 (EBNA-1). Animals immunized with either the first epitope of 60 kDa Ro or the cross-reactive EBNA-1 epitope progressively develop autoantibodies binding multiple epitopes of Ro and spliceosomal autoantigens. They eventually acquire clinical symptoms of lupus such as leukopenia, thrombocytopenia and renal dysfunction. These data support the hypothesis that some humoral autoimmunity in human lupus arises through molecular mimicry between EBNA-1 and lupus autoantigens and provide further evidence to suspect an etiologic role for Epstein-Barr virus in SLE.     

Opposite role of Ras in tumor necrosis factor-alpha-induced cell cycle regulation: competition for Raf kinase

We previously reported an epitope presenting vector, pCI, a derivative of a human invariant chain (Ii) expression vector, in which the class II associated invariant chain peptide (CLIP, Ii p89-101) could be substituted with antigenic peptides. In the current study, we used this vector to develop a new expression cloning system to identify CD4+ T cell epitopes. We inserted double-stranded oligo DNAs of randomized sequences into this vector and prepared an epitope-presenting library which loads randomized 13-mer peptides onto HLA class II molecules coexpressed in COS-7 cells. Utilizing this library, we isolated a cross-reactive epitope recognized by a glutamic acid decarboxylase (GAD) 65-autoreactive T cell clone established from a patient with insulin-dependent diabetes mellitus. Although the newly identified epitope (PVQLSNQWHVVGATF) was far different from the original epitope, GAD65 p116-128 (NILLQYVVKSFDR), it did have the capacity to stimulate the T cell clone comparable to that of the original GAD epitope. Our system may be applicable not only for identifying of cross-reactive epitopes for CD4+ T cells of known specificity, but also for detection of epitopes stimulatory for CD4+ T cells the epitopes of which are unknown.     

The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor.

Susceptibility to experimental autoimmune myasthenia gravis (EAMG), which is induced in mice by injection of purified Torpedo nicotinic acetylcholine receptor (TAChR), is influenced by the I-A locus products, which restrict presentation of AChR Th epitopes. The bm12 mutation of the I-Ab molecule in the C57BL/6 strain, which is highly susceptible to EAMG, yields the EAMG resistant mutant B6.C-H-2bm12 (bm12). We investigated here the consequences of the bm 12 mutation on the CD4+ response to the TAChR alpha subunit. Upon immunization with TAChR, CD4+ cells became sensitized to TAChR and anti-AChR antibodies were produced in both bm12 and C57BL/6 strains. Overlapping synthetic peptides, corresponding to the complete sequence of TAChR alpha subunit, were used to identify Th epitopes. CD4+ cells from C57BL/6 mice recognized peptides T alpha 150-169, T alpha 181-200, and T alpha 360-378. CD4+ cells from bm12 mice did not respond to any synthetic sequence. Upon injection of the three C57BL/6 Th epitope peptides, either individually or as a pool, CD4+ cells from C57BL/6 mice recognized each peptide and TAChR. Therefore they recognized epitopes similar or identical to those originated from TAChR processing. CD4+ cells from bm12 mice injected with the same peptides responded to T alpha 360-378 strongly, to a lesser extent to T alpha 181-200, never to peptide T alpha 150-169. Only CD4+ cells sensitized against the T epitope peptide T alpha 181-200 responded to TAChR. We tested if lack of response to T alpha 150-169, and the low response to T alpha 181-200, was due to inability of the I-Abm12 molecule to present the T epitope peptides. bm12 and C57BL/6 APC were used to present the T epitope peptides to specifically sensitized CD4+ cells from C57BL/6 mice. All T epitope peptides were presented by bm12 APC, although T alpha 150-169 was presented less efficiently than by C57BL/6 APC. Resistance to EAMG induced by the bm12 mutation may be due to the change in the epitope repertoire of AChR-specific Th cells, and lack of recognition of otherwise immunodominant Th epitopes. For at least one epitope this might be due to absence of potentially reactive, specific CD4+ clones.     

A SmD peptide induces better antibody responses to other proteins within the small nuclear ribonucleoprotein complex than to SmD protein via intermolecular epitope spreading

Autoantibody response against the small nuclear ribonucleoprotein (snRNP) complex is a characteristic feature of systemic lupus erythematosus. The current investigation was undertaken to determine whether activation of SmD-reactive T cells by synthetic peptides harboring T cell epitopes can initiate a B cell epitope spreading cascade within the snRNP complex. T cell epitopes on SmD were mapped in A/J mice and were localized to three regions on SmD, within aa 26-55, 52-69, and 86-115. Immunization with synthetic peptides SmD(31-45), SmD(52-66), and SmD(91-110) induced T and B cell responses to the peptides, with SmD(31-45) inducing the strongest response. However, only SmD(52-66) immunization induced T cells capable of reacting with SmD. Analysis of sera by immunoprecipitation assays showed that intermolecular B cell epitope spreading to U1RNA-associated A ribonucleoprotein and SmB was consistently observed only in the SmD(52-66)-immunized mice. Surprisingly, in these mice, Ab responses to SmD were at low levels and transient. In addition, the sera did not react with other regions on SmD, indicating a lack of intramolecular B cell epitope spreading within SmD. Our study demonstrates that T cell responses to dominant epitope on a protein within a multiantigenic complex are capable of inducing B cell responses to other proteins within the complex. This effect can happen without generating a good Ab response to the protein from which the T epitope was derived. Thus caution must be taken in the identification of Ags responsible for initiating autoimmune responses based solely on serological analysis of patients and animals with systemic autoimmune disorders.     

T cell studies in a peptide-induced model of systemic lupus erythematosus

We have previously reported that immunization with a peptide mimetope of dsDNA on a branched polylysine backbone (DWEYSVWLSN-MAP) induces a systemic lupus erythematosus-like syndrome in the nonautoimmune BALB/c mouse strain. To understand the mechanism underlying this breakdown in self tolerance, we examined the role of T cells in the response. Our results show that the anti-foreign and anti-self response induced by immunization is T cell dependent and is mediated by I-E(d)-restricted CD4(+) T cells of the Th1 subset. In addition, generation of the critical T cell epitope requires processing by APCs and depends on the presence of both DWEYSVWLSN and the MAP backbone. The breakdown in self tolerance does not occur through cross-reactivity between the T cell epitope of DWEYSVWLSN-MAP and epitopes derived from nuclear Ags. In this induced-model of SLE, therefore, autoreactivity results from the activation of T cells specific for foreign Ag and of cross-reactive anti-foreign, anti-self B cells. Despite the fact that tissue injury is mediated by Ab, the critical initiating T cell response is Th1.     

Differential presentation of glutamic acid decarboxylase 65 (GAD65) T cell epitopes among HLA-DRB1*0401-positive individuals.

Glutamic acid decarboxylase 65 (GAD65) is one of the major autoantigens in type 1 diabetes. We investigated whether there is variation in the processing of GAD65 epitopes between individuals with similar HLA backgrounds and whether the processing characteristics of certain immunogenic epitopes are different in distinct APC subpopulations. Using DR401-restricted T cell hybridomas specific for two immunogenic GAD65 epitopes (115-127 and 274-286), we demonstrate an epitope-specific presentation pattern in human B-lymphoblastoid cell lines (B-LCL). When pulsed with the GAD protein, some DRB1*0401-positive B-LCL, which presented GAD65 274-286 epitope efficiently, were unable to present the GAD65 115-127 epitope. However, all B-LCL presented synthetic peptides corresponding to either GAD epitope. In addition, when pulsed with human serum albumin, all cell lines gave equal stimulation of a DR4-restricted human serum albumin-specific T hybridoma. GAD65-transfected cell lines displayed the same presentation phenotype, showing that lack of the presentation of the 115-127 epitope was not due to inefficient uptake of the protein. Blood mononuclear adherent cells, B cells, or dendritic cells derived from the same individual displayed the same presentation pattern as observed in B cell lines, suggesting that the defect most likely is genetically determined. Therefore, individual differences in Ag processing may result in the presentation of distinct set of peptides derived from an autoantigen such as GAD65. This may be an important mechanism for the deviation of the immune response either into a regulatory pathway or into an inflammatory autoimmune reactivity.     

Identification of an epitope of type 1 streptococcal M protein that is shared with a 43-kDa protein of human myocardium and renal glomeruli.

The localization of opsonic and tissue-cross-reactive epitopes within the amino terminus of type 1 streptococcal M protein was investigated by using murine mAb raised against synthetic peptides of type 1 M protein. Two mAb (IIIA2 and IIIB8) reacted with epitopes located within amino acid residues 1-12 of type 1 M protein. These antibodies opsonized type 1 streptococci and did not cross-react with human kidney and heart tissue. Another mAb (IC7) reacted with mesangial cells of renal glomeruli and human myocardium. The cross-reactive epitope of mAb IC7 was localized to position 13-19, indicating that it is not the same epitope as the previously described vimentin-cross-reactive epitope at position 23-26 of type 1 M protein. In Western blots of mesangial cell and myocardial proteins, mAb IC7 cross-reacted with a 43-kDa protein. Neither vimentin nor actin inhibited the binding of mAb IC7 to the cross-reactive protein, as determined by Western blot or immunofluorescence inhibition tests. These results provide evidence that type 1 M protein contains at least one autoimmune epitope shared with both human glomeruli and myocardium.     

A major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein autoantigen

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180-205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher's exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.