Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3(+) regulatory T cells specific for the ubiquitous RNA-binding nuclear antigen La/Sjögren's syndrome (SS)-B.

RNA-binding nuclear antigens are a major class of self-antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)-B (La), is controlled by CD4(+) T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa-specific CD4(+) T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte-replete recipient mice expressing hLa as a neo-self-antigen. After initial antigen-specific cell division, hLa-specific donor CD4(+) T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)-10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3-negative donor cells demonstrated that accumulation of hLa-specific regulatory T cells (Treg ) was due primarily to expansion of small numbers of donor Treg . Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3(+) donor Treg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co-stimulatory and co-inhibitory molecules than B cells. Adoptive transfer of hLa peptide-loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa-specific Treg . Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa-specific T cells impaired FoxP3(+) donor T cell accumulation. Therefore, peripheral expansion of Treg specific for an RNA-binding nuclear antigen is mediated by antigen-presenting pDC in a type 1 IFN-dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA-binding nuclear antigens.

An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus.

An unknown environmental agent has been suspected to induce systemic lupus erythematosus (lupus) in man. Prompted by our recent immunochemical findings, we sought evidence for an association between Epstein-Barr virus infection and lupus. Because the vast majority of adults have been infected with Epstein-Barr virus, we chose to study children and young adults. Virtually all (116 of 117, or 99%) of these young patients had seroconverted against Epstein-Barr virus, as compared with only 70% (107 of 153) of their controls (odds ratio 49.9, 95% confidence interval 9.3-1025, P < 0. 00000000001). The difference in the rate of Epstein-Barr virus seroconversion could not be explained by serum IgG level or by cross-reacting anti-Sm/nRNP autoantibodies. No similar difference was found in the seroconversion rates against four other herpes viruses. An assay for Epstein-Barr viral DNA in peripheral blood lymphocytes established Epstein-Barr virus infection in the peripheral blood of all 32 of the lupus patients tested, while only 23 of the 32 matched controls were infected (odds ratio > 10, 95% confidence interval 2.53-infinity, P < 0.002). When considered with other evidence supporting a relationship between Epstein-Barr virus and lupus, these data are consistent with, but do not in themselves establish, Epstein-Barr virus infection as an etiologic factor in lupus.

Conserved features of Y RNAs: a comparison of experimentally derived secondary structures.

In this study, phylogenetically conserved structural features of the Ro RNP associated Y RNAs were investigated. The human, iguana, and frog Y3 and Y4 RNA sequences have been determined previously and the respective RNAs were subjected to enzymatic and chemical probing to obtain structural information. For all of the analyzed RNAs, the probing data were used to compose secondary structures, which partly deviate from previously predicted structures. Our results confirm the existence of two stem structures, which are also found at similar positions in hY1 and hY5 RNA. For the remaining parts of hY3 and hY4 RNA the secondary structures differ from those previously proposed based upon computer predictions. What might be more important is that certain parts of the RNAs appear to be flexible, i.e., to adopt several conformations. Another striking feature is that a characteristic pyrimidine-rich region, present in every Y RNA known, is single-stranded in all secondary structures. This may suggest that this region is readily available for base pairing inter-actions with other cellular nucleic acids, which might be important for the as yet unknown function of the RNAs.

Y3 is the most conserved small RNA component of Ro ribonucleoprotein complexes in vertebrate species.

YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions.

Genes for murine Y1 and Y3 Ro RNAs have class 3 RNA polymerase III promoter structures and are unlinked on mouse chromosome 6.

Murine YRNAs, which are components of the conserved Ro ribonucleoprotein (RNP) complex, have been identified by enzymatic RNA sequencing. Mouse Y1 (mY1) and Y3 (mY3; originally named mY2) RNAs share 97 and 95% identity to the human Y1 and Y3 RNAs, respectively. TATA-like sequences, Proximal Sequence Elements, and octamer sequences, which are upstream promoter element motifs indicative of Class 3 RNA Polymerase III (RNAPIII) transcribed genes, are found upstream of both the putative mY1 and mY3 coding regions. Further, these elements are strikingly conserved both in sequence and position relative to known Class 3 genes and to human YRNA genes. Inhibition of transcription in vitro by 200 micrograms/ml but not 1 microgram/ml of alpha-amanitin indicates transcription of the mouse YRNA genes by RNAPIII. Southern blot of C57BL/6J and Mus spretus murine genomic DNA with mY1 and mY3 gene-specific probes suggests that these genes are single copy in the mouse genome. Finally, gene mapping with a (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei mouse interspecific backcross DNA panel localizes the mY1 gene to the distal end of mouse chromosome 6, close to the motheaten (me) autoimmunity locus. The mY3 gene maps to the proximal end of mouse chromosome 6 very close to the T cell receptor beta locus, in a region homologous to human chromosome 7 where the human YRNA genes have been mapped.

Regulatory T cells and systemic lupus erythematosus.

CD4+CD25+ regulatory T cells (Treg) constitute an important mechanism of peripheral immune tolerance. Organ-specific autoimmune conditions, such as thyroiditis and insulin-dependent diabetes mellitus have been attributed to a breakdown of this tolerance mechanism. However, this T-cell subset has not been well studied in patients and mice with systemic lupus erythematosus (SLE; lupus). The information that has been gathered so far using new tools that discriminate Treg from activated T cells indicates that reduced numbers of Treg may exist in patients with lupus. In addition, potential defects in SLE Treg function have been documented in humans and mice. Our group has demonstrated equivalent proportions of thymic Treg in lupus prone and normal mice. We therefore propose that Treg function in SLE is the more important factor to address in future studies of murine lupus. Recent studies have shown that Toll-like receptor (TLR) ligation can result in an abrogation of Treg-mediated suppression; specifically ligation of TLR-2, -4, -8 and -9. We address this new information about TLRs and Treg and propose a model for Treg tolerance breakdown to nucleic acid-binding SLE autoantigens.

Neo-epitopes are required for immunogenicity of the La/SS-B nuclear antigen in the context of late apoptotic cells.

Mechanisms responsible for the induction of anti-nuclear autoantibodies (ANA) following exposure of the immune system to an excess of apoptotic cells are incompletely understood. In this study, the immunogenicity of late apoptotic cells expressing heterologous or syngeneic forms of La/SS-B was investigated following subcutaneous administration to A/J mice, a non-autoimmune strain in which the La antigenic system is well understood. Immunization of A/J mice with late apoptotic thymocytes taken from mice transgenic (Tg) for the human La (hLa) nuclear antigen resulted in the production of IgG ANA specific for human and mouse forms of La in the absence of foreign adjuvants. Preparations of phenotypically healthy cells expressing heterologous hLa were also immunogenic. However, hLa Tg late apoptotic cells accelerated and enhanced the apparent heterologous healthy cell-induced anti-La humoral response, while non-Tg late apoptotic cells did not. Subcutaneous administration of late apoptotic cells was insufficient to break existing tolerance to the hLa antigen in hLa Tg mice or to the endogenous mouse La (mLa) antigen in A/J mice immunized with syngeneic thymocytes, indicating a requirement for the presence of heterologous epitopes for anti-La ANA production. Lymph node dendritic cells (DC) but not B cells isolated from non-Tg mice injected with hLa Tg late apoptotic cells presented immunodominant T helper cell epitopes of hLa. These studies support a model in which the generation of neo-T cell epitopes is required for loss of tolerance to nuclear proteins after exposure of the healthy immune system to an excess of cells in late stages of apoptosis.

Transcriptional modulation of TCR, Notch and Wnt signaling pathways in SEB-anergized CD4+ T cells.

Gene expression changes in CD4 + Vbeta8+ T cells energized by in vivo exposure to staphylococcal enterotoxin B (SEB) bacterial superantigen compared to CD4 + Vbeta8+ non-energic T cells were assessed using DNA microarrays containing 5184 murine complementary DNAs. Anergy in splenic T cells of SEB-immunized BALB/c mice was verified by dramatically reduced proliferative capacity and an 8 x overexpression of GRAIL mRNA in CD4 + Vbeta8+ T cells taken from mice 7 days after injection. At an Associative t-test threshold of P<0.0005, 96 genes were overexpressed or detected only in anergic T cells, while 256 genes were suppressed or not detected in anergic T cells. Six of eight differential expressions tested using real-time quantitative PCR were validated. Message for B-Raf was detected only in non-anergic cells, while expression of the TCR signaling modulator Slap (Src-like adapter protein) and the TCR zeta-chain specific phosphatase Ptpn3 was enhanced. Modulation of multiple genes suggests downregulation of Wnt/beta-catenin signaling and enhanced Notch signaling in the anergic cells. Consistent with previous reports in a non-superantigen in vivo anergy model, mRNA for CD18 and the transcription factor Satb1 (special AT-rich-binding protein 1) was increased in SEB-energized T cells. This is the first report of global transcriptional changes in CD4+ T cells made anergic by superantigen exposure.

Photosensitivity diseases: cutaneous lupus erythematosus.

Ultraviolet radiation plays an important role in the induction of lesions in many patients with cutaneous lupus. In the photosensitive subset of lupus, subacute cutaneous lupus, the effects of ultraviolet radiation likely act in concert with specific autoantibodies, particularly anti-Ro-related autoantibodies, to produce lesions. Potential effects of ultraviolet radiation on the induction of cutaneous lupus, and the potential interplay of specific autoantibodies with ultraviolet radiation are discussed. The steps involved in the induction of cutaneous lupus lesions by ultraviolet radiation have not been fully elucidated. Recent advances in phototesting and analysis of the genetics of lupus should clarify the events leading to photosensitive cutaneous lupus lesions.

T-helper cell tolerance to ubiquitous nuclear antigens.

Systemic autoimmune diseases are characterized by the development of antinuclear autoantibodies. In order to understand the immunologic events leading to the development of such antibodies, knowledge of mechanisms of immune tolerance to nuclear antigens is required. By utilizing adoptive T-cell transfer strategies with transgenic mouse models expressing nuclear neo-self antigens, T-cell tolerance to the lupus-related nuclear antigens human La and nRNP A has been demonstrated. These findings also indicate the existence in normal animals of autoreactive B cells continuously presenting nuclear antigen, suggesting that nuclear antigens are not sequestered from the immune system. Investigations of CD4+ T-cell tolerance to non-nuclear antigens have revealed a number of mechanisms that protect the host from autoreactivity, including autoreactive T-cell deletion, regulatory T-cell development and anergy induction. Recent studies using T-cell receptor and neo-self nuclear antigen transgenic mice are revealing the importance of such mechanisms in maintaining tolerance to nuclear antigens. Mechanisms of tolerogenic antigen presentation, identification of tolerogenic antigen source(s) and the pathways leading to loss of tolerance to nuclear antigens in systemic autoimmune disease states are currently being sought.

Fine specificity of the autoimmune response to the Ro/SSA and La/SSB ribonucleoproteins.

The fine specificity of the Ro and La proteins has been studied by several techniques. In general, there is agreement in a qualitative sense that autoantibodies bind multiple epitopes. For some specific antibody binding, different studies agree quantitatively, for instance, the binding of the carboxyl terminus of 60-kd Ro as described by 2 studies using different techniques and the presence of an epitope within the leucine zipper of 52-kd Ro. In addition, there is general agreement about the location of a prominent epitope at the RRM motif region of the La molecule. On the other hand, the many specific epitope regions of the molecules differ among these studies. These discrepancies are likely the result of using different techniques, sera, and peptide constructs as well as a result of inherent advantages and disadvantages in the individual approaches. Several theories concerning the origin of not only the antibodies, but also the diseases themselves, have been generated from studies of the fine specificity of antibody binding. These include a theory of a primordial foreign antigen for anti-Ro autoimmunity, molecular mimicry with regard to La and CCHB, as well as the association of anti-Ro with HLA. These remain unproven, but are of continuing interest. An explanation for the association of anti-60-kd Ro and anti-52-kd Ro in the sera of patients has sprung from evaluating antibody binding. Data demonstrating multiple epitopes are part of a large body of evidence that strongly suggests an antigen-driven immune response. This means that the autoantigens are directly implicated in initiating and sustaining autoimmunity in their associated diseases. A number of studies have investigated the possibility of differences in the immune response to these antigens in SS and SLE sera. While several differences have been reported, none have been reproduced in a second cohort of patients. Furthermore, none of the reported differences may be sufficiently robust for clinical purposes, such as distinguishing between SS with systemic features and mild SLE, although some might be promising. For instance, in at least 3 groups of SLE patients, no binding of residues spanning amino acids 21-41 of 60-kd Ro has been found. Meanwhile, 1 of those studies found that 41% of sera from patients with primary SS bound the 60-kd Ro peptide 21-41. Perhaps future studies will elaborate a clinical role of such a difference among SS and SLE patients. Study of the epitopes of these autoantigens has, in part, led to a new animal model of anti-Ro and anti-La. Non-autoimmune-prone animals are immunized with proteins or peptides that make up the Ro/La RNP. Such animals develop an autoimmune response to the entire particle, not just the immunogen. This response has been hypothesized to arise from autoreactive B cells. In another, older animal model of disease, the MRL-lpr/lpr mouse, B cells have recently been shown to be required for the generation of abnormal, autoreactive T cells. Thus, there are now powerful data indicating that B cells that produce autoantibodies are directly involved in the pathogenesis of disease above and beyond the formation of immune complexes. Given that the autoreactive B cell is potentially critical to the underlying pathogenesis of disease, then studying these cells will be crucial to further understanding the origin of diseases associated with Ro and La autoimmunity. Hopefully, an increased understanding will eventually lead to improved treatment of patients. Progress in the area of treatment will almost surely be incremental, and studies of the fine specificity of autoantibody binding will be a part of the body of basic knowledge contributing to ultimate advancement. In the future, the animal models will need to be examined with regard to immunology and immunochemistry as well as genetics. The development of these autoantibodies has not been studied extensively because upon presentation to medical care, virtually all patients have a full-

Cognate T cell help is sufficient to trigger anti-nuclear autoantibodies in naive mice.

The mechanisms involved in the initiation of anti-nuclear autoantibodies are unknown. In this study, we show that one factor allowing anti-nuclear autoantibodies to develop is the incomplete nature of immune tolerance to many of these proteins. Immune responses in mice toward the ubiquitous nuclear autoantigen La/SS-B are much weaker than responses to the xenoantigen, human La (hLa; 74% identical). However, in transgenic (Tg) mice expressing hLa, the Ab response to this neo-autoantigen was reduced to a level resembling the weak autoimmune response to mouse LA: Partial tolerance to endogenous La autoantigen was restricted to the T compartment because transfer of CD4(+) T cells specific for one or more hLa determinants into mice bearing the hLa transgene was sufficient to elicit production of anti-hLa autoantibodies. Notably, only hLa- specific T cells from non-Tg mice, and not T cells from hLa Tg mice, induced autoantibody production in hLa Tg mice. These findings confirm partial Th tolerance to endogenous La and indicate the existence in normal animals of autoreactive B cells continuously presenting La nuclear AG: Therefore, the B cell compartment is constitutively set to respond to particular nuclear autoantigens, implicating limiting Th responses as a critical checkpoint in the development of anti-nuclear autoantibodies in normal individuals.

Epitope mimics and determinant spreading: pathways to autoimmunity.

Infectious microorganisms have evolved molecules which mimic the host in order to aid in their undetected propagation. In response, mammalian hosts have evolved a highly diverse immune repertoire designed to eradicate rapidly changing pathogens. The generation of diversity in the immune repertoire results in potentially damaging self cross-reactivities which require multiple regulatory controls to keep autoreactive lymphocytes in check. Here, we review how molecular mimicry at the T cell level might be important in the development of systemic autoimmunity.

The ultrastructural localization of 60-kDa Ro protein and human cytoplasmic RNAs: association with novel electron-dense bodies.

The 60-kDa Ro ribonucleoprotein is an important target of humoral autoimmune responses. However, the ultrastructural locations of the 60-kDa Ro protein and its associated small cytoplasmic RNAs (Y RNAs) have not been previously determined, and the functions of the Ro protein and RNAs are not known. In this study, the cellular locations of the 60-kDa Ro protein and the Ro Y1 and Y4 RNAs are determined by immunoelectron microscopy and in situ hybridization electron microscopy, respectively. Both Ro protein and Y RNAs are concentrated in discrete areas of the nucleoplasm, nucleolus, and cytoplasm of cultured cells and human skin sections. The 60-kDa Ro protein and Y RNAs are also present diffusely in the cytoplasm, where they occur in ribosome-rich regions, and in the nucleus. The presence of Ro ribonucleoprotein components in nucleoli and in ribosome-rich cytoplasmic areas suggests a potential for the involvement of Y RNAs and/or 60-kDa Ro protein in ribosome synthesis, assembly, or transport. Double labeling experiments show that Ro protein and Y RNAs colocalize in the nucleoplasm, nucleolus, and cytoplasm. In addition, aggregates of Y RNA occur unassociated with 60-kDa Ro protein, and aggregates of 60-kDa Ro protein occur unassociated with Y RNA. Aggregates of both Ro protein and Y RNAs label previously unreported nuclear and cytoplasmic electron-dense bodies. We propose that these distinctive Ro-associated electron-dense bodies may represent structure(s) important for cellular transport and/or Ro function.

Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis.

Y RNAs are small 'cytoplasmic' RNAs which are components of the Ro ribonucleoprotein (RNP) complex. The core of this complex, which is found in the cell nuclei of higher eukaryotes as well as the cytoplasm, is composed of a complex between the 60 kDa Ro protein and Y RNAs. Human cells contain four distinct Y RNAs (Y1, Y3, Y4 and Y5), while other eukaryotes contain a variable number of Y RNA homologues. When detected in a particular species, the Ro RNP has been present in every cell type within that particular organism. This characteristic, along with its high conservation among vertebrates, suggests an important function for Ro RNP in cellular metabolism; however, this function has not yet been definitively elucidated. In order to identify conserved features of Y RNA sequences and structures which may be directly involved in Ro RNP function, a phylogenetic comparative analysis of Y RNAs has been performed. Sequences of Y RNA homologues from five vertebrate species have been obtained and, together with previously published Y RNA sequences, used to predict Y RNA secondary structures. A novel RNA secondary structure comparison algorithm, the suboptimal RNA analysis program, has been developed and used in conjunction with available algorithms to find phylogenetically conserved secondary structure models for YI, Y3 and Y4 RNAs. Short, conserved sequences within the Y RNAs have been identified and are invariant among vertebrates, consistent with a direct role for Y RNAs in Ro function. A subset of these are located wholly or partially in looped regions in the Y3 and Y4 RNA predicted model structures, in accord with the possibility that these Y RNAs base pair with other cellular nucleic acids or are sites of interaction between the Ro RNP and other macromolecules.

Molecular chaperones are targets of autoimmunity in Ro(SS-A) immune mice.

We have used a murine model of experimental anti-Ro(SS-A) autoimmunity to dissect additional intermolecular interactions between the 52-kD Ro (Ro52) and 60-kD Ro (Ro60) autoantigens and molecular chaperones. Immune responses to members of the heat shock protein hsp70 and hsp90 families were measured by immunoblotting and ELISA in sera from mice immunized and boosted with purified recombinant Ro52, Ro60 and La (SS-B). All Ro52 and Ro60 immune sera immunoblotted the inducible glucose-regulated protein grp78 and hsp70 species but not constitutive hsc70 or hsp90. The kinetics of antibody production and reciprocal affinity purification experiments indicated that the grp78 and hsp70 responses were cross-reactive but distinct from immune responses to the primary Ro52 and Ro60 immunogens and the endoplasmic reticulum (ER)-resident chaperone calreticulin. No responses to molecular chaperones were detected in the La-immunized mice. Control immunizations indicated that the recruited grp78 and hsp70 responses were specific for the Ro proteins and not due to immunization with denatured protein. The rapid spreading of immunity to the inducible grp78 and hsp70 in Ro52- and Ro60-immunized mice suggests that these components may co-localize and physically associate under certain physiological conditions which may promote autoimmunization. The potential importance of the ER-resident chaperones grp78 and calreticulin is further supported by their co-localization with Ro in small apoptotic membrane blebs and the finding of a novel putative grp78 binding motif in the carboxyl-terminal region of Ro52.

Characterization and genomic sequence of the murine 60 kD Ro gene.

Autoantibodies binding 60 kD Ro (or SS-A) are commonly found in patients with systemic lupus erythematosus and Sjögren's syndrome. While many studies have examined the autoimmune response directed against this RNA-protein, its function is still uncertain. As part of a broad effort to better understand animal models of anti-Ro autoimmunity we have characterized the murine 60 kD Ro gene. Southern blot analysis of mouse genomic DNA suggests that the 60 kD Ro gene is a single copy gene. The complete sequence of the gene was determined from three overlapping genomic lambda phage clones (GenBank accession number AF065398). The murine 60 kD Ro gene spans approximately 23 kb and consists of 8 or 9 exons. DNA sequence analysis revealed the presence of multiple B1 repetitive units. It maps in synteny with the human 60 kD Ro gene. Therefore, the isolation and characterization of the 60 kD Ro gene will be instrumental for future studies on protein function and the role this protein plays in the development of autoimmune responses.

Induction of autoimmunity by multivalent immunodominant and subdominant T cell determinants of La (SS-B).

We investigated the consequences of altering the form and valence of defined autodeterminants on the initiation and spreading of experimentally induced La/Ro autoimmunity. Anti-La and Ro (SS-A) Ab responses were monitored following immunization of healthy mice with defined immunodominant and subdominant T cell determinants of the La (SS-B) autoantigen synthesized as either monomeric or multiple antigenic (MAP) peptides. Abs to mouse La (mLa) developed faster and were of higher titer in mice immunized with the subdominant mLa25-44 MAP compared with mice immunized with the 25-44 monomer. Rapid intermolecular spreading of the autoimmune response to 60-kDa Ro was observed in AKR/J mice immunized with mLa25-44 MAP, but not in mice immunized repeatedly with monomeric peptide. A/J mice immunized and boosted with the known tolerogenic mLa287-301 determinant delivered as monomeric peptide failed to develop Abs to either intact mLa or mLa287-301 peptide. However, immunization with the multivalent mLa287-301 peptide led to the rapid production of high titer mLa autoantibodies associated with a proliferative T cell response to the mLa287-301 peptide. The data suggested that the enhanced immunogenicity of MAPs was not due to augmented Ag presentation or T cell stimulation. However, MAP-, but not monomer peptide-, containing immune complexes were potent substrates for Ab-dependent fixation of complement. These results demonstrate that the form of Ag responsible for inducing autoimmunity can profoundly influence the nature and magnitude of the immune response. Thus, molecular mimicry of tolerogenic and nontolerogenic self determinants might trigger autoimmunity under conditions of altered valence.

Determinant spreading: lessons from animal models and human disease.

Spreading of the immune response is a common theme in organ-specific and systemic autoimmune diseases. We evaluated whether some of the mixed antinuclear antibody patterns characteristic of systemic autoimmunity might be the result of determinant spreading from a single initiating event. Immunisation of healthy mice with individual protein components of the La/Ro ribonucleoprotein (RNP) targeted in systemic lupus erythematosus and primary Sjögren's syndrome induced autoantibodies recognising Ro60 (SS-A), Ro52 (SS-A) and La (SS-B) and in some cases the molecular chaperones calreticulin and Grp78. The endogenous antigen(s) driving determinant spreading might be derived from physiological apoptosis which could explain the involvement of some chaperone proteins in the autoimmune response. Diversified anti-La/Ro antibody responses were initiated by challenge with a single subdominant T epitope of La even though some self epitopes of La were efficiently tolerised. The pattern of autoantibody responses in primary Sjögren's syndrome was strongly influenced by HLA class II phenotype which we speculate controls activation of T cells recognising defined peptides from the La/Ro RNP. In this way, HLA class II alleles may be critical in influencing initiation and spreading of systemic autoimmune reactions. Molecular mimicry of such determinants by exogenous agents might readily initiate spreading of an autoimmune response in genetically susceptible hosts.

Characterization of human telomerase complex.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called "telomeres" to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.