Galectin-3 binding protein links circulating microparticles with electron dense glomerular deposits in lupus nephritis.

OBJECTIVE: A high level of galectin-3-binding protein (G3BP) appears to distinguish circulating cell-derived microparticles in systemic lupus erythematosus (SLE). The aim of this study is to characterize the population of G3BP-positive microparticles from SLE patients compared to healthy controls, explore putative clinical correlates, and examine if G3BP is present in immune complex deposits in kidney biopsies from patients with lupus nephritis. METHODS: Numbers of annexin V-binding and G3BP-exposing plasma microparticles from 56 SLE patients and 36 healthy controls were determined by flow cytometry. Quantitation of microparticle-associated G3BP, C1q and immunoglobulins was obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Correlations between microparticle-G3BP data and clinical parameters were analyzed. Co-localization of G3BP with in vivo-bound IgG was examined in kidney biopsies from one non-SLE control and from patients with class IV (n = 2) and class V (n = 1) lupus nephritis using co-localization immune electron microscopy. RESULTS: Microparticle-G3BP, microparticle-C1q and microparticle-immunoglobulins were significantly (P < 0.01) increased in SLE patients by LC-MS/MS. Three G3BP-exposing microparticle populations could be discerned by flow cytometry, including two subpopulations that were significantly increased in SLE samples (P = 0.01 and P = 0.0002, respectively). No associations of G3BP-positive microparticles with clinical manifestations or disease activity were found. Immune electron microscopy showed co-localization of G3BP with in vivo-bound IgG in glomerular electron dense immune complex deposits in all lupus nephritis biopsies. CONCLUSIONS: Both circulating microparticle-G3BP numbers as well as G3BP expression are increased in SLE patients corroborating G3BP being a feature of SLE microparticles. By demonstrating G3BP co-localized with deposited immune complexes in lupus nephritis, the study supports cell-derived microparticles as a major autoantigen source and provides a new understanding of the origin of immune complexes occurring in lupus nephritis.

Anti-dsDNA antibodies as a classification criterion and a diagnostic marker for systemic lupus erythematosus: critical remarks.

Antibodies to mammalian dsDNA have, for decades, been linked to systemic lupus erythematosus (SLE) and particularly to its most serious complication, lupus nephritis. This canonical view derives from studies on its strong association with disease. The dogma was particularly settled when the antibody was included in the classification criteria for SLE that developed during the 1970s, most prominently in the 1982 American College of Rheumatology (ACR), and recently in The Systemic Lupus International Collaborating Clinics (SLICC) classification criteria. There are several problems to be discussed before the anti-dsDNA antibody can be accepted without further distinction as a criterion to classify SLE. Old and contemporary knowledge make it clear that an anti-dsDNA antibody is not a unifying term. It embraces antibodies with a wide spectrum of fine molecular specificities, antibodies that are produced transiently in context of infections and persistently in the context of true autoimmunity, and also includes anti-dsDNA antibodies that have the potential to bind chromatin (accessible DNA structures) and not (specificity for DNA structures that are embedded in chromatin and therefore unaccessible for the antibodies). This critical review summarizes this knowledge and questions whether or not an anti-dsDNA antibody, as simply that, can be used to classify SLE.

Low diagnostic and predictive value of anti-dsDNA antibodies in unselected patients with recent onset of rheumatic symptoms: results from a long-term follow-up Scandinavian multicentre study.

OBJECTIVES: To verify the diagnostic accuracy of anti-double-stranded DNA (anti-dsDNA) antibodies detected by the Crithidia luciliae immunofluorescence test (CLIFT) in a cohort of unselected patients, referred to a rheumatologist due to recent onset of rheumatic symptoms. METHOD: A total of 1073 consecutive patients were screened for anti-nuclear antibodies (ANAs). Serum samples from 292 ANA-positive and 292 matching ANA-negative patients were tested three times for anti-dsDNA antibodies, using two different CLIFT kits (ImmunoConcepts(®) and Euroimmun(®)). An initial clinical diagnosis was made by rheumatologists unaware of the results. The diagnoses were updated after a median follow-up of 4.8 years. RESULTS: CLIFT was positive at least once in 60 patients but only 23 patients were CLIFT positive in all of the assays. Diagnosis of systemic lupus erythematosus (SLE) was made initially in 65 patients, of whom 24 (37%) were CLIFT positive. Many other diagnoses were observed among the CLIFT-positive patients. Overall, 16 (5.5%) ANA-negative patients were CLIFT positive. After approximately 5 years, the diagnosis of SLE remained unchanged in 63 patients (23 CLIFT positive) and altered in only two (one CLIFT positive). Among the 36 CLIFT-positive patients who were not diagnosed with SLE at study entry, only one developed SLE during the follow-up period. CONCLUSIONS: CLIFT was not reliable as a diagnostic tool in unselected patients with rheumatic symptoms. ANAs were of little value as a screening test before the CLIFT analysis. CLIFT had surprisingly low positive predictive value (PPV) for the diagnosis of SLE despite its high specificity. For non-SLE patients, being CLIFT positive poses little risk of developing SLE within 5 years.

European League Against Rheumatism recommendations for monitoring patients with systemic lupus erythematosus in clinical practice and in observational studies.

OBJECTIVES: To develop recommendations for monitoring patients with systemic lupus erythematosus (SLE) in clinical practice and observational studies and to develop a standardised core set of variables to monitor SLE. METHODS: We followed the European League Against Rheumatism (EULAR) standardised procedures for guideline development. The following techniques were applied: nominal groups, Delphi surveys for prioritisation, small group discussion, systematic literature review and two Delphi rounds to obtain agreement. The panel included rheumatologists, internists, dermatologists, a nephrologist and an expert related to national research agencies. The level of evidence and grading of recommendations were determined according to the Levels of Evidence and Grades of Recommendations of the Oxford Centre for Evidence-Based Medicine. RESULTS: A total of 10 recommendations have been developed, covering the following aspects: patient assessment, cardiovascular risk factors, other risk factors (osteoporosis, cancer), infection risk (screening, vaccination, monitoring), frequency of assessments, laboratory tests, mucocutaneous involvement, kidney monitoring, neuropsychological manifestations and ophthalmology assessment. A 'core set' of minimal variables for the assessment and monitoring of patients with SLE in clinical practice was developed that included some of the recommendations. In addition to the recommendations, indications for specific organ assessments that were viewed as part of good clinical practice were discussed and included in the flow chart. CONCLUSIONS: A set of recommendations for monitoring patients with SLE in routine clinical practice has been developed. The use of a standardised core set to monitor patients with SLE should facilitate clinical practice, as well as the quality control of care for patients with SLE, and the collection and comparison of data in observational studies.

Circulating chromatin-anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis.

Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i) presence of EDS containing chromatin fragments and IgG in both organs in nephritic patients, (ii) chromatin fragments possessed high affinity for dermo-epidermal laminins and collagens, (iii) glomerular immune complex deposits did not predict similar interstitial deposits in skin, although such complexes were present in capillary lumina in glomeruli and skin of all nephritic individuals. Thus, chromatin-IgG complexes accounting for lupus nephritis seem to reach skin through circulation, but other undetermined factors are required for these complexes to deposit within skin membranes.

Development of lupus nephritis is associated with qualitative changes in the glomerular collagen IV matrix composition.

Lupus nephritis is associated with thickening of the glomerular extracellular membranes. Distribution of collagen IV alpha-chains in the glomerular basement membrane in kidneys of lupus-prone B/W mice has been examined in this study. The results are indicative of a qualitative change in the collagen IV matrix occurring around the time of development of proteinuria, with an embryonic alpha1/alpha2 isoform replacing the normal glomerular basement membrane (GBM). These changes mimic alterations seen in Alport syndrome and coincide with an increase in collagenolytic activity within the glomerulus. It has been hypothesized that alterations in collagen matrix synthesis represent compensatory responses to an increase in GBM proteolysis and could represent an important step in the pathogenesis of nephritis through the formation of a dysfunctional glomerular filter. Also, aberrations in the collagen matrix composition could contribute to the deposition of autoantibodies within the glomerulus.

Nucleosomes possess a high affinity for glomerular laminin and collagen IV and bind nephritogenic antibodies in murine lupus-like nephritis.

AIM: Lupus nephritis is closely associated with in vivo autoantibody-binding to glomerular membrane-associated electron-dense structures (EDS). The biochemical nature and cellular origin of EDS are controversial, and definitive characterisation needs to be performed. METHODS: By using the terminal transferase biotin-dUTP nick end-labelling (TUNEL) assay at the electron microscopic level, we have traced extracellular chromatin within the glomerular basement membranes of nephritic (NZBxNZW)F1 mice. The TUNEL assay was subsequently used in combination with standard immune electron microscopy (IEM). To analyse why chromatin particles associate with membranes, we determined the affinity of nucleosomes and DNA for glomerular laminin, collagen IV and the mesangial matrix proteoglycan perlecan by surface plasmon resonance. RESULTS: This intra-assay colocalisation TUNEL IEM demonstrated that autoantibodies fully colocalised with extracellular TUNEL-positive chromatin observed as EDS in glomerular membranes, similar to results obtained by the same technique applied to human lupus nephritis. Most importantly, these data validate the murine variant of lupus nephritis as a model to study origin of extracellular chromatin as a key element in human lupus nephritis. Kinetic analyses demonstrated that nucleosomes had a high affinity for collagen IV and laminin, but not for perlecan. CONCLUSION: Collectively, these results provide firm evidence that dominant target structures for nephritogenic autoantibodies are constituted by TUNEL-positive chromatin associated with glomerular capillary and mesangial matrix membranes at high affinity.

Diagnostic impact of contemporary biomarker assays for rheumatoid arthritis.

OBJECTIVE: The impetus towards early treatment for patients with rheumatoid arthritis (RA) requires more reliable disease markers than the non-specific immunoglobulin M (IgM) rheumatoid factor (RF). To determine the accuracy of newer biomarkers for RA testing for antibody against cyclic citrullinated peptides (anti-CCP Ab), IgA- and IgG-RF and cartilage oligomeric matrix protein (COMP) levels, measured by enzyme-linked immunosorbent assay (ELISA), were compared with IgM-RF isotyping. METHODS: Serum samples were investigated in patients with an established diagnosis of RA (n = 54), ankylosing spondylitis (AS) (n = 36), and non-inflammatory conditions (n = 18) (cohort A), and in 234 consecutive outpatients in a blinded fashion (cohort B). Non-parametric analysis of areas under the curve (AUC) of receiver operator characteristics were performed. RESULTS: The presence of anti-CCP Ab had the highest accuracy (96%) in distinguishing RA patients in cohort A and cohort B (accuracy 83%), and in both cohorts combined (accuracy 87%). This was related to the high specificity of anti-CCP Ab for RA (95-96%), even though IgM-RF was the most sensitive test (87-96%). Sensitivity (15-48%) and specificity (66-69%) of COMP as a marker for RA was low. Combining results of anti-CCP Ab and IgM-RF or any of the other assays did not increase the diagnostic accuracy for RA. CONCLUSION: The presence of anti-CCP Ab is the most accurate biomarker for RA in both selected and unselected cohorts, while the COMP assay is not very useful in RA diagnosis. Combining assays for anti-CCP Ab and IgM-RF or IgA-RF does not enhance RA diagnosis.

Glomerular apoptotic nucleosomes are central target structures for nephritogenic antibodies in human SLE nephritis.

Antibodies to double-stranded (dsDNA) are associated with systemic lupus erythematosus (SLE) and directly involved in human lupus nephritis. Information about their glomerular target antigens is inconsistent, and whether availability of target antigens, antibody specificity or avidity are nephritogenic parameters, is not determined. In this study, we analyzed renal tissue from anti-dsDNA antibody-positive lupus patients with nephritis by morphological and immunological assays, including immune electron microscopy (IEM) and colocalization IEM, an EM-based confocal microscopy assay. IEM demonstrated that antibody deposits were confined to electron dense structures (EDS) in glomerular membranes. These autoantibodies colocalized with nucleosome-binding anti-dsDNA/-histone/-transcription factor antibodies. To confirm the colocalization IEM-data, we developed a colocalization terminal deoxynucleotidyl-transferase (TdT) biotin-dUTP nicked end-labeled (TUNEL) IEM assay where extracellular DNA was traced by TdT-mediated introduction of biotinylated nucleotides and autoantibodies by IEM. Results consistently demonstrated that DNA colocalized with autoantibodies in glomerular membrane-associated EDS. The colocalization IEM and colocalization TUNEL IEM assays thus demonstrate that intra-glomerular membrane-associated nucleosomes are targeted by anti-dsDNA autoantibodies in human lupus nephritis. The data provide a new approach to understand basic molecular and immunological processes accounting for antibody-mediated nephritis in human SLE.

Rheumatoid factor by laser nephelometry and Waaler-Rose assay: prognostic value in patients with recent-onset rheumatoid arthritis.

OBJECTIVE: To evaluate the prognostic value of rheumatoid factor (RF), detected in the Waaler-Rose agglutination assay and by nephelometry, in patients with recent-onset rheumatoid arthritis (RA). METHODS: Consecutive patients with new-onset RA between 1993 and 1997 were followed for a median period of 4.7 years. Clinical data at baseline and drug use during the disease course were recorded. Outcome parameters studied were disease process, damage (erosions, joint surgery, extra-articular manifestations, and new co-morbidity), and death. Cut-off levels for RF were >40 IU/mL (nephelometry) and titres 1:160 (Waaler-Rose haemagglutination). RESULTS: RF tests were negative by both methods in 22% of RA patients (RF- group), while 33% were RF positive by nephelometry only (RF+ group) and 45% were positive by Waaler-Rose and nephelometry (RF++ group). Baseline clinical and laboratory findings as well as the number of subsequently used disease-modifying anti-rheumatic drugs (DMARDs), the number of patients starting and the time spent on steroid therapy were similar in the three RF groups. Odd ratios for death (n = 23), erosions (n = 62), and serious extra-articular disease manifestations (EAMs) (n = 13) as well as patient survival, erosion-free or surgery-free survival rates did not differ between the RF groups. Only rheumatoid nodules were more frequent in RF++ patients. CONCLUSION: The baseline presence of RF by either Waaler-Rose or nephelometry was not associated with differences in drug therapy, morbidity other than rheumatoid nodules, or mortality in RA patients in the first 5 years of disease. Being immunoglobulin M (IgM) RF positive thus had little impact on RA patient outcome.

Interleukin-2, but not interleukin-15, is required to terminate experimentally induced clonal T-cell anergy.

It has been demonstrated that T cells stimulated with nucleosome-polyomavirus T-antigen (self-nonself) complexes, but not nucleosomes, activate autoimmune nucleosome-specific T cells. As these cells may be naïve, such observations do not show that anergic T cells are reactivated. To understand the regulation of autoimmunity, this is important to assess, and this is the focus of this study. T-cell anergy was induced by antigen stimulation in the presence of antibodies to the costimulatory molecules CD80/CD86. Requirements for the reactivation of anergic T cells were analysed by the ability of antigen and interleukin-2 (IL-2) or IL-15 to increase T-cell proliferation and IL-2 transcription. Data demonstrate that stimulation of T cells with T-antigen and anti-CD80/86 antibodies promotes long-lasting clonal T-cell anergy. While T-antigen did not reactivate anergic T cells, proliferation and upregulation of IL-2 gene transcription was initiated by stimulation with antigen, costimulation and IL-2 added to the cultures. Proliferation per se was not sufficient to promote the reactivation of anergic T cells, as both IL-2 and IL-15 induced proliferation, while antigen and IL-2, but not IL-15, upregulated IL-2 mRNA levels. These data demonstrate that the innate immune system and IL-2 are central to the initiation and termination of T-cell anergy.

Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity.

BACKGROUND: The presence of "anti-DNA antibodies in abnormal titres" is a well established criterion for SLE classification, but there is no agreement on the performance of this test. OBJECTIVE: To study the correlation between clinical findings and five different solid and solution phase anti-DNA antibody assays. METHODS: 158 consecutively collected ANA positive sera were studied in a double blind fashion. Anti-DNA antibodies were determined by different solid phase assays (ssDNA-, dsDNA- specific ELISA, EliA anti-dsDNA assay, Crithidia luciliae assay), and by an experimental solution phase anti-DNA assay using biotinylated pUC18 plasmid, human, calf thymus, and E coli DNA. Antibody affinity was determined by surface plasmon resonance. Clinical data were obtained independently of the laboratory analyses and later related to the anti-dsDNA findings. RESULTS: Anti-dsDNA antibodies were most frequently detected by ELISA, but were not specific for SLE as they were present in up to 30% of other disease groups. Those detected by the Crithidia luciliae assay were predictive for SLE, while antibodies binding in solution phase ELISA using the pUC18 correlated strongly with the Crithidia luciliae assay. Surface plasmon resonance analysis showed that antibody binding to pUC18 was not due to higher relative affinity for dsDNA in general, but apparently to specificity for that plasmid DNA. Serum samples from three patients with lupus nephritis were positive in both pUC18 solution phase and Crithidia luciliae assays. CONCLUSIONS: Assay principle selection is decisive for the detection of clinically significant anti-DNA antibodies. Revision of the anti-DNA antibody criterion in the SLE classification may be needed.

Anti-DNA antibody subpopulations and lupus nephritis.

As a consequence of increased insight into the cellular and molecular mechanisms responsible for induction of B cell and T cell autoimmunity to DNA and nucleosomes, there is an obvious need to reconsider the dogma stating that anti-dsDNA antibodies serve as marker antibodies for SLE and also that anti-dsDNA antibodies per se are responsible for the initiation of lupus nephritis. Given that the potential to produce anti-dsDNA antibodies is an inherent property of the normal immune system and that few anti-DNA antibodies have nephritogenic potential, we must try to solve the problem whether it is avidity for DNA, specificity for unique DNA structures or cross-reactivity with non-DNA molecules, that make such antibodies pathogenic and thus potential markers for SLE and lupus nephritis. In this review, we will summarize contemporary problems related to these questions; (1) try to focus on phenotypic differences with respect to the ability to produce anti-dsDNA antibodies between individuals suffering from SLE and those not belonging to this diagnostic group, and (2) to describe differences between pathogenic and non-pathogenic anti-dsDNA antibodies.

Simian virus 40 large T-antigen, but not small T-antigen, trans-activates the human cytomegalovirus major immediate early promoter.

Cytomegalovirus infection is a major cause of morbidity in immunocompromised patients. The major immediate early promoter/enhancer (MIEP) of the human cytomegalovirus controls the expression of the immediate early genes 1 and 2 which play a central role both in primary and reactivated human cytomegalovirus (HCMV)-infections. Our previous studies have shown that co-infection of A549 cells with human cytomegalovirus and human polyomavirus BK resulted in enhanced expression of the immediate early genes 1 and 2 and that the early gene products of BK virus trans-activated the MIEP. However, neither the MIEP sequences required for mediating this trans-activation, nor the contribution of the individual BK virus early gene products were examined. The MIEP contains multiple binding sites for the transcription factors CREB, AP1, Sp1 and NFkappaB, which may mediate polyomavirus large T- or small t-antigens-induced promoter activation. Transient transfection studies in A549 cells demonstrated that SV40 large T-antigen, but not small t-antigen, trans-activated MIEP activity approximately 9-fold. Mutations in individual binding motifs in the context of the complete MIEP did not impair traits-activation by large T-antigen. The level of induction of a truncated MIEP consisting of a single set of CRE/AP1, NFkappaB, and Sp1 binding motifs by large T-antigen was reduced 2-fold compared to the full length MIEP. Extended truncations diminished trans-activation by large T-antigen. To determine the contribution of a single binding motif in the trans-activation by large T-antigen, a CRE/AP1, an NFkappaB, an Sp1, or a non-consensus Sp1-motif, respectively, was linked to the MIEP TATA-sequence respecting the natural spacing between the two transcription regulatory elements. Only the MIEP TATA-box with the correctly spaced non-consensus Sp1 binding site (GT-motif) was stimulated by large T-antigen. These results suggest that an isolated non-consensus Sp1-motif is important for trans-activation of the MIEP by large T-antigen, but that other cis-acting elements can compensate for this element in the context of the whole MIEP.

Antinuclear antibody screening in this new millennium: farewell to the microscope?

ANA testing by immunofluorescence technique (F-ANA) is nowadays still performed in much the same way as 45 years ago when the test was introduced. Due to its low specificity the F-ANA test has a poor predictive value for systemic autoimmune diseases and in addition has proven difficult to standardise. In the meantime, many of the nuclear and cytoplasmatic auto-antigens, related to specific types of autoimmune disease, have been characterised and can be tested for in specific ELISA assays (E-ANA). These assays are in large part automated and enable the large volume testing required, by the current attitude, to use ANA-testing for its high negative predictive value in the exclusion of systemic autoimmune disease. In addition, E-ANA assays give specific results for clinically relevant autoantibodies, while its test repertoire can be altered at any given time to reflect changes in current thinking on relevant auto-antigens. Thus, we suggest that the unspecific F-ANA test should no longer be considered the gold standard for the detection of clinically relevant autoantibodies.

Differences in the reactivity of CD4+ T-cell lines generated against free versus nucleosome-bound SV40 large T antigen.

Previous results have revealed a strong correlation between polyomavirus BK reactivation and disease activity and antinuclear auto-antibody production in the human autoimmune disease systemic lupus erythematosus. BK virus establishes a latent infection in most humans, and reactivation requires the production of the DNA-binding large T antigen. Experimentally induced expression of the polyomavirus SV40 large T antigen in mice induces both an immune response to large T antigen and autoimmune response to nuclear antigens and antinuclear antibody production. Previous results have indicated that human T-antigen-specific CD4+ T-cell lines are stimulated equally by free, soluble and nucleosome-bound T antigen. This study was designed to determine how antigen processing of nucleosomes containing bound SV40 large T antigen may affect the specificity and response characteristics of experimentally induced T-antigen-specific CD4+ T cells. The results indicated that CD4+ T-cell lines generated from mice immunized with soluble, free T antigen responded very poorly in response to stimulation with T antigen bound to nucleosomes. CD4+ T-cell lines generated from mice immunized with nucleosomes that had bound T antigen in situ responded to both free and nucleosome-bound T antigen. The T-antigen-specific, CD4+ memory T cells induced by latent polyomavirus infections in humans may be uniquely suited to initiate autoimmunity to nuclear antigens upon virus reactivation.

A longitudinal study of human cytomegalovirus serology and viruria fails to detect active viral infection in 20 systemic lupus erythematosus patients.

In this study, we investigated whether active human cytomegalovirus infection could be detected in 20 systemic lupus erythematosus (SLE) patients over a one-year observation period by polymerase chain reaction on serial urine specimens and by monitoring of IgG and IgM HCMV-specific antibody profiles in serial serum samples. Of 788 urine samples analysed for the presence of human cytomegalovirus DNA, only 2 specimens (0.25%) collected from two different patients contained genuine human cytomegalovirus sequences as determined by polymerase chain reaction and subsequent sequencing of the PCR products. These two patients had one positive sample out of 36 samples or 40 samples, respectively. Nineteen of the patients (95%) possessed IgG antibodies against human cytomegalovirus, while 9 (45%) produced IgM antibodies. However, none of the patients showed signs of an active virus infection as judged by the stable anti-HCMV IgG or IgM antibody levels during the observation period, nor was any correlation between disease activity and HCMV serology/viruria observed. Of single serum samples of 26 age- and sex-matched blood donors, 21 (81%) were HCMV IgG positive and 1 (3.8%) was IgM seropositive. In conclusion, our data fail to establish an active human cytomegalovirus infection in SLE patients.

Termination of human T cell tolerance to histones by presentation of histones and polyomavirus T antigen provided that T antigen is complexed with nucleosomes.

OBJECTIVE: To investigate whether polyomavirus T antigen linked to histones through nucleosome-T antigen complexes has the potential to terminate histone-specific T cell anergy. METHODS: Blood mononuclear cells from healthy individuals were used as the source to establish T cell lines initiated and maintained by T antigen, histones, nucleosome-T antigen complexes, or nucleosomes. Proliferative responses of these lines to T antigen, histones, and nucleosomes were determined. RESULTS: Whereas T cell lines could be established using T antigen or T antigen-nucleosome complexes, histones or nucleosomes did not have this potential. However, T cell lines selected by T antigen-nucleosome complexes responded subsequently to histones and nucleosomes. Identical results were obtained with murine and human nucleosomes, provided that they were complexed with T antigen. CONCLUSION: T antigen-specific T cells possess the potential to proliferate when interacting with an antigen-presenting cell that presents T antigen. In the presence of T antigens complexed with nucleosomes, T antigen-specific T cells offer bystander help that may terminate histone-specific T cell anergy. These T cells may progress into functional, autoimmune T cells if histones are properly presented.

T cell lines specific for polyomavirus T-antigen recognize T-antigen complexed with nucleosomes: a molecular basis for anti-DNA antibody production.

We have previously demonstrated that in vivo expression of the polyomavirus DNA-binding T-antigen initiated production of IgG antibodies to T-antigen and to DNA, but not to a panel of autoantigens not related to nucleosomes, indicating an antigen-selective T cell-dependent B cell response. In this study, we demonstrate that CD4-positive T cells from both normal and systemic lupus erythematosus (SLE) patients readily proliferate in response to pure T-antigen, and also to T-antigen in complex with nucleosomes. T-antigen-specific T cell lines from both normal individuals and SLE patients proliferate in response to nucleosome-T-antigen complexes, but not to nucleosomes or histones. B cells co-cultured with T-antigen-specific T cells and stimulated with nucleosome-T-antigen complexes produce anti-T-antigen and anti-DNA antibodies, indicating that such CD4-positive T cells have the potential to interact with B cells specific for individual components of nucleosome-T-antigen complexes. Thus, a non-self DNA-binding protein like polyomavirus T-antigen may initiate and maintain an antibody response to DNA when T-antigen is actively expressed.