Efficacy and safety of subcutaneous tabalumab in patients with systemic lupus erythematosus: results from ILLUMINATE-1, a 52-week, phase III, multicentre, randomised, double-blind, placebo-controlled study.

OBJECTIVES: Evaluate efficacy and safety of tabalumab, a human IgG4 monoclonal antibody that binds and neutralises membrane and soluble B-cell activating factor (BAFF) versus placebo plus standard of care (SoC) in patients with systemic lupus erythematosus (SLE). METHODS: This phase III, 52-week study randomised 1164 patients with moderate-to-severe SLE (Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index ≥6 at baseline). Patients received SoC plus subcutaneous injections of tabalumab or placebo, starting with a loading dose (240 mg) at week 0 and followed by 120 mg every two weeks (120 Q2W, n=387), 120 mg every four weeks (120 Q4W, n=389) or placebo Q2W (n=388). PRIMARY ENDPOINT: proportion of patients achieving SLE Responder Index 5 (SRI-5) response at week 52. RESULTS: Similar proportions of patients in each group achieved SRI-5 response at week 52 (120 Q2W: 31.8%; 120 Q4W: 35.2% and placebo: 29.3%). Key secondary endpoints were not met. In a sensitivity analysis not excluding patients who decreased antimalarials or immunosuppressants, SRI-5 response was achieved with 120 Q4W (37.0% vs 29.8% placebo; p=0.021), but not 120 Q2W (34.1%; p=0.171). Significant reductions in anti-dsDNA antibodies, increases in C3 and C4, and reductions in total B cells and immunoglobulins were observed with tabalumab. No differences were observed between treatment groups in percentage of deaths (120 Q2W: 0.8%; 120 Q4W: 0.5%; placebo: 0.5%), serious adverse events (AEs) (range 11.1-14.4%) or treatment-emergent AEs (range 81.1-82.3%). CONCLUSIONS: Tabalumab had biological activity-changes in anti-dsDNA, complement, B cells and immunoglobulins-consistent with BAFF pathway inhibition. Key clinical efficacy endpoints did not achieve statistical significance. Safety profiles were similar with tabalumab and placebo. TRIAL REGISTRATION NUMBER: NCT01196091.

Effect of reduced sulfur compounds on the fermentation of phosphoric acid pretreated sugarcane bagasse by ethanologenic Escherichia coli.

The addition of reduced sulfur compounds (thiosulfate, cysteine, sodium hydrosulfite, and sodium metabisulfite) increased growth and fermentation of dilute acid hydrolysate of sugarcane bagasse by ethanologenic Escherichia coli (strains LY180, EMFR9, and MM160). With sodium metabisulfite (0.5mM), toxicity was sufficiently reduced that slurries of pretreated biomass (10% dry weight including fiber and solubles) could be fermented by E. coli strain MM160 without solid-liquid separation or cleanup of sugars. A 6-h liquefaction step was added to improve mixing. Sodium metabisulfite also caused spectral changes at wavelengths corresponding to furfural and soluble products from lignin. Glucose and cellobiose were rapidly metabolized. Xylose utilization was improved by sodium metabisulfite but remained incomplete after 144 h. The overall ethanol yield for this liquefaction plus simultaneous saccharification and co-fermentation process was 0.20 g ethanol/g bagasse dry weight, 250 L/tonne (61 gal/US ton).

T cells in the pathogenesis of systemic lupus erythematosus.

The role of T cells in the pathogenesis of systemic lupus erythematosus (SLE) is reviewed with a focus on autoantigen-specific T cells in SLE. The initial clue to a role for T cells in SLE was histopathologic studies demonstrating extensive infiltration of T cells at the sites of inflammation. Later studies, showing association between HLA polymorphisms and specific autoantibodies, directly implicated a role for T cells in autoantibody production. More recently, we and others have identified and characterized autoantigen-specific T cells in SLE. We review these studies on the role of autoantigen-specific T cells in SLE and present new findings on the molecular characterization of T cell immunity to Sm-B, Sm-D and U1-70kD small nuclear ribonucleoprotein (snRNP) autoantigens.

Structural analysis of TCRalpha and beta chains from human T-Cell clones specific for small nuclear ribonucleoprotein polypeptides Sm-D, Sm-B and U1-70 kDa: TCR complementarity determining region 3 usage appears highly conserved.

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are systemic autoimmune diseases that are characterized by the presence of autoantibodies reactive with U small nuclear RNP (snRNP) autoantigens. Both B and T cells are important in the pathogenesis of the disease, and T- and B-cell immunity against snRNP polypeptides have been shown to be linked in vivo. Currently, several alternative hypotheses for the pathogenesis of these diseases have been proposed. These include loss of tolerance, modified self-antigens, molecular mimicry and nondirected immune activation. To help distinguish between the various models of disease pathogenesis, we have characterized the T-cell receptor (TCR) CDR3 from a large panel of well-characterized human T-cell clones and lines specific for individual snRNP polypeptides. The results presented here reveal highly restricted TCR usage across patients by the snRNP-reactive T cells based on the deduced amino acid sequence of the CDR3 loop. These data support the hypothesis that T-cell responses against self antigens in SLE and MCTD are antigen driven and that there are a limited number of T-cell epitopes present on the snRNP autoantigens.

T cell epitope mapping of the Smith antigen reveals that highly conserved Smith antigen motifs are the dominant target of T cell immunity in systemic lupus erythematosus.

B cell and T cell immunity to the Smith Ag (Sm) is a characteristic feature of systemic lupus erythematosus (SLE). We have shown that T cell immunity against Sm can be detected in SLE patients, and that T and B cell immunity against Sm are linked in vivo. TCR usage by Sm-reactive T cells is highly restricted and characteristic of an Ag-driven immune response. Sm is a well-characterized complex Ag consisting of proteins B1, B2, D1, D2, D3, E, F, and G. A unique feature of all Sm proteins is the presence of homologous motifs, Sm motif 1 and Sm motif 2. We used limiting dilution cloning and synthetic peptide Ags to characterize the human T cell immune response against Sm in seven SLE patients. We sought to determine the precise antigenic peptides recognized, the common features of antigenic structure recognized, and the evolution of the T cell response against Sm. We found there was a highly restricted set of Sm self-peptides recognized by T cells, with three epitopes on Sm-B and two epitopes on Sm-D. We found that T cell immunity against Sm-B and Sm-D was encoded within the highly conserved Sm motif 1 and Sm motif 2, and that immunity against these epitopes appeared stable. The present study supports the concept that T cell immunity to Sm is an Ag-driven immune response directed against a highly restricted set of self-peptides, encoded within Sm motif 1 and Sm motif 2, that is shared among all Sm proteins.

The appearance of U1 RNP antibody specificities in sequential autoimmune human antisera follows a characteristic order that implicates the U1-70 kd and B'/B proteins as predominant U1 RNP immunogens.

OBJECTIVE: To observe the order of development of anti-U1 RNP peptide antibodies in humans. METHODS: Immunoblots against Jurkat cell lysates were performed on 5,882 serum samples from 3,668 patients referred on clinical grounds for RNP antibody testing to a reference laboratory between 1989 and 1999. In patients from whom multiple samples were drawn, we determined the order in which IgG antibodies to the U1 RNP peptides A, B'/B, C, D, and 70 kd appeared. RESULTS: One hundred sixty-three patients with serial samples were identified in whom antibodies to at least one U1 RNP peptide initially were not present but later appeared. The first RNP antibodies to appear were most often directed against the 70 kd and B'/B peptides (P < 0.01). Antibodies to the A and C peptides usually developed after other RNP peptide antibodies, and antibodies to D often emerged only after immunity to multiple other U1 RNP proteins had appeared. B'/B, but not 70 kd, was a frequent early target of spreading after initial immunity to other RNP peptides. CONCLUSION: Orderly patterns of emergence of U1 RNP peptide antibodies appear to exist in humans. Two peptides, 70 kd and B'/B, show characteristics of early immunogens in the development of human RNP immunity.

Immunization with a bacterial ATP-binding cassette transporter fragment suppresses autoimmunity and prolongs survival in MRL/lpr lupus-prone mice.

The present study was undertaken to better define the role of the U1 70 kDa antigen in a spontaneous murine model of systemic lupus erythematosus (SLE) by testing whether immunization with the U1 70 kDa polypeptide could alter the production of antibodies against U1 70 kDa or against other small nuclear ribonucleoproteins (snRNP), modify disease expression or alter survival. We found that, while immunization with a U1 70 kDa derived fusion protein (70 KFP) tended to delay the development of anti-snRNP antibodies in the sera of MRL/lpr mice, it had no effect on autoimmune-mediated renal disease or survival. Unexpectedly, it was found that MRL/lpr mice immunized with a 367 amino acid fragment of a bacterial ATP-binding cassette transporter, MFP, had prolonged survival compared to saline injection or U1 70 kDa immunization and that this was associated with a delay in the onset of SLE-like proliferative glomerulonephritis. This is the first study, to our knowledge, in which a bacterial ATP-binding cassette transporter was shown to be beneficial in treating a murine model of SLE. We report that MFP significantly prolonged longevity in the MRL/lpr murine model of SLE compared to saline injection or 70 KFP immunization and that improved survival was associated with a delay in the onset of SLE-like glomerulonephritis.

Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells.

The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.

Analysis of T cell receptors specific for U1-70kD small nuclear ribonucleoprotein autoantigen: the alpha chain complementarity determining region three is highly conserved among connective tissue disease patients.

The U1-70kD autoantigen is a major target of B cell responses in patients with connective tissue diseases (CTD). T cell responses are important in the pathogenesis of CTD, however little is known about autoantigen-specific T cells in these diseases. We have recently proven that U1-70kD-reactive human T cells exist. To further characterize these autoreactive T cells, U1-70kD-reactive T cell clones have been generated from patients with CTD using either a recombinant fusion protein or synthetic peptides spanning the U1-70kD polypeptide. T cell receptors (TCR) isolated from the U1-70kD-reactive T cell clones were sequenced and the third complementarity-determining region (CDR3) compared to determine if a common motif was present. mAb blocking of antigen-induced proliferation was done to determine the HLA restriction element used in recognition of the U1-70kD autoantigen by T cells. The results presented here indicate that TCRAV CDR3 usage is highly restricted among U1-70kD autoantigen-specific human T cells clones derived from CTD patients with distinctive structural features. Furthermore, the recognition of the U1-70kD autoantigen occurs in the context of HLA-DR.

Long-term outcome in mixed connective tissue disease: longitudinal clinical and serologic findings.

OBJECTIVE: To determine the long-term clinical and immunologic outcomes in a well-characterized cohort of 47 patients with mixed connective tissue disease (MCTD), including reactivity with U small nuclear RNP (snRNP) polypeptides. METHODS: Patients were followed up over a period of 3-29 years with immunogenetic and systematic clinical and serologic analysis. Sera were analyzed for reactivity with snRNP polypeptides U1-70 kd, A, C, B/B', and D, for anti-U1 RNA, and for anticardiolipin antibodies (aCL). RESULTS: The typical core clinical features of MCTD tended to develop over time; features of inflammation as well as Raynaud's phenomenon and esophageal hypomotility diminished, while pulmonary hypertension, pulmonary dysfunction, and central nervous system disease persisted, following treatment. A favorable outcome was observed in 62% of patients; 38% had continued active disease or had died, with death associated with pulmonary hypertension and aCL. All patients had autoantibodies to the U1-70 kd polypeptide of snRNP, and most were positive for anti-U1 RNA. An orderly progression of intramolecular spreading of autoantibody reactivity against snRNP polypeptides was observed, as was the novel finding of "epitope contraction" followed by disappearance of anti-snRNP autoantibodies during prolonged remission. CONCLUSION: These patients demonstrated the typical immunogenetic, clinical, and serologic findings of MCTD, and the condition rarely evolved into systemic lupus erythematosus or systemic sclerosis. The majority of patients had favorable outcomes, with pulmonary hypertension being the most frequent disease-associated cause of death. Intramolecular spreading of autoantibody reactivity against snRNP polypeptides was observed, followed by "epitope contraction" and ultimate disappearance of anti-snRNP autoantibodies during prolonged disease remission.

T cell receptor beta-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease.

OBJECTIVE: To determine the structure of T cell receptors (TCR) used by Sm-B-reactive human T cell clones, to map T cell epitopes on the Sm-B autoantigen, and to determine the HLA restriction element used in the recognition of Sm-B by T cells. METHODS: Sm-B-reactive T cell clones were generated from patients with connective tissue disease by using either a recombinant fusion protein or synthetic peptides. The TCR structure was defined with the use of polymerase chain reaction and DNA sequencing. Synthetic peptides were used to map T cell epitopes on Sm-B. HLA restriction element usage was defined by using monoclonal antibody blocking. RESULTS: Usage of the TCR third complementarity-determining region (CDR3) was highly restricted among Sm-B autoantigen-specific human T cell clones. Only amino acids 48-96 of the Sm-B2 autoantigen were recognized by T cells, and this occurred in the context of HLA-DR. CONCLUSION: TCR CDR3 gene usage is highly conserved by Sm-B autoantigen-specific T cell clones, and this appears to be related to the recognition of a limited number of T cell epitopes on the Sm-B autoantigen presented in the context of HLA-DR.

Cytokine mRNA expression in the B/W mouse model of systemic lupus erythematosus--analyses of strain, gender, and age effects.

To gain a better understanding of inherent gender-related effects on autoimmunity, cytokine genes were examined in female and male New Zealand Black X New Zealand White (B/W) mice, which are a murine model of systemic lupus erythematosus (SLE). In preliminary studies, semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a trend for B/W spleen cell interferon gamma (IFN-gamma) mRNA in B/W female spleen cells to exceed that of males. This difference was obliterated following concanavalin A (Con A) stimulation. Spleen cells from B/W mice of both sexes were then examined at 6, 18, and 27 weeks of age, and results were compared with matched groups of nonautoimmune DBA/2 mice. Pooled splenocytes from all 12 groups of animals were compared simultaneously for expression of mRNA specific for IFN-gamma, interleukin 4 (IL-4) and interleukin 6 (IL-6). Strain was a potent influence on cytokine transcripts. In unstimulated splenocytes from female and male B/W mice, there was a notable trend for IFN-gamma and IL-6 mRNA expression to exceed transcripts from nonautoimmune DBA/2 mice. When comparisons were carried out by gender, a highly significant increase of IFN-gamma transcripts was apparent in B/W females compared to B/W males at the age of 27 weeks. Following Con A incubation, strain and gender differences were eliminated. IL-4 transcript expression was similar in all pools of cells, and age was not an important factor in expression of any transcript. This study represents the first examination of multiple cytokine transcripts in lymphoid cells from B/W mice. In this hormone-sensitive model of SLE, strain and gender determined in vivo expression of IFN-gamma and IL-6 mRNA.

Analysis of human T cell and B cell responses against U small nuclear ribonucleoprotein 70-kd, B, and D polypeptides among patients with systemic lupus erythematosus and mixed connective tissue disease.

OBJECTIVE: To analyze T and B cell reactivity with U small nuclear RNP (snRNP) 70-kd, B, and D polypeptides among patients with connective tissue disease (CTD) and to examine the functional characteristics of snRNP-reactive T cell clones. METHODS: We used an snRNP enzyme-linked immunosorbent assay and immunoblotting to characterize antibodies in patients' sera. We used human recombinant fusion proteins 70 kd, B, and D to stimulate and clone snRNP-reactive T cells from CTD patients. We analyzed the cell surface phenotype, antigenic specificity, and cytokine profiles of T cell clones. RESULTS: Patients showed T cell responsiveness to snRNP polypeptides that paralleled their autoantibody reactivities. A total of 256 clones were generated, and clones were identified which were specific for the 70-kd, B, or D polypeptides. Clones expressed a T helper cell phenotype, and were found to produce substantial quantities of both interleukin-4 (IL-4) and interferon-gamma, and lesser quantities of IL-2 and IL-6. CONCLUSION: These results show that CTD patients have clonable circulating snRNP-reactive T cells that parallel the specificity of snRNP-reactive antibodies in their sera. The snRNP-reactive T cells exhibit a helper cell phenotype and produce cytokines which are important in B cell help and differentiation.

T cell receptor V beta usage in murine experimental autoimmune thyroiditis.

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg induced experimental autoimmune thyroiditis (EAT) in which the thyroid infiltrate consists primarily of mononuclear cells (MNC) (lymphocytic EAT). MTg-sensitized spleen cells cultured with MTg together with anti-IL2R antibody induce a granulomatous form of EAT in which the thyroid is infiltrated by MNC in addition to PMNs, histiocytes, and multinucleated giant cells. CD4+ T cells are the primary effector cells for both forms of EAT. The presence of specific T cell receptor (TCR) V beta families in the thyroid infiltrate was examined by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) amplification of mRNA. At the time of maximal disease severity, cells infiltrating the thyroid expressed primarily V beta 8, V beta 4, V beta 11, and V beta 14 as determined by flow cytometry. RT-PCR confirmed these findings and also detected several additional V beta gene families, including V beta 1, V beta 2, V beta 6, V beta 13, and V beta 15; V beta 3, V beta 10, and V beta 12 were also detected in some, but not all, experiments. There were no differences in the V beta T cell repertoires in thyroids of mice with lymphocytic vs granulomatous EAT. RT-PCR analysis of intrathyroidal MNC 11 days after cell transfer showed TCR V beta mRNA transcripts to be primarily restricted to V beta 4, V beta 11, and V beta 14, whereas the predominant thyroid-infiltrating T cell 21 days after cell transfer was V beta 8+. Depletion of V beta 8+ T cells in recipient mice did not reduce EAT severity. TCR V beta usage shifted predominantly to V beta 4+, V beta 11+, or V beta 14+ T cells of both CD4+ and CD8+ T cell subsets. These results indicate that multiple V beta TCR are expressed in thyroids of mice with EAT.

Analysis of anti-U1 RNA antibodies in patients with connective tissue disease. Association with HLA and clinical manifestations of disease.

OBJECTIVE: To determine the prevalence of anti-U1 RNA antibodies in connective tissue disease (CTD) patients and evaluate immunogenetic and clinical features of patients possessing these antibodies. METHODS: RNA immunoprecipitation was used to analyze patient and healthy control sera for the presence of anti-R1 RNA antibodies. Enzyme-linked immunosorbent assay and immunoblotting were used to determine small nuclear RNP (snRNP) polypeptide antibodies. HLA polymorphisms were determined by microcytotoxicity and DNA typing. RESULTS: Anti-U1 RNA IgM and IgG antibodies were found in 60% of anti-RNP positive patients. All of the anti-U1 RNA positive patients had anti-70K, and most had anti-A, (U1)snRNP polypeptide antibodies. HLA-DR2/DR4, as well as Raynaud's phenomenon and synovitis, were significantly increased in the anti-U1 RNA positive group. CONCLUSION: The presence of anti-U1 RNA antibodies correlates with anti-70K and anti-A polypeptide antibodies. In addition, the anti-U1 RNA positive CTD patient group is immunogenetically and clinically distinctive from the anti-U1 RNA negative patient group.

Molecular and serologic analysis of HLA genes and immunoglobulin allotypes in IgA nephropathy.

We studied a large North American Caucasian population of patients with biopsy proven IgA nephropathy for polymorphisms of HLA-A,B,C, HLA-DR, HLA-DQ and HLA-DP using a combination of serologic phenotyping and polymerase chain reaction sequence specific oligonucleotide probe (PCR-SSOP) hybridization genotyping. We also examined patients for polymorphisms of immunoglobulin by determining Gm and Km allotypes. When compared to healthy local controls there was an apparent decrease in HLA-DR5 (DRw11, DRw12) suggesting that this allele had a protective effect on disease susceptibility. None of the previously reported HLA-DQ associations found among Japanese or british Caucasian patients were found among this large North American Caucasian population. The HLA-DPB1*0601 genotype was increased among patients, but this was not significant when corrected for multiple comparisons. There were no differences in the distribution of Gm or Km allotypes among patients versus controls, regardless of whether they were stratified into those with progressive or non-progressive renal disease. Taken together, these findings suggest that there is substantial genetics heterogeneity in susceptibility to IgA nephropathy among different ethnic and/or geographically distinct populations.

Human T cell clones reactive against U-small nuclear ribonucleoprotein autoantigens from connective tissue disease patients and healthy individuals.

SLE and mixed connective tissue disease (MCTD) are characterized by the presence of high titers of autoantibodies against uridine-rich RNA-small nuclear ribonucleoprotein (snRNP) Ag. Because the presence of such snRNP-reactive autoantibodies has recently been shown to be associated with polymorphisms of HLA, this study was undertaken to determine whether snRNP-reactive T cells could be identified and characterized from patients. PBMC were stimulated with affinity-purified snRNP Ag and cloned by limiting dilution in the presence of rIL-2 and rIL-4, snRNP-reactive human T cell clones were generated from three patients and two healthy blood donors who possessed disease-associated HLA genotypes. The cell surface phenotype of clones determined by flow cytometry was CD3+, CD4+, CD45RO+, TCR V alpha beta+. TCR V beta analysis, performed using V beta-specific primers and polymerase chain reaction, revealed that the T cell lines generated were clonal; a limited number of TCR V beta genes were expressed among the clones tested. All clones tested by mAb blocking of Ag-induced proliferation were restricted by HLA-DR. Several T cell clones were identified that were specific for B'/B or D polypeptides. These results demonstrate that snRNP-reactive T cells can be isolated from SLE and MCTD patients in vitro, and that Ag-driven expansion of such T cells could play a role in the immunopathogenesis of these diseases in vivo.

U1-70-kd autoantibody-positive mixed connective tissue disease in children. A longitudinal clinical and serologic analysis.

OBJECTIVE: To obtain longitudinal data on the clinical, serologic, and immunogenetic features of children with mixed connective tissue disease (MCTD). METHODS: Eleven children with MCTD were followed up for a mean of 9.8 years. Enzyme-linked immunosorbent assay and immunoblotting were used to analyze sera for autoantibodies to small nuclear ribonucleoprotein polypeptides. HLA types were determined in 9 patients, by microcytotoxicity and DNA typing. RESULTS: All 11 children had anti-U1-70-kd auto-antibodies. Six of 9 were positive for HLA-DR2, 4 of 9 for HLA-DR4, and 9 of 9 for either HLA-DR2 or DR4. Outcomes were favorable with no functional impairment in 8 of the 11 children and were poor in 3. CONCLUSION: The frequency of HLA-DR2/DR4 is increased among children with anti-U1-70-kd autoantibody positive MCTD.