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While virus-specific antibodies are broadly recognized as correlates of protection, virus-specific T cells are important for direct clearance of infected cells. Failure to generate hepatitis B virus (HBV)-specific antibodies is well-known in patients with end-stage renal disease. However, whether and to what extent HBV-specific cellular immunity is altered in this population and how it influences humoral immunity is not clear. To address it, we analyzed HBV-reactive T cells and antibodies in hemodialysis patients post vaccination. 29 hemodialysis patients and 10 healthy controls were enrolled in a cross-sectional study. Using multiparameter flow cytometry, HBV-reactive T cells were analyzed and functionally dissected based on granzyme B, interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and IL-4 expression. Importantly, HBV-reactive CD4+ T cells were detected not only in all patients with sufficient titers but also in 70% of non-responders. Furthermore, a correlation between the magnitude of HBV-reactive CD4+ T cells and post-vaccination titers was observed. In summary, our data showed that HBV-reactive polyfunctional T cells were present in the majority of hemodialysis patients even if humoral immunity failed. Further studies are required to confirm their in vivo antiviral capacity. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols., Graphical Abstract, Despite HBV vaccination failure in end-stage renal disease (ESRD), HBV-reactive T cells are generated in about 70% of ESRD-vaccinees. Further, the non-responder group demonstrated polyfunctional HBV-reactive CD4+ T cells suggesting their potential anti-viral capacities. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols.

Objective To unravel the mechanisms that control bony ankylosis in ankylosing spondylitis (AS). Methods Histomorphologic and histomorphometric analyses were performed on zygapophyseal joints obtained from 18 patients with AS, 9 patients with osteoarthritis (OA), and 10 cadaver donors without a rheumatic disease (controls). The proteoglycan content of the cartilage was determined by Safranin O staining and the chondrocyte apoptosis according to caspase 3 expression. Results AS joints were categorized into 3 groups according to the morphology of the joint surfaces and joint space: group 1 were joints with an open joint space, group 2 were joints with cartilaginous fusion, and group 3 were joints with bony fusion of the joint surfaces. Progressive loss of the joint space from group 1 joints to group 3 joints suggests that this grouping corresponds to sequential stages of joint remodeling. Cartilage thickness and subchondral bone plate thickness declined from group 1 to group 3 (P < 0.01). Increased chondrocyte apoptosis rates were found in groups 1 and 2 (P < 0.05), while in group 3, a reduction in the proteoglycan content was found (P < 0.001). Bone marrow replacement and invasion of the subchondral bone plate by fibrous tissue was found predominantly in AS joints in group 2. Conclusion Cartilage degeneration, indicated by cartilage thinning, enhanced chondrocyte apoptosis, and proteoglycan loss, and subchondral bone thinning, promoted by invasion of the subchondral bone plate by a fibrous tissue originating from the bone marrow, are hallmarks of joint remodeling in AS.

Objective The interleukin-12 (IL-12) family of cytokines has been suggested to play a critical role in inflammatory autoimmune diseases, and recent studies analyzing peripheral blood and synovial fluid from patients with spondyloarthritides suggest that IL-23 might be a proinflammatory factor in these disorders. This study was undertaken to investigate the presence and source of IL-23 in the spines of patients with ankylosing spondylitis (AS). Methods The frequency of IL-23–positive and IL-12–positive cells within the subchondral bone marrow and within fibrous tissue replacing normal bone marrow in facet joints of patients with AS was analyzed by immunohistochemistry. The origin of IL-23–positive cells was determined by double staining of CD163+ macrophages, CD68+ macrophages, CD1a+ dendritic cells, tryptase-positive mast cells, myeloperoxidase-positive cells, CD20+ B cells, and CD3+ T cells. Findings in 28 facet joints from 22 AS patients were compared with those in 20 facet joints from 13 patients with osteoarthritis (OA) and 10 normal control specimens. Results The frequency of IL-23–positive cells in subchondral bone marrow from the joints of AS patients (mean ± SD 42.50 ± 32.81/high-power field [hpf]) was significantly increased compared to that in subchondral bone marrow from OA patients (OA 15.63 ± 29.90/hpf) (P = 0.0017) or controls (19.36 ± 16.8/hpf) (P = 0.03). Myeloperoxidase-positive cells and, to a lesser extent, macrophages and dendritic cells were found to be the origin of IL-23 in the bone marrow. In AS and OA patients, the frequency of IL-23–positive cells was significantly higher than that of IL-12–positive cells (P < 0.001 in both patient groups). Within fibrous tissue from AS and OA facet joints, IL-23 was predominantly produced by CD163+ macrophages (mean ± SD 0.64 ± 0.59/hpf and 4.36 ± 3.4/hpf, respectively) and CD68+ macrophages (2.3 ± 0.65/hpf and 6.54 ± 4.1/hpf, respectively). Conclusion IL-23 is expressed in the subchondral bone marrow and in fibrous tissue replacing bone marrow in facet joints of patients with AS. It might have a role in inflammatory processes and in chronic changes in AS joints, which makes it an interesting potential therapeutic target in this disease.

Objective NSAIDs are first-line therapy in axial SpA (axSpA). The proportion of patients responding to NSAIDs and differences between AS and non-radiographic axSpA (nr-axSpA) in this regard have not been studied in detail to date. The aim of this study was to examine the proportion of patients with AS and nr-axSpA responding to NSAIDs according to current treatment recommendations. Methods Consecutive anti-TNF-naive patients with nr-axSpA and AS (n = 50 each) were included if their BASDAI score was ⩾4 without having received maximal NSAID doses. In case of a BASDAI score ⩾4 1 week later, another NSAID was prescribed. For the next 3 weeks, continuous intake of maximal doses was recommended but patients could reduce doses in case of intolerance or improvement. MRI of the SI joints was performed at baseline and week 4. Results All outcomes except for CRP and MRI scores improved significantly after 4 weeks of NSAIDs, with no difference between axSpA subgroups. An Assessment of SpondyloArthritis international Society 40% (ASAS40) response and partial remission rates were 35 and 16% at week 4, respectively. At the same time point, a BASDAI score ⩾4 was still present in 44% of patients, 30% of which had reduced NSAID doses, partly due to intolerance (38%). Only 13% of all patients had continuously taken NSAIDs at the maximal dosage, but there was no difference in the efficacy outcome compared with those who had taken reduced doses. Conclusion AS and nr-axSpA patients had similar response rates to NSAIDs while objective signs of inflammation did not change over 4 weeks. Only a minority of patients was willing to take maximal doses of NSAIDs, and ⩾40% patients remained candidates for TNF blockers. These results may influence future trial designs.

Recent studies using magnetic resonance imaging have suggested that the subchondral bone marrow and the entheses are the sites which are primarily involved in the peripheral and axial inflammation found in patients with spondyloarthritides. Histopathological analyses indicated that the typical morphological features at these sites reflect an inflammation (osteitis) at the bone cartilage interface and in the subchondral bone marrow. This finding implies that synovitis may be of minor importance, especially in comparison to other inflammatory joint diseases such as rheumatoid arthritis. Here, we summarize current available knowledge on synovial involvement in inflammatory processes related to SpA.

Objective Histologic studies have shown B cell clusters in the subchondral bone marrow of the spine of patients with ankylosing spondylitis (AS). An immunotherapy targeting B cells in AS is therefore of interest. We undertook this study to examine the efficacy and safety of rituximab in patients with AS refractory to nonsteroidal antiinflammatory drugs in whom previous treatment with tumor necrosis factor α (TNFα) blockers either had not been tried or had failed. Methods In this phase II clinical trial, 1,000 mg rituximab was administered intravenously at baseline and at week 2 in 20 patients with active AS. Ten of these patients had never received TNF blockers, and treatment with TNF blockers had failed in the other 10 patients. The primary end point was a 20% improvement in disease activity at week 24 according to the criteria of the Assessment of SpondyloArthritis international Society (an ASAS20 response). Results Seventy-five percent of the patients were male, 90% were HLA–B27 positive, their mean age was 39.7 years, and their mean disease duration was 16.8 years. Patients had active disease, defined as a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score ≥4. While there was no clear response at week 24 in the group in whom TNF blockers had failed (30% had achieved an ASAS20 response, 10% had achieved an ASAS40 response, none had achieved partial remission according to the ASAS criteria, and none had achieved 50% improvement on the BASDAI [a BASDAI50 response] beyond an expected placebo response), we observed a good improvement in the TNF blocker–naive group at week 24 (50% had achieved an ASAS20 response, 40% had achieved an ASAS40 response, 30% had achieved partial remission according to the ASAS criteria, and 50% had achieved a BASDAI50 response). Conclusion Although rituximab does not seem to be effective in patients with AS that does not respond to TNF blockers, it had significant efficacy in TNF blocker–naive patients. Therefore, further controlled trials with B cell–directed therapies should be performed in TNF blocker–naive AS patients in the future.

Background Imaging has an essential role in the new spondyloarthritis (SpA) classification criteria for axial but not for peripheral manifestations. We evaluated the impact of imaging findings for identification and treatment decisions in patients with peripheral spondyloarthritis (pSpA) and controls (non-SpA). Methods Patients with pSpA (Assessment of SpA international Society criteria, n=30) and non-SpA (n=30), aged

Objective Analysis of the histopathologic features of hip arthritis in patients with ankylosing spondylitis (AS) has revealed accumulation of infiltrating mononuclear cells in the bone end plate and presence of hyaline articular cartilage that is not found in areas of total cartilage destruction. This study was undertaken to assess whether chondrocytes attract lymphocytes and whether cartilage chondrocytes from patients with AS have the potential to directly stimulate T cells in an HLA-restricted manner. Methods Human HLA–B27+ T cell lines, specific for the Epstein-Barr virus–derived peptide EBNA258–266, and autologous chondrocytes, serving as nonprofessional antigen-presenting cells (APCs), were available for use in a model system to study chondrocyte functions in femoral head joint cartilage of patients with AS. Peptide functionality of cytotoxic T cells was assessed by flow cytometry, and cellular interactions were detected by fluorescence confocal microscopy. Results When maintained in an alginate matrix, chondrocytes isolated from the femoral heads of patients with AS constitutively expressed type II collagen and CD80. When pulsed with the EBNA258–266 peptide, autologous chondrocytes functioned as APCs and, specifically, induced interferon-γ production in CD8+ T cells. In mixed chondrocyte–T cell cultures, cell–cell contacts were dependent on the presence of the EBNA258–266 peptide. T cells adjacent to chondrocytes produced perforin and granzyme B; both molecules were found in focal aggregates, a prerequisite for antigen-specific lysis of target cells. Conclusion Antigen presentation through human chondrocytes allows the stimulation of peptide-specific CD8+ T cells. These results indicate that human chondrocytes can act as nonprofessional APCs, and suggest that there is an interferon-γ–triggered autocrine loop of immune cell–mediated chondrocyte activation in the already inflamed environment. Thus, local HLA-dependent activation of peptide-specific cytotoxic CD8+ T cells by chondrocytes might contribute to inflammatory processes in the spondylarthritides.

We previously suggested that fibroblast-rich granulation tissue eroding the subchondral bone is instrumental in the joint remodeling that occurs in ankylosing spondylitis (AS). The purpose of this study was to determine if this granulation tissue also carries bone-forming capabilities, which we approached by searching for bone-forming cells (hypertrophic chondrocytes, osteoblasts) in its vicinity. We also assessed adipogenic tissue transformation, which has been suggested to be an intermediate feature in AS bone formation based on imaging studies.The facet joints of AS patients, osteoarthritis (OA) patients, and autopsy subjects (controls) were screened for subchondral granulation tissue. We searched for hypertrophic chondrocytes by assessing RUNX-2, type X collagen, and matrix metalloproteinase 13 (MMP-13) expression, for osteoblasts by analyzing RUNX-2, CD56, and type I collagen expression, as well as for signs of new bone formation. Adipocytes and lipid accumulation were assessed in Safranin O-stained sections.In the joints of AS and OA patients, RUNX-2-positive cells were found to be lining the granulation tissue. These cells coexpressed type I collagen but lacked type X collagen and MMP-13 expression, confirming their osteoblastic nature. In 91% of AS joints and in 20% of OA joints (P 0.05), we observed foci of new bone formation at contact zones between the granulation tissue and the cartilage. Joints containing bony spots showed greater replacement of the adjacent bone marrow by granulation tissue than did joints without bone formation (P 0.05). The granulation tissue often contained adipocytes and lipid accumulations. Replacement of the subchondral bone marrow by fat tissue was also frequently found but was not associated with new bone formation.The subchondral granulation tissue carries osteoblasts, which promote new bone formation, leading to intraarticular ankylosis of the facet joints in AS.

Background: CD4+ T cell responses to the G1 domain of aggrecan in patients with ankylosing spondylitis (AS) were recently reported. Whether such an immune response can be seen in the CD8+ subpopulation has not yet been determined. Objective: To determine if HLA-B27 restricted G1-specific CD8+ T cells are present in AS and to analyse immunodominant CD8+ T cell epitopes. Methods: Peripheral blood mononuclear cells of 45 patients with AS were stimulated with overlapping 18-mer peptides covering the whole G1 protein. Results were compared with those for patients with rheumatoid arthritis (RA) and healthy controls. For epitope analysis, G1-specific interferon gamma positive (IFNγ+) T cells were isolated by magnetic activated cell sorting. After in vitro expansion, CD8+ T cells were restimulated with 14 subpools of G1 peptides. T cells responding to G1 peptide subpools were quantified by flow cytometry according to IFNγ secretion. Predicted peptides were subsequently confirmed by stimulation with single peptides. Results: G1-specific CD8+ T cell responses were found in 29/45 (64%) patients with AS, 18/35 (51%) patients with RA, but not in healthy controls. Five CD8+ T cell epitopes were identified as immunodominant in five patients. However, the T cell response was not HLA-B27 restricted. Nonamer peptides with an HLA-B27 binding motif did not induce a T cell response. Conclusion: A G1 peptide-specific CD8+ T cell response is present in AS but also in patients with RA. It does not seem to be HLA-B27 restricted. Whether such a response has a role in the pathogenesis of AS needs clarification.

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.

Although non-obese diabetic (NOD) mice spontaneously develop T cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self-Ag. Furthermore, as autoimmunity has been postulated to result from T cell responses directed toward self-peptides that bind with low affinity to NOD I-A(g7) MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic beta cells. We identified LACK peptides that were the targets of T cells in LACK-immunized NOD mice while binding to I-A(g7) with low affinity. While CD4(+) T cells from NOD mice secreted IFN-gamma, IL-4, IL-5 and IL-10 in response to LACK, those from LACK-expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK-expressing Tg mice exhibited self-reactive CD4(+) T cells with impaired proliferation capabilities. Hence, even self-peptides that bind to I-A(g7) with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that T cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4+ T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4+ T cells with a defined MHC/peptide specificity.

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.

Involvement of the axial skeleton is a characteristic feature of spondyloarthritis (SpA). Asymmetrical arthritis and enthesitis of the peripheral joints are found in about 30–60% of patients.1–3 A 52-year-old patient with a history of psoriasis reported inflammatory back pain, painful ankles and knees and severe morning stiffness on admission. The physical examination revealed tender entheses at different locations: upper thoracic spine, lumbar spinous processes, both iliac crests and posterior superior iliac spines. Furthermore, there was tenderness of both sacroiliac joints, both knees and both ankles without joint swelling. HLA-B27 antigen was negative. The sedimentation rate (70/76) and levels of C reactive protein (1.48 mg/dl) were increased. The Bath Ankylosing Spondylitis Activity Index was 6.0, and the Bath Ankylosing Spondylitis Functional Index was 4.6. Radiographs of the pelvis showed bilateral grade 3 sacroiliitis. Thus, ankylosing spondylitis (AS) associated with psoriasis was diagnosed. Treatment on admission comprised methotrexate (15 mg/week) and prednisolone (5 mg/day). Whole-body MRI (WB-MRI) was performed on a special 1.5 T scanner (Avanto, Siemens, Germany) for evaluation of the extent of suspected widespread inflammatory lesions. Images of the entire body, excluding the hands, were acquired in a head-to-toe scan using parallel imaging and a whole-body surface coil …

Objective In Yersinia enterocolitica-triggered reactive arthritis (Yersinia ReA), the synovial T cell response is primarily directed against bacterial components, which are mostly unknown. This study was performed to investigate the synovial proliferative T cell response to a panel of recombinant Yersinia antigens in patients with Yersinia ReA and in controls. Methods Synovial fluid mononuclear cells (SFMC) were obtained from 4 patients with Yersinia ReA and from 14 patients with arthritides of different etiology. SFMC were stimulated with 5 recombinant Yersinia antigens (the 19-kd urease β subunit, 13-kd ribosomal L23 protein, 32-kd ribosomal L2 protein, 18-kd outer membrane protein H, and Y enterocolitica heat-shock protein 60 [hsp60]), and with human, Chlamydia trachomatis, and Borrelia burdorferi hsp60. Three T cell clones specific for Y enterocolitica hsp60 were generated from 1 patient with Yersinia ReA. Antigen-induced cytokine release was measured by enzymelinked immunosorbent assay. Results SFMC from all 4 patients with Yersinia ReA responded to each of the Yersinia antigens except the 13-kd protein. These antigens were also recognized by SFMC from a subgroup of patients with undifferentiated arthritis (n = 4), but not by SFMC from other patients with arthritis of different etiology (n = 10). Y enterocolitica hsp60 induced the strongest proliferative response in all cases. Two types of hsp60-reactive T cell clones could be obtained. One clone responded to all hsp60 variants, including the human variant, and showed a type 2 T helper (Th2)-like cytokine-secretion pattern. In contrast, another clone with specificity for the bacterial hsp60 proteins, but not the human equivalent, reacted with a more Th1-like pattern. Conclusion In Y enterocolitica-triggered ReA, at least 4 immunodominant T cell antigens exist, which might be used in lymphocyte proliferation assays to identify patients with Yersinia ReA. The hsp60 is a strong antigen, inducing both bacteria-specific and potentially autoreactive CD4+ T cells of both the Th1 and Th2 type.

Introduction: The molecular mechanisms of syndesmophyte formation in ankylosing spondylitis (AS) are yet to be characterised. Molecules involved in bone formation such as Wnt proteins and their antagonists probably drive syndesmophyte formation in AS. Methods: This study investigated sequential serum levels of functional dickkopf-1 (Dkk1), a potent Wnt antagonist involved in bone formation in arthritis, by capture ELISA with its receptor LRP6 in 65 AS patients from the German Spondyloarthritis Inception Cohort. Dkk1 levels were then related to structural progression (syndesmophyte formation) as well as sclerostin and C-reactive protein (CRP) levels. Results: Functional Dkk1 levels were significantly (p=0.025) higher in patients with no syndesmophyte growth (6.78±5.48 pg/ml) compared with those with syndesmophyte growth (4.13±2.10 pg/ml). Dkk1 levels were highly correlated to serum sclerostin levels (r=0.71, 95% CI 0.53 to 0.82; p

Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease β-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease β-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs.

Objective Osteocytes are considered to be sensors of bone damage and regulators of bone mass by specifically expressing sclerostin, an inhibitor of bone formation. The contribution of osteocytes in regulating local bone remodeling in arthritis is unknown. The aim of this study was to investigate the role of osteocytes as contributors to bone remodeling in ankylosing spondylitis (AS). Methods Sclerostin expression and osteocyte death were assessed by immunohistochemistry in joints derived from patients with AS, patients with rheumatoid arthritis (RA), and patients with osteoarthritis (OA), as well as from control subjects. In addition, the serum level of sclerostin was assessed by enzyme-linked immunosorbent assay in healthy subjects and patients with AS; this assessment included the longitudinal correlation of sclerostin serum levels and radiographic progression in the spine of patients with AS. Results Sclerostin expression was confined exclusively to osteocytes. Whereas the majority of osteocytes in healthy individuals and patients with RA were sclerostin positive, expression was significantly reduced in patients with OA and was virtually absent in patients with AS. Moreover, serum levels of sclerostin were significantly lower in patients with AS than in healthy individuals. Importantly, low serum sclerostin levels in patients with AS were significantly associated with the formation of new syndesmophytes (P = 0.007). Conclusion Sclerostin expression is impaired in patients with AS, suggesting a specific alteration of osteocyte function in this disease. A low serum level of sclerostin in the setting of AS is linked to increased structural damage, emphasizing the role of sclerostin in the suppression of bone formation.

To the Editor: We thank Pacheco-Tena, et al 1 for their letter. … Address correspondence to Dr. H. Appel, Charite Universitatsmedizin Berlin, Campus Benjamin Franklin, Rheumatology, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: heiner.appel{at}charite.de

Objective Zygapophyseal joints of the spine are often affected in ankylosing spondylitis (AS). In this study, we undertook a systematic immunohistologic evaluation of the immunopathology of the zygapophyseal joints in patients with advanced AS. Methods We obtained zygapophyseal joints from 16 AS patients undergoing polysegmental correction of kyphosis and from 10 non-AS controls (at autopsy). Immunohistologic analysis of the bone marrow was performed by analyzing the number of infiltrating T cells (CD3, CD4, CD8), B cells (CD20), osteoclasts (CD68), bone marrow macrophages (CD68), and microvessel density (CD34) per high-power field. Results Zygapophyseal joints from 6 of 16 AS patients, but from none of the controls, exhibited 2 or more CD3+ T cell aggregates, signifying persistent inflammation. Interstitial CD4+ and CD8+ T cells were significantly more frequent in AS patients compared with non-AS controls (P = 0.002 and P = 0.049, respectively). While there was no clear difference between the number of CD20+ B cells in AS patients overall compared with controls, there was a significant difference when persistently inflamed joints from patients with AS were compared with joints without active inflammation from patients with AS or joints from controls (both P = 0.03). Microvessel density in bone marrow from AS patients with active inflammation was significantly higher than that in bone marrow from controls. Conclusion This immunohistologic study of bone marrow from zygapophyseal joints demonstrates persistent inflammation in the spine of patients with AS, including those with longstanding disease. The findings of increased numbers of T cells and B cells and neoangiogenesis suggest that these features play a role in the pathogenesis of AS.

Objective Previous histopathologic and magnetic resonance imaging studies suggest that the subchondral bone marrow might be the primary site of inflammation in patients with ankylosing spondylitis (AS) and that this might be reflected by inflammation found in hip joints. The aim of this study was to conduct an immunohistologic assessment of the bone–cartilage interface and subchondral bone marrow in AS patients with hip arthritis. Methods We collected femoral heads from patients with AS, osteoarthritis (OA), and rheumatoid arthritis (RA) who were undergoing hip replacement. The subchondral bone marrow and bone–cartilage interface were assessed immunohistochemically by evaluating infiltrating T cells, microvessel density, and osteoclasts. Areas of the femoral head surface with and without cartilage were assessed separately. Results At sites with surface cartilage, we found subchondral infiltration of CD3+ T cell aggregates at significantly higher numbers in AS patients as compared with OA patients, but not RA patients. At sites of complete cartilage destruction, the frequency of CD3+ T cell aggregates was significantly reduced as compared with sites with cartilage on the surface in AS patients, but not in RA patients. Similar differences were found for CD4+ and CD8+ T cells. Only at sites with surface cartilage, but not those without, angiogenesis and osteoclastic foci in the subchondral bone marrow in AS patients were significantly increased as compared with RA patients and with OA patients. Conclusion These findings suggest that the subchondral bone marrow and bone–cartilage interface are primary sites of inflammation in AS and that cartilage might be necessary for the induction of inflammation.

Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.

Objective Several hypotheses have been proposed to explain the strong association between HLA–B27 and ankylosing spondylitis (AS). Among these, the arthritogenic peptide theory proposes that certain B27 subtype alleles bind specific arthritogenic peptide(s) due to their unique amino acid anchor residues. Cartilage antigens have been discussed as candidate targets for the immune response in AS. The recognition of HLA–B27–peptide complexes by self-reactive CD8+ T cells might contribute to joint-specific tissue damage. Therefore, we investigated the presence of autoreactive CD8+ T cells specific for cartilage-derived peptides in patients with AS. Methods An HLA–B27–binding prediction program and a proteasome-cutting prediction program for the human 20S proteasome were used to screen 18 human cartilage proteins for potentially immunogenic nonamer peptides. The peptides identified were used to stimulate peripheral blood mononuclear cells from 20 HLA–B27–positive patients with AS and synovial fluid (SF) mononuclear cells from 7 HLA–B27–positive patients with AS. Activation of T cells was measured by antigen-specific intracellular cytokine staining and quantified by flow cytometry. Results From the screening analysis, we identified 121 nonamer peptides. Of these, 1 peptide derived from type II collagen and 1 from type VI collagen were stimulatory for peripheral blood CD8+ T cells in only 1 of 20 patients. However, in 4 of 7 SF samples the same type VI collagen–derived nonamer peptide stimulated SF CD8+ T cells, but none of the other peptides was stimulatory. This CD8+ T cell response could be blocked by an anti–HLA–B27 antibody, confirming an HLA–B27–restricted immune response. Conclusion Our findings suggest that cartilage-directed cellular autoimmunity might play an important role in joint-specific tissue damage in patients with AS. Future research is necessary to determine whether the identified peptide is of pathogenetic relevance.

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Reactive arthritis (ReA) mostly presents as an asymmetrical oligoarthritis, usually affecting the leg joints.1 It is a T cell dependent inflammatory disease following infections with various enteropathic bacteria—for example, Yersinia enterocolitica 0:3 , Salmonella enteritidis, Shigella fexneri, or microbes pathogenic for the urogenital tract such as Chlamydia trachomatis. 2 Identification of the structure of antigens of triggering microbes which drive immune responses in the synovial fluid would be of great interest. We recently described two major antigens, the Ye 19 kDa and the Ye HSP60 inducing synovial T cell proliferation in patients with Ye triggered ReA.3–7 They seemed to overcome the specificity problem of proliferation assays with T cells cultured from the site of inflammation to ReA triggering bacteria, because the local immune response to whole bacteria …

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8+ cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8+ T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8+ T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8+ T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.

Introduction Recent data about radiographic progression during treatment with tumor necrosis factor-alpha (TNF-α) blocker agents in patients with ankylosing spondylitis (AS) have prompted an intensive discussion about the link between inflammation/bone destruction and new bone formation and the order of events. Therefore, we analysed parameters of cartilage degradation, neoangiogenesis, and new bone formation in different cohorts of patients with axial spondyloarthritis with and without treatment with TNF-α blocker agents. Method TNF-α blocker-naïve AS patients were investigated for serum levels of metalloproteinase-3 (MMP-3) (n = 71), vasoendothelial growth factor (VEGF) (n = 50), and bone-specific alkaline phosphatase (BALP) (n = 71) at baseline and after 1 and 2 years. This was compared with 34 adalimumab-treated patients with axial spondyloarthritis (22 AS and 12 non-radiographic axial spondyloarthritis patients) before and after 36 to 52 weeks of treatment. Results There were no significant changes in serum levels of MMP-3 (P > 0.05), VEGF (P > 0.05), and BALP (P > 0.05) in a large cohort of TNF-α blocker-naïve AS patients followed for 2 years. In contrast, adalimumab-treated spondyloarthritis (AS and non-radiographic axial spondyloarthritis) patients had a significant decrease of VEGF (P < 0.001) and MMP-3 (P = 0.022) after 36 to 52 weeks of therapy. Most interestingly, the level of BALP increased significantly after 36 to 52 weeks of therapy (P < 0.001). A decrease in MMP-3 serum levels correlated significantly to an increase of BALP (r = -0.398, P = 0.02). In the case of VEGF, there was a negative correlation without significance (r = -0.214, P > 0.05). Conclusions Rising levels of BALP and the negative correlation between MMP-3 and BALP in spondyloarthritis patients with TNF-α blocker treatment indicate that new bone formation in AS occurs if inflammation is successfully treated and might be part of a healing process.

Ankylosing spondylitis (AS) is a chronic inflammatory disease which affects primarily the sacroiliac joints and the spine. In patients with active disease, magnetic resonance imaging (MRI) of the spine shows areas of bone marrow edema, the histopathological equivalent of which is unknown. In this study we correlate inflammation in the spine of patients with AS as revealed by histological examination with bone marrow edema as detected by MRI. We have compared the histopathological findings of zygapophyseal joints from 8 patients with AS (age: 30 to 64, disease duration 7 to 33 years) undergoing spinal surgery with findings in MRI. For histopathological analysis, we quantified infiltrates of CD3+, CD4+ and CD8+ T cells as well as CD20+ B cells immunohistochemically. Bone marrow edema was evaluated in hematoxylin and eosin stained sections and quantified as the percentage of the bone marrow area involved. All patients with AS showed interstitial mononuclear cell infiltrates and various degrees of bone marrow edema (range from 10% to 60%) in histopathological analysis. However, in only three of eight patients histopathological inflammation and edema in the zygapophyseal joints correlated with bone marrow edema in zygapophyseal joints of the lumbar spine as detected by MRI. Interestingly, two of these patients showed the highest histological score for bone marrow edema (60%). This first study correlating histopathological changes in the spine of patients with AS with findings in MRI scans suggests that a substantial degree of bone marrow inflammation and edema is necessary to be detected by MRI.

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8+ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8+ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8+ T cells could be increased by stimulating CD8+ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8+ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27+ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the β chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-γ and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

Introduction In ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS. Methods We assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic proteins (BMPs) and negative regulators (dickkopf-1 (DKK-1)), sclerostin, (wingless inhibitory factor 1 (wif-1)) of chondrocyte hypertrophy in the cartilage of facet joints from patients with AS or osteoarthritis (OA) and from autopsy controls (CO) by immunohistochemistry. Sex determining region Y (SRY)-box 9 (Sox9) and type II collagen (COL2) expression was assessed as indicators of chondrocyte integrity and function. Results The percentage of hypertrophic chondrocytes expressing Runx2, COL10, MMP13, osteocalcin or beta-catenin was significantly increased in OA but not in AS joints compared to CO joints. Frequencies of sclerostin-positive and DKK-1-positive chondrocytes were similar in AS and CO. In contrast, wif-1- but also BMP-2- and BMP-7-expressing and Sox9-expressing chondrocytes were drastically reduced in AS joints compared to CO as well as OA joints whereas the percentage of COL2-expressing chondrocytes was significantly higher in AS joints compared to CO joints. Conclusions We found no evidence for chondrocyte hypertrophy within hyaline cartilage of AS joints even in the presence of reduced expression of the wnt inhibitor wif-1 suggesting that chondrocyte hypertrophy is not a predominant pathway involved in joint fusion and remodeling in AS. In contrast, the reduced expression of Sox9, BMP-2 and BMP-7 concomitantly with induced COL2 expression rather point to disturbed cartilage homeostasis promoting cartilage degeneration in AS.