Rheumatology-Rhumatologie

Objective To evaluate the role of immune cells and their effector cytokines in the pathogenesis and progression of knee osteoarthritis (OA) in matched OA synovial fluid (SF) and synovial tissue sam...

Aims Rheumatoid arthritis (RA) is a systematic autoimmune disorder, characterized by synovial inflammation, bone and cartilage destruction, and disease involvement in multiple organs. Although nume...

Via Krishan Maggon

Arthritis Rheumatol. Author manuscript; available in PMC 2016 Dec 28. Published in final edited form as: doi: 10.1002/art.39458 PMCID: PMC5195265 NIHMSID: NIHMS835582 PMID: 26473401 Enthesitis New Insights Into Pathogenesis, Diagnostic Modalities, and Treatment The publisher's final edited version of this article is available free at Arthritis Rheumatol See other articles in PMC that cite the published article. Introduction Enthesitis is a central feature of spondyloarthritis (SpA). Although enthesitis has traditionally been considered to be a focal insertional disorder, advanced imaging and pathologic findings suggest that enthesitis is a diffuse process with effects on adjacent bone and soft tissue. As a result of repeated biomechanical stress, it appears that microdamage at the enthesis triggers an inflammatory response in the synovium, leading to synovitis. Along with mechanical stress, exogenous bacteria may play a role in activating the immune response, especially in genetically predisposed individuals whose major histocompatibility locus encodes the class I molecule HLA–B27. Recent studies in animal models suggest that autoimmunity against versican and fibrocartilage proteins, and bone morphogenetic protein (BMP) signaling play roles in enthesitis development. Finally, interleukin-23 (IL-23) has been implicated in enthesitis with inflammatory effects mediated through IL-17 and tumor necrosis factor (TNF), and new bone formation driven by IL-22. Although prior therapeutic choices were limited to nonsteroidal antiinflammatory drugs (NSAIDs) and activity modification, in recent years TNF inhibitors have proven to be useful. Further research on the effects of IL-22 and IL-23 blockade is needed to understand the effects on the treated patient. While enthesitis is underdiagnosed by physical examination alone, the use of ultrasound has proven to be highly sensitive for the detection of enthesitis, with utility in monitoring response to therapy, and will be an invaluable tool for assessing the efficacy of newer treatments. This review summarizes the substantial progress that has been made in addressing the pathophysiology, molecular mechanisms, genetic associations, clinical features, diagnostic modalities, and treatment of enthesitis. Definitions and evolution of the enthesis concept Historic definition Although the adjective “enthetic” derives from the ancient Greek word “enthetikos,” meaning “introduced into the body from without,” in the nineteenth century the adjective was increasingly used to refer to diseases that were “implanted into the body from external sources” (1). It was not until the twentieth century that the term “enthesis” was used as it is today, referring to focal insertional abnormalities at sites of bony attachments to tendons, ligaments, fascia, muscles, or joint capsules (2,3). The first suggestion that the enthesis is centrally affected in SpA was made by Ball in 1971 and was substantiated after a review of pathologic tissues from both patients with rheumatoid arthritis (RA) and patients with ankylosing spondylitis (AS), where he noted the presence of a unique inflammatory enthesopathy that could help to distinguish SpA from RA (2). Broadening the definition of enthesis with the concept of the “enthesis organ” Magnetic resonance imaging (MRI) and ultrasound findings have suggested that enthesopathy encompasses pathologic changes extending to the adjacent bone and soft tissues (4). Likewise, it has been argued that this entity should be considered an “enthesis organ” encompassing not only the enthesis itself, but also the fibrocartilage, bursa, fat pad, adjacent trabecular bone networks, and deeper fascia (5) (Figure 1). Representing areas where hard and soft tissues meet, entheses are sites of concentrated stress with effects not only on the bony attachment interface and the enthesis itself, but also on these neighboring tissues (4–7). Entheses and mechanical stress The concept of an enthesis organ was extended to that of a synovioentheseal complex (8,9), which refers to the relationship between the proinflammatory synovium and the avascular enthesis. In contrast to other skeletal locations, the enthesis is a site of repetitive biomechanical forces. High biomechanical stress at the enthesis triggers an inflammatory cascade with cytokine production by infiltrating monocytes and lymphocytes in the adjacent synovial tissue, resulting in an articular inflammatory response, and clinically leading to synovitis adjacent to attachment sites (8,9). Support for this theory of a dynamic response to biomechanical stress at the enthesis originates from animal models. In one experiment, botulinum toxin A injection delayed fibrocartilage development, suggesting that enthesis development is sensitive to mechanical environmental factors (10). In a mouse model that overexpresses TNF, enthesitis was reduced when the hind legs of the mice were made non–weight-bearing through tail suspension (11). Those authors proposed that triggering of mechanoreceptors via the MAPK pathway stimulates the production of inflammatory mediators. As a result of biomechanical stress, adjacent bone reacts with formation of surface spurs or enthesophytes, observed both radiographically and on histologic examination (12). In early disease, there is destruction of superficial fibrocartilage, with vascular invasion and inflammatory cell infiltration, predominantly with macrophages (13). This leads to another important microanatomical feature, which is the presence of blood vessels at sites where synovium, subchondral bone, and bone marrow are close to each other. In early experiments using labeled phosphorus, Ball identified capillary-like vessels that pass through the enthesis to the marrow (2). Later studies described the presence of vascular channels penetrating cortical bone in the knees of mice adjacent to the cruciate ligaments with associated subclinical changes, including subchondral bone damage and microcyst formation. In the rat adjuvant-induced arthritis model, vascular channels provided a site for inflammatory tissue entry and osteoclast activation (14). Whether enthesitis is a primary central lesion or a secondary process remains a matter of debate. Studies that have implicated enthesitis as the primary process include studies of TNF-transgenic mice, in which the earliest lesion appears to be in the enthesis (11). However, this may be model specific, and a number of reports have challenged the idea of enthesitis as the primary inflammatory lesion (15,16). In one study examining different stages of spontaneous tail spondylitis and peripheral arthritis in HLA–B27/hβ2m–transgenic mice, histologic samples displayed destructive synovitis with neutrophils and multinucleated giant cells rather than by enthesitis or osteitis (16). Among human studies examining biopsy specimens and MRIs of sacroiliac joints, synovitis and subchondral bone marrow changes were more prominent features while enthesitis was not (17,18). In a subsequent study, in patients with early untreated knee or ankle arthritis, analyses revealed a higher synovitis score by MRI in SpA than in RA, whereas there were no differences in the prevalence of enthesitis as assessed by perientheseal focal tissue, entheseal enhancement, and bone marrow edema (15). However, in light of substantial data in animal models highlighting 3 stages of tendon response to injury that have been defined by distinct pathologic changes, determining the initiating event in the enthesis may be confounded by the timing of the analysis (19–21). Contributing cellular and molecular mechanisms Genetic susceptibility It has long been known that AS susceptibility is largely genetically determined. The strongest genetic association is with the major histocompatibility complex (MHC)–encoded class I molecule, HLA–B27, and it is postulated that HLA–B27 contributes to ~40% of the overall risk for SpA (22). Protein misfolding of nascent HLA–B27 in the endoplasmic reticulum has been hypothesized to trigger an unfolded protein response with aberrant recognition by natural killer cell receptors (23). The HLA–B27–induced unfolded protein response in macrophages has been demonstrated in HLA–B27–transgenic rats and is associated with an increase in IL-23 production by these cells (24). Although HLA–B27 remains the dominant risk factor for susceptibility to the AS phenotype, other important influences of the MHC have been observed (25). More recently, Haroon et al (26) found a positive association of B*27:05:02 with enthesitis, dactylitis, and symmetric sacroiliitis in a cohort of psoriatic arthritis (PsA) patients, whereas B*44 haplotypes were associated with a decreased frequency of enthesitis, dactylitis, and joint fusion. Finally, investigators have recently focused on genes outside of the MHC region, such as ERAP1 and ERAP2, which code for aminopeptidases that are involved in MHC class I presentation (25,27). Although additional HLA class I and class II alleles have also been implicated, the scale and scope of gene identification to date have not yet matched the putative total genetic risk for SpA. Microbial factors Microbial infection with virulent organisms remote from affected joints, as well as gastrointestinal dysbiosis without a directly invading pathogen, are known features of certain phenotypes of SpA, and it has long been appreciated that microbial factors can lead to immune activation (28). Clinically, reactive arthritis (ReA) is known to follow infections with Chlamydia, Campylobacter, Shigella, or Yersinia. AS patients consistently have been found to have subclinical gut inflammation and increased gastrointestinal permeability (29,30). In animal models, HLA–B27–transgenic rats raised in germ-free environments do not develop intestinal inflammatory or peripheral joint disease, yet the disease recurs if rats are reconstituted with Bacteroides, supporting the role of gut flora in the development of joint inflammation (31). In a more recent study, colonoscopic biopsies of the terminal ileum of AS patients showed a discrete microbial signature as revealed by sequencing and quantitative polymerase chain reaction analysis of the 16S ribosomal RNA (16S rRNA) gene, exhibiting higher levels of 5 families of bacteria as compared to healthy controls (32). In that study there was no significant difference in the 16S rRNA copy number between patients with AS and controls, indicating that the observed differences were not due to bacterial overgrowth. It has been postulated that the combination of bacterial adjuvants and mechanical factors act synergistically to activate the immune response, particularly in genetically predisposed individuals (5). Fibrocartilage and versican autoimmunity A number of studies have indicated that autoimmunity against fibrocartilage proteins, including aggrecan, may underlie enthesitis and spondylitis (33). A model of SpA induced by immunizing BALB/c mice with the G1 globular domain of versican, leading to spondylitis and enthesitis, suggests that versican autoimmunity may also play a role in enthesitis (34). The inflammatory lesions are characterized by mononuclear cell infiltration at the entheseal insertions to the vertebrae, as is seen with AS, and are associated with angiogenesis which then progresses to cause destructive discitis (35). Role of bone morphogens In the DBA/1 mouse model, where mice develop spontaneously occurring arthritis that culminates in bone formation and joint ankylosis, male mice in crowded conditions developed arthritis in the hind paws that was entheseal, but not synovially based, with new bone formation driven by BMP-7 signaling (36). In that experiment, the incidence of arthritis was increased in mice that were caged together in crowded conditions, yet decreased when the mice were placed in larger cages (37). Thus, in addition to a genetic predisposition for enthesitis, this observation points to the role of environmental factors in the development of arthritis. Finally, immunohistochemical studies in SpA show increased synovial expression of BMP-2 and BMP-6, which is up-regulated by proinflammatory cytokines such as IL-1 and TNF, suggesting that synovial molecules contribute to chronic arthritis and joint ankylosis (36,38). Role of proinflammatory cytokines The role of IL-23 has been addressed as a major driver of cascades that lead to inflammation and bone remodeling in SpA. Alterations in AS susceptibility are related to the existence of single-nucleotide polymorphisms in the IL-23 receptor as demonstrated in genome-wide association studies, and serum levels of the IL-12/23 p40 subunit have been shown to be significantly higher in patients with PsA compared with controls (39,40). IL-23 is produced in the gut, suggesting that the intestinal mucosa is a key site of IL-23 production in SpA. Additionally, Chlamydia trachomatis also leads to induction of IL-23 via CHOP10. Taken together, these findings indicate that IL-23 is a pivotal cytokine and potentially central to the pathogenesis of SpA (41). Increased IL-17 expression by innate immune cells such as mast cells and neutrophils in SpA has been shown to target the facet joints and synovial tissue (42,43). In a subsequent set of investigations, Sherlock et al found that IL-23 could induce SpA by acting on an isolated population of CD3+CD4−CD8− entheseal resident lymphocytes, leading to increased expression of TNF and IL-6 in the enthesis. When IL-23 was overexpressed, mice developed enthesitis with inflammation, which spread into the adjacent synovium (41). Enthesitis was associated with new bone erosion. IL-23 promoted inflammation through IL-17 and TNF, whereas new bone formation was associated with overproduction of IL-22 (41,44) (Figure 2). Additional support for the role of IL-23 comes from the SKG mouse model, in which curdlan (β-1,3-glucan) injections induce enthesitis and dactylitis. Arthritis and spondylitis were IL-23 dependent and were transferable to SCID mouse recipients with CD4+ T cells (45). In this model, disease severity was dependent on the external microbial environment and the host immunogenetic background. More recent work illustrates the differential impact of microbiota on specific pathologic features of SpA; ileitis development, ileal IL-23 expression, and lymph node IL-17A production were microbiota dependent, but arthritis was not (46). In curdlan-treated SKG mice, enthesitis was specifically dependent on IL-17A and IL-22 (47). The role of up-regulation of the IL-23/Th17 pathway in promoting joint inflammation and bone turnover is further supported by recent murine studies, with inhibition of the PsA phenotype after neutralization of IL-17A (48,49). Clinical enthesitis in SpA SpA is by definition a heterogeneous group of clinical entities long recognized as having unique phenotypes that include AS, ReA, PsA, enteropathic arthritis, and what has traditionally been referred to as undifferentiated arthritis. However, with advances in imaging and careful long-term followup observations, it appears that these diseases share common features, including subclinical spinal and peripheral joint inflammation, along with associations with microbes and gene identifications. In attempting to develop a model for an underlying unifying anatomical basis for SpA, an “enthesitis-based model” has been proposed as the basis for the osteitis, periostitis, and new bone formation that are seen in SpA (5). The association between enthesitis and adjacent osteitis has been further supported by imaging and cadaver studies, primarily in patients with PsA (50–52). Regional sites Patients with SpA have a remarkable propensity for inflammation at certain enthesis sites that are ubiquitous and numerous. Clinically, peripheral enthesitis is observed not only in all forms of SpA, but particularly frequently in juvenile-onset SpA. A number of patients with juvenile SpA are classified as having enthesitis-related arthritis (ERA), a heterogeneous subtype that includes some patients who predominantly have enthesitis, enthesitis and arthritis, or juvenile AS. Compared to other subtypes of juvenile idiopathic arthritis, ERA is associated with worse function, worse quality of life, and increased pain (53,54). Enthesitis can be seen in 33–58% of patients with ReA and may be the only clinical manifestation in some whose disease has been triggered by an enteric infection (55). In SpA, the entheses of the lower extremities are more frequently involved than those of the upper limbs, and the heel is the most frequent site (55). In addition to the Achilles and plantar fascia insertions, identified sites of enthesitis include muscle attachments to the greater and lesser trochanters, the insertion of the quadriceps tendon at the upper patellar pole, the insertions of the patellar ligament at the lower patellar pole and the tibial tubercle, acromial and clavicular insertions of the deltoid muscle, and the insertions of the flexor and extensor tendons at the phalanges (55–57). It is unknown why there is a predilection for the entheses at the lower parts of the lower limbs, although it has been hypothesized that this may be due to the length, anatomy, and higher mechanical load at these sites. Given the presumed role of repetitive biomechanical forces discussed above, it is not surprising that in patients with longstanding AS, those with occupational activities that required more bending, twisting, and stretching had more functional limitations and radiographic damage than those whose jobs required little or no dynamic flexibility (58). A recently published computer-based method that fully quantified syndesmophyte heights and volumes on computed tomography scans has revealed that syndesmophytes grow at different rates over time in AS patients, suggesting that mechanical factors local to the disc space may influence syndesmophyte formation (59). Clearly, there are sites that are not associated with SpA despite being sites of significant biomechanical stress, and perhaps it is the compressive and shear force nature of the stress as well as the putative role of antigen expression adjacent to the enthesis that may underlie this apparent discrepancy (5). Additionally, it cannot be discounted that the increased detection of enthesitis at the lower limbs is explained by the accessibility of these sites to ultrasound. Diagnostic criteria and outcome measures Enthesitis is often underdiagnosed in the clinic; clinical assessment and quantification of peripheral enthesitis in daily practice lacks sensitivity and specificity (56,60,61). Although both the Amor criteria (62) and the European Spondylarthropathy Study Group criteria (63) for SpA include peripheral enthesitis, there are limitations to these criteria with regard to the exact quantification of enthesitis. Two clinical methods have been designed and often implemented for evaluating enthesopathy in AS: Mander’s Entheseal Index and the Maastricht Ankylosing Spondylitis Enthesitis Score (MASES) (64,65). Both rely on pain elicited by local pressure of entheseal points. The intraarticular and deep location of entheseal insertions, however, makes quantification of enthesitis by physical examination alone difficult, and not surprisingly, these scoring systems have only moderate sensitivity and specificity for predicting positive sonogram results, depending on the entheseal site (66). Imaging of the enthesis Because of the clinical limitations described above and the poor sensitivity of markers of inflammation, it is necessary to rely on typical abnormalities seen on various imaging techniques to diagnose SpA. Plain radiographs are limited by their inability to show inflammation or soft tissue changes, although late chronic bony changes such as enthesophyte formation or occasional erosions can be seen at the attachment of the Achilles tendon or plantar aponeurosis. More sensitive methods such as ultrasound and MRI, which are useful in their ability to detect both inflammatory and chronic changes in enthesitis at both early and late stages, can be used. MRI MRI has changed the way we approach both the diagnosis and classification of SpA; it is particularly useful in detecting spinal disease in early AS when conventional radiographs are still normal (67). The use of fat-suppressed, fat-saturated, and water-sensitive MRI sequences has demonstrated that the extracapsular inflammation of joints quite often represents enthesitis with variable degrees of soft tissue and bone marrow edema (68,69) (Figures 3–5). The typical appearance of enthesitis on MRI includes soft tissue inflammatory changes outside the joint capsule and perientheseal bone marrow edema (70). Recent studies have examined the utility of whole-body MRI, which has shown promise in the detection of subclinical axial and peripheral enthesitis (71). Of course, MRI has limitations; structures that make up entheses have a low signal on conventional MRI, with low water accumulation in the areas where fibroblasts are tightly cross-linked. MRI is further limited by its cost and availability, and therefore ultrasound remains the preferred modality for the detection of enthesitis both in the clinical setting as well as in research. Ultrasound Ultrasound has indeed proven to be a highly useful and sensitive tool in the evaluation of enthesitis and improves the ability of the clinical examination to detect enthesopathy. In one study of 92 patients with PsA, ultrasound was useful in detecting subclinical entheseal involvement, independent of clinical examination and symptoms (72). In another study of 600 lower limb entheses, at least 1 ultrasound sign of enthesopathy was detected in 60% of clinically asymptomatic cases of enthesitis, thus demonstrating a higher sensitivity than physical examination (73). Ultrasound may be most useful in the early diagnosis of SpA, and likewise, entheseal abnormalities can be detected prior to overt clinical disease. Nevertheless, in an older cross-sectional single-center study of 51 SpA patients and 24 controls, neither MRI nor power Doppler ultrasound (PDUS) discriminated between SpA and controls (74). In a prospective single-center cohort study of 118 patients with symptoms suggestive of SpA conducted by D’Agostino and colleagues (57), vascularization at cortical bone detected by PDUS of at least one enthesis provided good predictive value for diagnosing SpA with a sensitivity of 76.5% and a specificity of 81.3%. Indeed, PDUS is a sensitive and reliable technique used to detect increased blood flow in the enthesis revealing neovascularity and subclinical active inflammation (56,75) (Figure 6). Recent studies have indicated that ultrasound may accurately predict which patients will go on to develop SpA (51,57,75). In one investigation, ultrasound examination of Achilles erosions correlated with objective activity-based measurements of SpA outcomes, and was sensitive to change (76). In the study by D’Agostino and colleagues described above, vascularized enthesis as detected by PDUS combined with Amor’s criteria proved to be the only independent contributors to a diagnosis of SpA (57). Finally, ultrasound may be used to monitor response to therapeutic interventions. A few studies have illustrated improvement in enthesitis shown on ultrasound after the use of TNF antagonists (77,78). In one investigation of 327 patients with active SpA who were treated with anti-TNF therapy for 6 months, cumulative entheseal morphologic abnormalities, intraenthesis and perienthesis, and bursitis were all significantly decreased on PDUS after 6 months of treatment (77). In another study, D’Agostino et al monitored regression of enthesitis using PDUS after treatment with infliximab (78), providing confirmatory evidence for the utility of ultrasound in a clinical research setting. Treatment of enthesitis Historically, treatment of clinical enthesitis had been limited to NSAIDs. Continuous use of NSAIDs not only controls symptoms of disease, but may also slow progression of bony changes in AS (79,80). Therefore, Assessment of SpondyloArthritis international Society/European League Against Rheumatism guidelines place optimal NSAID therapy as a cornerstone of the management plan for AS (81). Treatment with TNF inhibitors is indicated in patients that do not respond to NSAID therapy. TNF inhibition with adalimumab, etanercept, infliximab, and golimumab has been shown to be efficacious in the treatment of enthesitis (82–87). Olivieri et al (88) have reported that adalimumab and etanercept are effective treatments of MRI-documented refractory heel enthesitis, with progressive improvement of bone edema in a 6-month period (88). Agents that block IL-23 have the potential to inhibit both inflammation and altered bone remodeling, although further analysis of the effect of IL-22 and IL-23 blockade on bone pathologies in animal models and patients with PsA are needed to address this important therapeutic issue (89). Entheseal inflammation in a passive-transfer model of collagen antibody-induced arthritis was reduced by an antibody to the p19 subunit of IL-23, which was also associated with the down-regulation of several inflammatory mediators, such as IL-6 and IL-1β, and genes such as Rankl, Ctsk, and matrix metalloproteinases known to be involved in bone erosion (41). Both ustekinumab, a monoclonal antibody directed against the common p40 subunit of IL-12 and IL-23, and secukinumab, a human anti–IL-17A monoclonal antibody, have already demonstrated promise in PsA, with significant improvements in enthesitis (90,91). Apremilast, an oral inhibitor of phosphodiesterase 4, which increases cAMP and thus modulates multiple proinflammatory mediators, has demonstrated efficacy in PsA, with significant improvements in the severity of both enthesitis and dactylitis evidenced by reductions in MASES over a 52-week period (92). Finally, bisphosphonates may also have a role in peripheral enthesitis felt to be refractory to NSAID therapy. In a 6-month randomized controlled comparison of intravenous pamidronate treatment of NSAID-refractory AS, patients treated with pamidronate showed symptomatic improvement with significant reductions in Bath Ankylosing Spondylitis Functional Index and Bath Ankylosing Spondylitis Disease Activity Index measurements together with regression of periarticular osteitis documented by MRI with gadolinium (93). Treatment of patients with SpA enthesitis with currently available agents has not had universal success. In placebo-controlled trials of methotrexate and leflunomide in PsA, enthesitis measures were not assessed (94,95). In a randomized controlled trial, sulfasalazine was not effective for enthesitis (96). Other agents that have not demonstrated clinical efficacy in AS include tocilizumab, and lymphocyte-targeted therapies such as abatacept (97,98). Rituximab only showed modest therapeutic efficacy in SpA (99,100). Conclusions In summary, investigations and clinical observations uniformly point out with increasing clarity that the enthesis is much more than a simple attachment site. A number of studies have shown that it functions as a unit comprising adjacent tissues, including bone and fibro-cartilage linked to synovium, and serves as a way of dissipating stress over a wide area. Inflammation at the enthesis manifests in the adjacent synovium presumably via immunity to common antigens or via release of proinflammatory cytokines at the enthesis. Although work by Benjamin and McGonagle (9) suggests that the enthesis is the primary SpA lesion, the precise role of the enthesis in early stages of disease, especially regarding issues of cause or effect, remains an area of continued debate and discovery. Improved imaging modalities may in the future be able to detect enthesitis at different stages of disease. However, this will require a clinically diverse and large sample size to help address this question. Inflammation at the enthesis is likely modulated by multiple factors. A more complete role for genetic predisposition will require additional advances in gene sequencing and discovery. Repeated biomechanical stress with the resultant inflammatory response regulated by IL-17, IL-22, and IL-23 now provide clues as to why certain areas of the body are affected, and perhaps why others are not. The spine itself (the clinical hallmark of the disease) remains inaccessible to traditional enthesitis-focused research methodologies thus far. However, newer imaging techniques are on the horizon. Further examination into the role of the inflammatory mediators, including IL-17, IL-22, and IL-23 as well as potentially others, in driving enthesitis and bone formation will be important to direct our attention toward future therapeutic targeted pathways in patients with SpA. ​ Acknowledgments Supported in part by the NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases grant P01-AR-052915 and National Center for Advancing Translational Sciences grant UL1-TR-000124). The authors wish to thank Joseph Robinson, MD (Cedars-Sinai Medical Center Department of Radiology) for assistance with MRI acquisition and interpretation. 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JOURNAL ARTICLE Establishment of anti-C1q monoclonal antibodies to measure serum C1q levels discriminating disease severity subsets of rheumatoid arthritis within 5 years of onset Chikako Yukioka, Kosuke Ebina, Yasunori Shimaoka, Masao Yukioka, Hideki Yoshikawa, Ken Nakata, Takahiro Ochi Modern Rheumatology 2019 September 19, : 1-5 Objectives: To establish anti-C1q monoclonal antibodies which can measure serum C1q levels discriminating disease severity subsets of rheumatoid arthritis (RA) within 5 years of onset. Methods: In this multi-centre, longitudinal, observational study, 122 RA patients [102 females, baseline age 58.5 years, rheumatoid factor (RF) positivity 78.7%, serum C-reactive protein (CRP) 1.2 mg/dl, and concomitant methotrexate (MTX) 4.9 mg/week (29.5%)] within 5 years of onset (disease duration 21.0 months) were enrolled from 1985 to 2000. Patients were not treated by more than 8 mg/week of MTX or biologics which may strongly affect the course of joint destruction. Disease severity at 10-15 years of onset was classified according to the number of destructed joints of overall 68 joints on plain radiographs (36 patients were mild RA group involving only peripheral joints and 86 were severe RA group involving large axial joints). Baseline serum C1q levels were evaluated by ELISA with newly developed 4 monoclonal anti-C1q antibodies, and compared between two groups as well as conventional RA disease activity markers. Results: There were no significant differences between two groups in baseline conventional RA disease activity markers such as RF, erythrocyte sedimentation rate, CRP, and matrix metalloproteinase-3. However, compared to mild RA group, severe RA group showed higher baseline serum C1q levels (μg/ml) evaluated by anti-C1q monoclonal antibodies of no.33 (104.8 ± 22.3 vs. 118.3 ± 19.3; p  = .0024), no. 40 (102.6 ± 21.9 vs 121.2 ± 22.3; p  = .000069), no. 54 (102.1 ± 22.5 vs. 119.3 ± 26.9; p  = .00052), and no. 76 (105.6 ± 21.8 vs. 122.6 ± 26.4; p  = .00043). Receiver operating characteristic curve analysis revealed that in patients with serum C1q levels of ≥110.5 μg/ml (measured by antibody no. 40), 78.9% (75/95) belonged to severe RA group. Conclusion: Measuring serum C1q levels of RA within 5 years of onset by newly developed anti-C1q antibodies may be useful in predicting the prognosis of disease severity evaluated by the extent of joint destruction. Discussion You are not logged in. Sign Up or Log In to join the discussion. 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Abstract Background Synovial membrane inflammation is common in osteoarthritis (OA) and increases cartilage injury. However, synovial fluid and histology studies suggest that OA inflammatory respon...

Mast cells are present in limited numbers in normal human synovium, but in rheumatoid arthritis and other inflammatory joint diseases this population can expand to constitute 5% or more of all synovial cells.

Abstract Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1β. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum–Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

To identify a synovial fluid (SF) biomarker profile characteristic of individuals with an inflammatory osteoarthritis (OA) endotype. A total of 48 knees (of 25 participants) were characterized for an extensive array of SF biomarkers quantified by Rules Based Medicine using the high-sensitivity...

Despite ground-breaking clinical success in the treatment of different cancers, immune checkpoint inhibitors can cause profound inflammatory and immune-related adverse events. Autoimmune inflammatory arthritis following immune checkpoint inhibitor treatment has been reported; however, to date, no...

Cellular signatures define pathobiological endotypes in early rheumatoid arthritis, informing prognosis and treatment.

The aim of this global collaboration was to develop a consensual set of items for the analysis of synovial biopsies in clinical practice and translational research through the EULAR Synovitis Study Group (ESSG) and OMERACT Synovial Tissue Biopsy Group.

We aimed to explore the accuracy of physical examination (PE) to detect the synovial and extra-synovial pathologies in psoriatic arthritis (PsA) in comparison to ultrasonography (US). Twenty-nine PsA patients with hand pain were included in the study.

Via Krishan Maggon

The ontogeny of macrophages in most organs has already been established. Owing to the limited number and inaccessibility of synovial macrophages (SMs), the origin of SMs has not been fully elucidated.

Via Krishan Maggon

Objective Genetic associations imply a role for CD8+ T cells and the interleukin‐23 (IL‐23)/IL‐17 axis in psoriatic arthritis (PsA) and other spondyloarthritides (SpA). IL‐17A+CD8+ (Tc17) T cells a...

Via Krishan Maggon

KEY POINTS TCR-γδ tetramer identified ligand expression by flow cytometry. TCR-γδ ligands were induced on activated monocytes or T cells. Bioinformatics combined with mass spectrometry produced an overlapping list of 16 candidate ligands. Visual Abstract Abstract Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1β. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum–Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression. This article is featured in In This Issue, p.2353 Introduction Full understanding of γδ T cell biology has been handicapped by ignorance of the ligands for most TCR-γδ. γδ T cells reside at mucosal and epithelial barriers and often accumulate at sites of inflammation with autoimmunity, infections, or tumors (1). Evidence suggests that γδ T cells provide protection against infections with bacteria, viruses, and protozoans and are generally beneficial in autoimmunity (1–17). In addition, a role for γδ T cells in the immune response against tumors in humans is evident from a seminal study reporting that intratumoral γδ T cells are the most favorable prognostic immune population across 39 cancer types in humans (18). γδ T cells are often highly lytic against transformed proliferative cells, infected cells, and infiltrating CD4+ T cells in inflammatory arthritis (9, 17, 19). They can produce a variety of cytokines including IFN-γ, TNF-α, and IL-17 (20), as well as insulin-like growth factor-1 (IGF1) and keratinocyte growth factor (KGF) that promote epithelial wound repair (21). These collective studies indicate that a principal function of γδ T cells is in response to tissue injury of various causes. It is, thus, not surprising that γδ T cells are often suggested to react to host components that are upregulated or exposed during proliferation or cell injury (22). As such, γδ T cells may function in tissue homeostasis and immunoregulation as much as in protection from infection. Yet in the vast majority of cases, little if anything is known regarding the nature of these self-components or whether they actually engage the TCR-γδ. Whereas αβ T cells recognize proteins that are processed into peptides and presented on MHC molecules, the few proposed ligands for γδ T cells suggest that they recognize mostly intact proteins directly, without MHC restriction. This makes them highly attractive for immunotherapy. Despite the elaborate mechanisms that αβ T cells and B cells use to prevent autoreactivity, γδ T cells have been frequently reported to respond to autologous proteins. Furthermore, in contrast to other lymphocytes that maximize the potential diversity of their receptors, γδ T cells frequently show limitations in their diversity. Thus, human γδ T cells comprise a subset of Vδ2 T cells, the predominant γδ in peripheral blood that respond to prenyl phosphates and certain alkyl amines (23–25), and Vδ1 T cells, which do not respond to these compounds and often accumulate at epithelial barriers and sites of inflammation (1). A similar limited repertoire occurs in the mouse in which Vγ5Vδ1 cells colonize the epidermis, and a Vγ6Vδ1 subset colonizes the tongue, lung, and female reproductive tract (21, 26). This restricted repertoire implies that TCR-γδ ligands may also be limited. This may provide for a more rapid response and perhaps explain why, in contrast to αβ T cells and B cells, it is difficult to generate Ag-specific γδ T cells by immunization with a defined Ag. Various ligands for γδ T cells have been proposed, although only a few have been confirmed to bind to TCR-γδ, and these lack any obvious similarity in structure. γδ T cells for which ligands have been identified include the murine γδ T cell clone G8, which recognizes the MHC class I–like molecules T10 and T22 (27), γδ T cells from mice infected with HSV that recognize herpes glycoprotein gl (28), a subset of murine and human γδ T cells that bind the algae protein PE (20), a human γδ T cell clone G115 that recognizes ATP synthase complexed with ApoA-1 (28), a human γδ T cell clone (Vγ4Vδ5) from a CMV-infected transplant patient that recognizes endothelial protein C receptor (EPCR) (29), and some human Vδ1 T cells that recognize CD1d-sulfatide Ags (30). However, to date no systematic process has been reported for determining the spectrum of human TCR-γδ ligands. To provide an unbiased approach for the identification of candidate ligands for human γδ T cells, we produced a biotinylatable form of a soluble TCR-γδ (sTCR-γδ) from a synovial Vδ1 γδ T cell clone of a Lyme arthritis patient. The tetramerized sTCR-γδ was used in flow cytometry to identify various cell types that expressed candidate ligands. Initial analysis of 24 tumor cell lines identified a set of nine ligand-positive tumors, enriched for those of epithelial and fibroblast origin, and 15 ligand-negative tumors, largely of hematopoietic origin. In addition, ligand was not expressed by primary monocytes or T cells, although each could be induced to express ligand following their activation. Ligand expression was sensitive to trypsin digestion, revealing the protein nature of the ligands, and was also reduced by inhibition of glycolysis. These findings provide a framework and strategy for the identification of individual ligands for human synovial γδ T cells. Materials and Methods Production of a sTCR-γδ Human synovial γδ T cell clones from a Lyme arthritis patient were produced as previously described (9, 31). One of these clones, Bb15, was chosen for production of the sTCR-γδ using modification of a previously reported procedure (32, 33). Both TCR chains were produced as a single transcript in a baculovirus vector. The pBACp10pH vector used contains two back-to-back promoters, p10 and polyhedrin (Fig. 1A). The p10 promoter is followed by multiple cloning sites for the γ-chain, and the polyhedrin promoter is followed by multiple cloning sites for the δ-chain. Downstream of the γ-chain, we placed a hexa-His tag for nickel column purification, followed by a biotinylation sequence for tetramerization. The γ-chain and δ-chain were PCR amplified using high fidelity polymerase (Deep Vent Polymerase; New England Biolabs). Both TCR chain sequences were verified following the initial PCR amplification as well as after insertion into the pBACp10pH vector. Virus encoding the sTCR-γδ was generated by cotransfection of Sf21 moth cells using the Sapphire baculovirus DNA and Transfection kit (Orbigen) with the sTCR pBACp10pH construct. Virus was harvested 6 d later and used as primary stocks (P1 stock). Two additional rounds of viral amplification, P2 and P3, were completed using midlog phase Sf21 cells (∼1.6 × 106 cells/ml) allowed to adhere for 1 h before infecting at a multiplicity of infection of 0.01 or 0.1 with P1 and P2 stock, respectively. After 72 h of infection, culture medium was clarified by centrifugation (1000 × g for 10 min) and filtration (VacuCap 90PF 0.8/0.2 μm Supor membrane filter units; Pall, Westborough, MA) before storing in the dark at 4°C until use. Protein production occurred in 12-l batches of midlog phase (∼1.6 × 106 cells/ml) Hi5 cells growing in suspension (0.5 l of culture in 1 l spinner flasks) and infected with P3 stock at a 1:50 dilution. Following 72 h of infection, cells were removed by centrifugation and filtration as described above. The filtered supernatant (∼12 l) containing secreted sTCR-γδ was concentrated to ∼100 ml before dialyzing against 1 l of nickel column loading buffer (20 mM NaPhosphate buffer, pH 7.4, 20 mM imidazole, 0.5 M NaCl) using a Pellicon diafiltration system with two 10K MWCO membranes (MilliporeSigma, Burlington, MA) back down to ∼100 ml. After system flushing, the final sample volume was ∼200 ml. It was then loaded onto loading buffer–equilibrated His-Trap HP columns (GE Healthcare, Little Chalfont, U.K.) at 100 ml per 2 × 5 ml columns. Columns were washed with at least 10 column volumes of loading buffer until baseline absorption was achieved. Bound proteins were eluted using a gradient from 20 to 500 mM imidiazole over 20 column volumes. Elution was monitored by absorbance at 280 nM, and 1 ml fractions were collected. Fractions containing the target protein were identified using SDS-PAGE gel analysis using Coomassie Blue. High purity (>95%) sTCR-γδ fractions were pooled, dialyzed against PBS (pH 7.4), and frozen at −80°C until used in future studies. Yields were typically ∼1.0–2.5 mg/l of culture. Purified sTCR-γδ was then biotinylated using a biotin-protein ligase system (Avidity) and tetramerized with streptavidin-PE (BioLegend) for FACS staining. Verification of TCR-γδ protein was confirmed by SDS-PAGE gel analysis using Coomassie Blue as well as immunoblot using Abs to Vδ1 or Cγ (Endogen). Flow cytometry Cells were stained with either sTCR-γδ-PE (10 μg/ml) or negative controls that included streptavidin-PE (10 μg/ml), IgG-PE (10 μg/ml) (BioLegend), or a sTCRαβ-PE (a kind gift of Dr. M. Davis). Additional surface staining of T cells consisted of CD4, CD8, CD19, and CD25 (BioLegend). Live–Dead staining (BD Bioscience) was used to eliminate dead cells from analysis. Samples were run on an LSRII flow cytometer (Becton Dickinson). Purification and activation of human monocytes and T cells and cell lines Human monocytes were purified from human PBMC using CD14-labeled magnetic beads, followed by column purification (Miltenyi Biotec) and then cultured in RPMI complete medium with 10% FCS in the absence or presence of either a Borrelia burgdorferi sonicate (10 μg/ml) or LPS (1 μg/ml; Sigma-Aldrich) for 18 h. To some cultures were added TNF-α (10 ng/ml) (BioLegend), anti-TNF-α (10 μg/ml) (BioLegend), IL-1β (10 pg/ml) (Invitrogen), or anti–IL-1β (5 μg/ml) (R&D Systems). Cells were then stained with the sTCR-γδ tetramer. T cells from PBMC were either used fresh or were activated with anti-CD3/anti-CD28 (each 10 μg/ml; BioLegend) + IL-2 (50 U/ml; Cetus) and propagated for 3 d. Cells were then stained with the sTCR-γδ tetramer. Human PBMC were obtained using an approved protocol from The University of Vermont Human Studies Committee. Verified cell lines were obtained from American Type Culture Collection. CHO cells deficient for glycosaminoglycans (GAGs) were derived as previously described (34). Bioinformatics analysis Expression profiling (35) based on Illumina RNAseq technology (36) was used to characterize the transcriptomes of 22 of the 24 tumor cell lines examined (excluding bronchoepithelial cell line and 2fTGH). Expression data for all known genes (37) were generated, and those genes whose representation in tetramer-positive cell lines was significantly higher than in negative cell lines were considered as candidate ligands. Mass spectrometry analysis Biotinylated sTCR-γδ was bound to avidin magnetic beads and then incubated with cell lysates from monocytes activated with B. burgdorferi sonicate. Magnetic beads alone, without TCR-γδ tetramer, with monocyte lysates served as a negative control. After 4 h, beads were washed five times, and bound proteins were then separated on polyacrylamide gels. Gel lanes for each sample type were cut into 12 identical regions and diced into 1-mm cubes. In-gel tryptic digestion was conducted on each region as previously described (38). Extracted peptides were subjected to liquid chromatography tandem mass spectrometry (38), except that the analysis was performed using an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Tandem mass spectra were searched against the forward and reverse concatenated human IPI database using SEQUEST, requiring fully tryptic peptides, allowing a mass tolerance of 2 Da and mass additions of 16 Da for the oxidation of methionine and 71 Da for the addition of acrylamide to cysteine. SEQUEST matches in the first position were then filtered by XCorr scores of 1.8, 2, and 2.7 for singly, doubly, and triply charged ions, respectively. Protein matches made with more than two unique peptides were further considered. This list had a peptide false discovery rate of <0.01%. Inhibition of glycolysis, transcription, translation, and endoplasmic reticulum–Golgi transport or trypsin or heparinases I–III treatment Inhibition of glycolysis was performed using the 2-deoxyglucose (2-DG, 5 mM; Sigma-Aldrich) for 48 h. Transcription and translation were inhibited using, respectively, actinomycin D (5 μg/ml; ICN Biomedicals) or cycloheximide (1 μg/ml; MilliporeSigma) for 18 h. Endoplasmic reticulum (ER)–Golgi transport was blocked using brefeldin A (1:1000) or monensin (1:1400) (BD Bioscience) for 18 h. Cell surface protein digestion was performed using trypsin (Invitrogen) (1×; 5–10 min, 37°C.). GAGs were removed from cells by treatment with heparinases I–III (2 μU/ml) for 30 min in RPMI 1640 with no serum. The reaction was then stopped by the addition of PBS–BSA. Statistical analysis The following statistical tests were used: unpaired Student t test when comparing two conditions, and one-way ANOVA with Sidak test for correction for multiple comparisons when comparing multiple variables across multiple conditions. Results Production of a human synovial sTCR-γδ We previously produced a panel of synovial Vδ1 γδ T cells from Lyme arthritis patients (9, 31). A representative clone, Bb15 (Vδ1Vγ9), was selected from which to clone its TCR-γδ. The pBACp10pH vector has been used previously to produce murine sTCR-γδ tetramers (33). It contains two back-to-back promoters, p10 and polyhedrin, in which the p10 promoter is followed by multiple cloning sites for inserting the γ-chain, and the polyhedrin promoter is followed by multiple cloning sites for inserting the δ-chain (Fig. 1A). Downstream of the γ-chain we placed a hexa-His tag for purification, followed by a biotinylation BRP sequence for tetramerization with streptavidin-PE. Protein production was undertaken in Hi5 cells followed by purification using His-Trap HP columns. Fractions were analyzed by SDS-PAGE, and those with protein of the correct size were pooled, with yields typically of 1–2 mg/l of culture. A sample sTCR-γδ preparation is shown in Fig. 1B, stained with Coomassie Blue, showing bands of the expected size for the heterodimer under nonreducing (59 kDa) and reducing conditions (30/28 kDa for the γ- and δ-chains, respectively). The protein was stained by immunoblot with Abs to either Vδ1 or Cγ (Fig. 1C) and also blocked anti-γδ Ab staining of the synovial γδ T cell clones (Fig. 1D). The purified sTCR-γδ was then biotinylated and tetramerized with streptavidin-PE for use by flow cytometry. As an additional measure of specificity, sTCR-γδ tetramer staining of a fibrosarcoma tumor cell line (2fTGH) could be inhibited by anti-γδ Ab but not control IgG (Fig. 1E). Finally, staining of 2fTGH cells with the sTCR-γδ tetramer was dose dependent but did not increase with increasing dose on a negative tumor line, Daudi (Fig. 1F). FIGURE 1. Production of human synovial sTCR-γδ. (A) pBACp10pH vector containing the δ-chain driven by the polyhedrin promoter and the γ-chain with hexa-His and biotinylation BRP sequences driven by the p10 promoter from γδ T cell clone Bb15 (Vγ9Vδ1). (B) Sample of nickel NTA column-purified sTCR-γδ analyzed by SDS-PAGE under reducing and nonreducing conditions, and stained with Coomassie Blue. (C) Immunoblot of sTCR-γδ stained with anti-Vδ1 or anti-Cγ. (D) γδ T cell clone Bb15 was stained with anti–TCR-γδ Ab in the absence or presence of competing sTCR-γδ. (E) The fibrosarcoma cell line 2fTGH was stained with the sTCR-γδ in the absence or presence of the indicated concentrations of anti-γδ Ab or control IgG. (F) Titration of sTCR-γδ staining of the positively staining tumor line 2fTGH or negatively staining line Daudi. Number inserts indicate percent positively staining cells. Findings are representative of three experiments. Expression of sTCR-γδ candidate ligand(s) varies among cell lines We initially used the sTCR-γδ tetramer to screen a panel of 24 cell lines from a variety of cell types. None of the cell lines stained with the negative controls (IgG-PE, avidin-PE, or sTCR-αβ tetramer-PE), but the sTCR-γδ tetramer gave a spectrum of staining in which nine cell lines were strongly positive and the other cell lines manifested low to undetectable surface staining (Fig. 2). Of interest was that the positive group was enriched for cell lines of epithelial and fibroblast origin, cell types known to exist where γδ T cells are often found, such as skin, intestines, and synovium. With this information, expression profiling (35) using available RNAseq was used to characterize the transcriptomes of 22 of the 24 tumor cell lines (RNAseq on the bronchoepithelial and 2fTGH were not available). Expression data for all known genes (37) were generated, and those genes whose representation in tetramer-positive cell lines was significantly higher than in negative cell lines were considered to be candidate ligands. This produced an initial list of candidate ligands for sTCR-γδ (Supplemental Table I). FIGURE 2. sTCR-γδ tetramer staining of a cell line panel. A panel of 24 diverse cell lines was stained with either sTCR-αβ or sTCR-γδ, gated on live cells, and examined by flow cytometry. Shown are examples of tumors representing either (A) positive staining or (B) negative staining with sTCR-γδ, with the complete list summarized below each example. Number inserts indicate mean fluorescence intensity of entire histogram. Findings are representative of four experiments. Candidate sTCR-γδ ligands are sensitive to trypsin and reduced by inhibition of transcription, translation, ER–Golgi transport, or removal of GAGs We treated the positively staining cell lines with trypsin and noted a complete disappearance of surface staining, as exemplified for bronchoepithelial cells in Fig. 3A. Similar results were observed with two additional tumor lines. This supports the view that the TCR-γδ ligand contains a protein component essential for recognition by the receptor. We also observed no increase in sTCR-γδ tetramer staining of cells (C1R or HeLa) expressing CD1a, b, c, or d, nor with MICA/B (data not shown). Thus, at present there is no evidence that the synovial Vδ1 TCR-γδ ligand is one of these MHC class I–like molecules, at least bound to endogenous molecules from these particular cell lines. FIGURE 3. sTCR-γδ ligand is sensitive to protease, blockers of ER–Golgi transport, translation, or transcription and contains GAGs. The human bronchoepithelial cell line was either untreated or treated with (A) trypsin for 15 min, (B) untreated or treated for 18 h with cycloheximide or actinomycin D, or (C) untreated or treated for 18 h with brefeldin A or monensin. Cells were then stained with sTCR-γδ tetramer. (D) The 2fTGH fibrosarcoma cell line, wild-type CHO cells, or GAG-deficient CHO cells were either untreated or treated with a combination of heparinases I–III for 30 min and then stained with sTCR-γδ tetramer. Number inserts indicate mean fluorescence intensity of entire histogram. Findings are representative of three experiments. We further determined that surface TCR-γδ ligand expression was reduced by inhibition of protein translation or transcription with, respectively, cycloheximide or actinomycin D (Fig. 3B). Surface ligand was also considerably reduced by inhibition of transport from the ER to Golgi using either brefeldin A or monensin (Fig. 3C). This further demonstrated the protein nature of candidate TCR-γδ ligands. Finally, we examined the extent to which GAGs contribute to ligand binding by TCR-γδ. This was tested in two ways. Initially, the ligand-positive fibrosarcoma cell line 2fTGH was either treated or not with heparinases I–III, which removes most GAGs. This considerably reduced sTCR-γδ tetramer staining (Fig. 3D). This was further supported by the observation that sTCR-γδ stained wild-type but not GAG-deficient CHO cells (Fig. 3D). sTCR-γδ ligands are expressed by activated monocytes In considering what primary cells might express ligand(s) for the sTCR-γδ, we first examined fresh monocytes, as we had observed previously that following their activation with B. burgdorferi or LPS, monocytes could activate the synovial γδ T cell clones (31). Consistent with these earlier findings, we observed that the sTCR-γδ tetramer did not stain freshly isolated human monocytes, but following 24 h activation with a sonicate of B. burgdorferi or LPS, there was a robust upregulation of sTCR-γδ tetramer staining (Fig. 4). The same cells did not stain with negative controls that included avidin-PE, IgG-PE, or a human sTCR-αβ tetramer-PE. Because activated monocytes are known to produce certain cytokines, particularly TNF-α and IL-1β, we examined the possible influence of these cytokines on ligand expression. Curiously, the low level of sTCR-γδ tetramer staining of fresh monocytes was reduced further with TNF-α, whereas ligand expression by Borrelia-activated monocytes was not affected by the further addition of TNF-α or blocking anti–TNF-α Ab (Fig. 4B). By contrast, IL-1β increased ligand expression by fresh but not activated monocytes, and blocking anti–IL-1β Ab partially inhibited ligand expression by activated monocytes (Fig. 4C). Thus, sTCR-γδ ligand expression appears to be partly regulated by certain monocyte-derived cytokines. FIGURE 4. TCR-γδ ligand is induced on human monocytes following activation. (A) Freshly isolated monocytes were either unstimulated or activated with B. burgdorferi or LPS for 18 h and then stained with the indicated reagents and analyzed by flow cytometry. (B and C) Fresh monocytes or monocytes activated with Borrelia were incubated in the presence of medium alone or TNF-α or blocking anti-TNF-α (B) or IL-1β or blocking anti–IL-1β (C). Number inserts indicate percent positively staining cells. Error bars represent SEM. Findings are representative of four experiments. Given the induction of sTCR-γδ ligand expression by activated monocytes, we prepared lysates from Borrelia-activated monocytes and then used the biotinylated sTCR-γδ complexed with avidin magnetic beads as a bait. Following incubation with the monocyte lysates, the sTCR-γδ was isolated by magnetic purification and washed five times; bound proteins were separated on polyacrylamide gels, and gel slices were subjected to trypsin digestion and analyzed by mass spectrometry. Avidin magnetic beads alone incubated with monocyte lysates served as a negative control. This analysis yielded 291 unique proteins (Supplemental Table II). When compared with the list produced by the RNAseq bioinformatics approach of the tumor lines, 16 proteins were found in common (Supplemental Table III). Of interest is that two of these, Annexin A2 and heat shock protein 70, have previously been proposed as γδ ligands (39–41). sTCR-γδ ligands are expressed by activated T cells We further analyzed freshly isolated PBL from three individuals of various ages (28–66). This consistently revealed that fresh CD8+ T cells exhibited negligible sTCR-γδ staining, whereas a subset of fresh CD4+ T cells manifested modest levels of sTCR-γδ staining (Fig. 5A). In contrast to the freshly isolated T cells, following 3 d activation with anti-CD3/CD28 + IL-2, we observed that a subset of both CD4+ and CD8+ T cells now displayed high levels of sTCR-γδ staining (Fig. 5B). Both the proportion of cells expressing ligand and the density was higher on activated CD4+ T cells compared with CD8+ T cells. Given that in vitro–activated proliferating T cells express sTCR-γδ ligand, we considered that the subset of fresh CD4+ T cells expressing ligand might also represent a proliferative subset. One of the most rapidly proliferative T cell subsets in vivo is T regulatory cells (Treg) (42). Treg can be identified as a subset of fresh CD4+ T cells expressing CD25. Indeed, when we subset fresh human CD4+ T cells based on CD25 expression, sTCR-γδ tetramer staining was again observed preferentially by the CD25+ subset (Fig. 5C). FIGURE 5. sTCR-γδ tetramer stains a subset of activated human T cells and Treg. PBL were stained with Abs to CD4 and CD8 as well as with sTCR-αβ tetramer-PE or sTCR-γδ tetramer-PE either (A) freshly isolated or (B) 3 d after activation with anti-CD3/CD28 + IL-2. Number inserts indicate the percentages of T cells staining negatively or positively with sTCR-γδ tetramer, as a portion of the total CD4+ or CD8+ subsets, as well as mean fluorescence intensity (MFI) in some cases. Findings are representative of six experiments. (C) Freshly isolated PBL were stained with anti-CD4, anti-CD25 or isotype control, and streptavidin-PE (SA-PE) or sTCR-γδ-PE. Shown are cells gated on CD4 expression. Number inserts indicate MFI of sTCR-γδ-PE staining for CD25+ and CD25− subsets. Findings are representative of two experiments. TCR-γδ ligand expression is partly dependent upon glycolysis The finding that fresh monocytes and T lymphocytes expressed low to negligible levels of sTCR-γδ ligand(s), but upregulated expression following activation, raised the possibility that this might reflect the known induction of glycolysis following activation of T cells, monocytes, or dendritic cells (43, 44) and the resultant synthetic capacity promoted by glycolysis (45). This notion is supported by the fact that ligand-expressing Treg are also highly glycolytic (42). We thus examined this question in two ways. First, we exposed activated T cells to 2-DG, an inhibitor of glycolysis. This reduced expression of both CD25 and sTCR-γδ ligand (Fig. 6A). Second, we distinguished between activated T cells on day 3 based on their expression of CD25, as this identifies cells responsive to IL-2 and are hence most glycolytic (45). As shown in Fig. 6B, CD25+ T cells expressed sTCR-γδ ligand whereas the CD25− subset was devoid of ligand expression. Of further note is that within the CD25+ subset, CD4+ T cells again expressed more ligand than CD8+ T cells (Fig. 6B). We extended this analysis to the ligand-positive tumor 2fTGH and observed that 2-DG also resulted in reduced ligand expression in these cells (Fig. 6C). FIGURE 6. TCR-γδ ligand expression parallels glycolysis. (A and B) PBL were activated with anti-CD3/CD28 + IL-2 in the absence or presence of 2-DG (5 mM). On day 3, cells were stained with Abs to CD4, CD8, CD25, and sTCR-γδ tetramer-PE. Shown in (A) are the levels of CD25 and TCR-γδ ligand without or with 2-DG. Shown in (B) is the expression of TCR-γδ ligand in CD4+ or CD8+ subsets based on surface CD25. (C) 2fTGH cells were cultured for 48 h in either regular medium or medium plus 2-DG (5 mM). Cells were then stained with TCR-αβ or TCR-γδ. Number inserts indicate mean fluorescence intensity (MFI) of sTCR-γδ-PE staining. Findings are representative of three experiments. Discussion To our knowledge, the current findings provide the first unbiased characterization of the spectrum of ligand expression for human synovial Vδ1 γδ T cells. The range of ligand expression may reflect the various locations and seemingly diverse functions attributed to γδ T cells. For example, ligand induction by B. burgdorferi– or LPS-activated monocytes parallels their known ability to activate synovial γδ T cell clones (9, 31). In addition, ligand expression by fresh CD4+ but not CD8+ T cells also correlates with our previous observations that Lyme arthritis synovial γδ T cells suppress by cytolysis the expansion of synovial CD4+ but not CD8+ T cells in response to B. burgdorferi (9). Finally, defining the spectrum of tumor cell types that express TCR-Vδ1 ligands may help explain which tumors contain Vδ1 γδ T cells and impact their effectiveness as immunotherapy. The collective findings are also most consistent with the view that γδ T cells respond to self-proteins as much as or possibly more than foreign proteins. Although these results were obtained using a sTCR-γδ tetramer from a single synovial γδ T cell clone, the fact that it shares a common Vδ1 chain found on most synovial γδ T cells (9), as well as γδ T cells found in intestinal epithelium (1, 10, 21), several tumors (18), and cells expanded in PBL following certain infections such as HIV (46, 47) and CMV (29), suggests the possibility that Vδ1 γδ T cells from these other sources may share a common physiology of ligand expression. Previous studies of ligands for murine and human γδ T cells have come largely from the identification of individual molecules that activate a specific γδ T cell clone (27–30). Although this has been successful in some instances, the current study applied a broader approach of using a sTCR-γδ tetramer in an unbiased fashion to identify the spectrum of ligand expression and how they are regulated. This approach also provided two independent methods by which to identify candidate ligands. One method used RNAseq transcriptome analysis from 22 tumor cell lines to match genes increased in positively staining tumors and decreased in negatively staining tumors. The second approach used the sTCR-γδ tetramer as a bait to bind ligands from lysates of activated monocytes and then identify the bound proteins by mass spectrometry. It is of considerable intertest that among these two sets of candidate ligands were 16 in common, two of which, Annexin A2 and heat shock protein 70, have been previously proposed as ligands for γδ T cells (39–41). By contrast, surface sTCR-γδ tetramer binding was eliminated by treatment with trypsin or removal of GAGs, and also suppressed by inhibition of ER–Golgi transport, suggesting the involvement of a combination of protein and GAGs in tetramer binding. Future studies will explore through knockdown and transfection methods whether any of the candidate ligands we have identified activate the original γδ T cell clone and the extent to which GAG/glycoprotein binding may or may not be a confounder. Although the findings thus far have not determined whether there is one or several synovial Vδ1 TCR-γδ ligands, they do provide a framework for understanding the distribution and regulation of ligand expression, which is critical for better understanding of γδ T cell biology. For example, γδ T cells have been implicated in the defense against a variety of infections (2–7), which is consistent with our finding that different TLR agonists induce TCR-γδ ligand expression on monocytes. Similar studies using a murine sTCR-γδ also found ligands induced with bacterial infection (21). In addition, γδ T cells have been found to generally alleviate various autoimmune models (12–15), which may be consistent with the expression of ligand by a subset of activated CD4+ T cells. The induction of TCR-γδ ligand expression by activation of primary monocytes or T cells, as well as ligand expression by a variety of highly proliferative tumor cell lines, suggested that the metabolic state of cells may influence their ability to express TCR-γδ ligands. Activation of monocytes and T cells is known to induce a metabolic switch to glycolysis to provide the synthetic capacity for proliferation (43, 44). In addition, Treg, which are known to be glycolytic in vivo (42), spontaneously expressed ligand. Moreover, most tumors are highly glycolytic, and the inhibition of glycolysis in these cells also reduced ligand expression. Collectively, these findings suggest that some γδ T cells may function to survey and regulate highly proliferative cells. It is of some interest that the cell lines bearing high levels of TCR-γδ ligand expression were enriched for those of epithelial and fibroblast origin, because Vδ1 γδ T cells are typically found at epithelial barriers, such as skin or intestinal epithelium, as well as in inflamed synovium, which is rich in fibroblasts (48). By contrast, sTCR-γδ ligand expression was noticeably absent from most cell lines of hematopoietic origin. The spectrum of cell line staining with the human synovial sTCR-γδ also bears considerable similarity to previous results using a murine sTCR-γδ, which strongly stained epithelial and fibroblast tumors, and less well tumors of hematopoietic origin (33). These same murine sTCR-γδ also stained macrophages activated by TLR2 or TLR4 stimuli, similar to our findings with monocytes activated by Borrelia or LPS (49). Furthermore, staining of macrophages by the murine sTCR-γδ was also not affected by the absence of β2-microgloublin, suggesting little or no contribution of ligand by classical or nonclassical MHC class I molecules. This agrees with our findings that the human synovial sTCR-γδ tetramer staining was not affected by the presence or absence of CD1 or MICA/B molecules. The findings in this study were made using primary cells and tumor cell lines. Future studies will attempt to extend these results to analyses of sTCR-γδ tetramer histologic staining of primary tissues as well as tumors and inflamed synovium to determine the spectrum of TCR-γδ ligand expression at these sites. Screening primary tumors for binding of sTCR-γδ tetramer may also help identify tumors that may benefit from immunotherapy with Vδ1 γδ T cells. In addition, identifying the ligands in inflamed synovium or intestinal epithelium will provide therapeutic strategies for manipulating the function of infiltrating γδ T cells. Disclosures The authors have no financial conflicts of interest. Acknowledgments We thank Dr. Roxana del Rio-Guerra for technical assistance with flow cytometry, as well as the Harry Hood Bassett Flow Cytometry and Cell Sorting Facility at The University of Vermont Larner College of Medicine. We thank Drs. Mark Davis and Naresha Saligrama for providing the human soluble TCR-αβ. We also thank the Vermont Genetics Network National Institutes of Health IDeA Networks of Biomedical Research Excellence program and the Vermont Center for Immunology and Infectious Diseases National Institutes of Health Centers of Biomedical Research Excellence program for support of the mass spectrometry facility. 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