Targeting B cells and plasma cells in autoimmune diseases: From established treatments to novel therapeutic approaches.

Autoimmune diseases are characterized by the recognition of self-antigens by the immune system, which leads to inflammation and tissue damage. B cells are directly and indirectly involved in the pathophysiology of autoimmunity, both via antigen-presentation to T cells and production of proinflammatory cytokines and/or autoantibodies. Consequently, B lineage cells have been identified as therapeutic targets in autoimmune diseases. B cell depleting strategies have proven beneficial in the treatment of rheumatoid arthritis (RA), systemic lupus erythematous (SLE), ANCA-associated vasculitis (AAV), multiple sclerosis (MS), and a wide range of other immune-mediated inflammatory diseases (IMIDs). However, not all patients respond to treatment or may not reach (drug-free) remission. Moreover, B cell depleting therapies do not always target all B cell subsets, such as short-lived and long-lived plasma cells. These cells play an active role in autoimmunity and in certain diseases their depletion would be beneficial to achieve disease remission. In the current review article, we provide an overview of novel strategies to target B lineage cells in autoimmune diseases, with the focus on rheumatic diseases. Both advanced therapies that have recently become available and more experimental treatments that may reach the clinic in the near future are discussed.

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis.

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-ß (IKKß, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Editorial for Special Issue "Advances in the Pathogenesis and Treatment of Immune-Mediated Inflammatory Diseases".

This Special Issue focuses on the rapidly evolving field of immune-mediated inflammatory diseases (IMIDs) and the achievements that were made over the last 10 years [...].

Splenic Architecture and Function Requires Tight Control of Transmembrane TNF Expression.

Soluble tumor necrosis factor (sTNF) is an important inflammatory mediator and essential for secondary lymphoid organ (SLO) development and function. However, the role of its transmembrane counterpart (tmTNF) in these processes is less well established. Here, the effects of tmTNF overxpression on SLO architecture and function were investigated using tmTNF-transgenic (tmTNF-tg) mice. tmTNF overexpression resulted in enlarged peripheral lymph nodes (PLNs) and spleen, accompanied by an increase in small splenic lymphoid follicles, with less well-defined primary B cell follicles and T cell zones. In tmTNF-tg mice, the spleen, but not PLNs, contained reduced germinal center (GC) B cell fractions, with low Ki67 expression and reduced dark zone characteristics. In line with this, smaller fractions of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells were observed with a decreased Tfh:Tfr ratio. Moreover, plasma cell (PC) formation in the spleen of tmTNF-tg mice decreased and skewed towards IgA and IgM expression. Genetic deletion of TNFRI or -II resulted in a normalization of follicle morphology in the spleen of tmTNF-tg mice, but GC B cell and PC fractions remained abnormal. These findings demonstrate that tightly regulated tmTNF is important for proper SLO development and function, and that aberrations induced by tmTNF overexpression are site-specific and mediated via TNFRI and/or TNFRII signaling.

NIK-IKK complex interaction controls NF-κB-dependent inflammatory activation of endothelium in response to LTßR ligation.

NF-κB-inducing kinase (NIK; also known as MAP3K14) is a central regulator of non-canonical NF-κB signaling in response to stimulation of TNF receptor superfamily members, such as the lymphotoxin-ß receptor (LTßR), and is implicated in pathological angiogenesis associated with chronic inflammation and cancer. Here, we identify a previously unrecognized role of the LTßR-NIK axis during inflammatory activation of human endothelial cells (ECs). Engagement of LTßR-triggered canonical and non-canonical NF-κB signaling promoted expression of inflammatory mediators and adhesion molecules, and increased immune cell adhesion to ECs. Sustained LTßR-induced inflammatory activation of ECs was NIK dependent, but independent of p100, indicating that the non-canonical arm of NF-κB is not involved. Instead, prolonged activation of canonical NF-κB signaling, through the interaction of NIK with IκB kinase α and ß (also known as CHUK and IKBKB, respectively), was required for the inflammatory response. Endothelial inflammatory activation induced by synovial fluid from rheumatoid arthritis patients was significantly reduced by NIK knockdown, suggesting that NIK-mediated alternative activation of canonical NF-κB signaling is a key driver of pathological inflammatory activation of ECs. Targeting NIK could thus provide a novel approach for treating chronic inflammatory diseases.

B Lineage Cells in ANCA-Associated Vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease that affects small sized blood vessels and can lead to serious complications in the lungs and kidneys. The prominent presence of ANCA autoantibodies in this disease implicates B cells in its pathogenesis, as these are the precursors of the ANCA-producing plasma cells (PCs). Further evidence supporting the potential role of B lineage cells in vasculitis are the increased B cell cytokine levels and the dysregulated B cell populations in patients. Confirmation of the contribution of B cells to pathology arose from the beneficial effect of anti-CD20 therapy (i.e., rituximab) in AAV patients. These anti-CD20 antibodies deplete circulating B cells, which results in amelioration of disease. However, not all patients respond completely, and this treatment does not target PCs, which can maintain ANCA production. Hence, it is important to develop more specific therapies for AAV patients. Intracellular signalling pathways may be potential therapeutic targets as they can show (disease-specific) alterations in certain B lineage cells, including pathogenic B cells, and contribute to differentiation and survival of PCs. Preliminary data on the inhibition of certain signalling molecules downstream of receptors specific for B lineage cells show promising therapeutic effects. In this narrative review, B cell specific receptors and their downstream signalling molecules that may contribute to pathology in AAV are discussed, including the potential to therapeutically target these pathways.

Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.

The lung-draining mediastinal lymph nodes (MLNs) are currently widely used to diagnose sarcoidosis. We previously reported that T-helper (Th) 17.1 cells are responsible for the exaggerated interferon-γ production in sarcoidosis lungs. In this study, we aimed to investigate 1) whether Th17.1 cells are also increased in the MLNs of sarcoidosis patients and 2) whether frequencies of the Th17.1 cells at diagnosis may correlate with disease progression.MLN cells from treatment-naive pulmonary sarcoidosis patients (n=17) and healthy controls (n=22) and peripheral blood mononuclear cells (n=34) and bronchoalveolar lavage fluid (BALF) (n=36) from sarcoidosis patients were examined for CD4+ T-cell subset proportions using flow cytometry.Higher proportions of Th17.1 cells were detected in sarcoidosis MLNs than in control MLNs. Higher Th17.1 cell proportions were found in sarcoidosis BALF compared with MLNs and peripheral blood. Furthermore, BALF Th17.1 cell proportions were significantly higher in patients developing chronic disease than in patients undergoing resolution within 2 years of clinical follow-up.These data suggest that Th17.1 cell proportions in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and could serve as a new therapeutic target.

Molecular mechanisms underpinning T helper 17 cell heterogeneity and functions in rheumatoid arthritis.

T helper 17 (Th17) cells are important mediators of immune responses against extracellular bacteria and fungi, and as such play critical regulatory roles in maintaining mucosal homeostasis. Conversely, Th17 cells and their effector molecules interleukin 17A (IL-17A), IL-17F, interferon (IFN)γ, tumor necrosis factor (TNF)α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are implicated in the pathology of rheumatoid arthritis (RA). Interactions between Th17 cells and other immune cells or stromal cells that are present in the synovial tissue during the earliest phases of the disease, may eventually lead to chronic inflammation, irreversible cartilage degradation and bone erosions. Recent evidence points towards Th17 cell plasticity as an essential contributing process in RA pathology, since Th17 cells are able to adopt a pathogenic phenotype under the influence of environmental, inflammatory and genetic factors. A remarkable feature of this pathogenic Th17 cell phenotype is the high production of GM-CSF and TNFα and the co-appearance of Th1 cell characteristics, such as transcription factor T-box 21 (T-bet) and IFNγ expression. Recently, much progress has been made in unravelling the mechanisms underlying Th17 cell plasticity and pathogenicity. Of interest, many of the environmental and inflammatory factors associated with RA pathology, such as pro-inflammatory mediators and cytokines, microbiome dysbiosis, metabolism and diet, obesity, vitamins, steroids and hormones are linked to the development of pathogenic Th17 cells. Moreover proteins encoded by established genetic risk factors for RA including CCR6, CD226, CSF2, EOMES, ETS1, GATA3, IL2, IL6R, IL23R, IKZF3, IRAK1, IRF4, IRF8, PRKCQ, PRDM1, RBPJ, RUNX1 and TAGAP are directly involved in Th17 cell differentiation and/or function. This review provides a detailed overview of the molecular mechanisms involved in the heterogeneity and pathogenicity of Th17 cells in the context of autoimmune diseases, with a focus on RA. Understanding these mechanisms creates great potential to identify and select novel therapeutic targets which could improve current therapies or lead to development of new treatment strategies in RA.

Loss of IL-22 inhibits autoantibody formation in collagen-induced arthritis in mice.

Interleukin 22 (IL-22) expression is associated with increased joint destruction and disease progression in rheumatoid arthritis (RA). Although IL-22 is considered a pro-inflammatory cytokine, its mechanism of action in RA remains incompletely understood. Here, we used the collagen-induced arthritis model in IL-22 deficient (IL-22(-/-) ) mice to study the role of IL-22 in RA. In spite of normal disease incidence, disease severity is significantly diminished in IL-22(-/-) mice. Moreover, pathogenicity of Th17 cells and development and function of B cells are unaffected. In contrast, splenic plasma cells, as well as serum autoantibody titers, are reduced in the absence of IL-22. At the peak of disease, germinal centers (GCs) are severely reduced in the spleens of IL-22(-/-) mice, correlating with a decline in GC B-cell numbers. Within the GC, we identified IL-22R1 expressing follicular dendritic cell-like stromal cells. Human lymphoid stromal cells respond to IL-22 ex vivo by inducing transcription of CXCL12 and CXCL13. We therefore postulate IL-22 as an important enhancer of the GC reaction, maintaining chemokine levels for the persistence of GC reactions, essential for the production of autoantibody-secreting plasma cells. Blocking IL-22 might therefore prevent immune-complex deposition and destruction of joints in RA patients.

The role and modulation of CCR6+ Th17 cell populations in rheumatoid arthritis.

The IL-17A producing T-helper-17 (Th17) cell population plays a major role in rheumatoid arthritis (RA) pathogenesis and has gained wide interest as treatment target. IL-17A expressing Th cells are characterized by the expression of the chemokine receptor CCR6 and the transcription factor RORC. In RA, CCR6+ Th cells were identified in peripheral blood, synovial fluid and inflamed synovial tissue. CCR6+ Th cells might drive the progression of an early inflammation towards a persistent arthritis. The CCR6+ Th cell population is heterogeneous and several subpopulations can be distinguished, including Th17, Th22, Th17.1 (also called non-classic Th1 cells), and unclassified or intermediate populations. Interestingly, some of these populations produce low levels of IL-17A but are still very pathogenic. Furthermore, the CCR6+ Th cells phenotype is unstable and plasticity exists between CCR6+ Th cells and T-regulatory (Treg) cells and within the CCR6+ Th cell subpopulations. In this review, characteristics of the different CCR6+ Th cell populations, their plasticity, and their potential impact on rheumatoid arthritis are discussed. Moreover, current approaches to target CCR6+ Th cells and future directions of research to find specific CCR6+ Th cell targets in the treatment of patients with RA and other CCR6+ Th cell mediated autoimmune diseases are highlighted.

Synovial fibroblasts directly induce Th17 pathogenicity via the cyclooxygenase/prostaglandin E2 pathway, independent of IL-23.

Th17 cells are critically involved in autoimmune disease induction and severity. Recently, we showed that Th17 cells from patients with rheumatoid arthritis (RA) directly induced a proinflammatory loop upon interaction with RA synovial fibroblasts (RASF), including increased autocrine IL-17A production. To unravel the mechanism driving this IL-17A production, we obtained primary CD4(+)CD45RO(+)CCR6(+) (Th17) cells and CD4(+)CD45RO(+)CCR6(-) (CCR6(-)) T cells from RA patients or healthy individuals and cocultured these with RASF. IL-1ß, IL-6, IL-23p19, and cyclooxygenase (COX)-2 expression and PGE2 production in Th17-RASF cultures were higher than in CCR6(-) T cell-RASF cultures. Cytokine neutralization showed that IL-1ß and IL-6, but not IL-23, contributed to autocrine IL-17A induction. Importantly, treatment with celecoxib, a COX-2 inhibitor, resulted in significantly lower PGE2 and IL-17A, but not IFN-γ, production. Combined celecoxib and TNF-α blockade more effectively suppressed the proinflammatory loop than did single treatment, as shown by lower IL-6, IL-8, matrix metalloproteinase-1 and matrix metalloproteinase-3 production. These findings show a critical role for the COX-2/PGE2 pathway in driving Th17-mediated synovial inflammation in an IL-23- and monocyte-independent manner. Therefore, it would be important to control PGE2 in chronic inflammation in RA and potentially other Th17-mediated autoimmune disorders.

Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice.

On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)-like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overexpressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca(2+) influx, nuclear factor (NF)-κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK.

IL-17/Th17 mediated synovial inflammation is IL-22 independent.

BACKGROUND: Interleukin (IL)-17A and Th17 cells are critically involved in T cell-mediated synovial inflammation. Besides IL-17A, Th17 cells produce IL-22. Recently, Th22 cells were discovered, which produce IL-22 in the absence of IL-17. However, it remains unclear whether IL-22 and Th22 cells contribute to T cell-mediated synovial inflammation. Therefore, we examined the potential of IL-22 and Th22 cells to induce synovial inflammation and whether IL-22 is required for T cell-mediated experimental arthritis. METHODS: Peripheral and synovial Th17 and Th22 cells were identified and sorted from patients with rheumatoid arthritis (RA). Co-culture experiments of these primary T cell populations with RA synovial fibroblasts (RASF) were performed. The in vivo IL-22 contribution to synovial inflammation was investigated by inducing T cell-mediated arthritis in IL-22 deficient mice and wild-type mice. RESULTS: Peripheral Th17 and Th22 cell populations were increased in patients with RA and present in RA synovial fluid. In T cell-RASF co-cultures, IL-22 in the presence of IL-17A had limited effects on IL-6, IL-8, matrix metalloproteinase-1 (MMP-1) and MMP-3 production. Furthermore, primary peripheral blood and synovial Th17 cells were more potent in the induction of these factors by RASF compared with Th22 cells. In line with this, similar synovial inflammation and disease severity was found between IL-22 deficient and wild-type mice in T cell-mediated experimental arthritis. CONCLUSIONS: These findings show that IL-17A/Th17 cell-mediated synovial inflammation is independent of IL-22 and Th22 cells. This implies that targeting IL-17A/Th17 cells, rather than IL-22/Th22 cells, should be the focus for treatment of T cell-mediated synovial inflammation.

A novel 3D spheroid model of rheumatoid arthritis synovial tissue incorporating fibroblasts, endothelial cells, and macrophages.

Objective: Rheumatoid Arthritis (RA) is a progressive and systemic autoimmune disorder associated with chronic and destructive joint inflammation. The hallmarks of joint synovial inflammation are cellular proliferation, extensive neoangiogenesis and infiltration of immune cells, including macrophages. In vitro approaches simulating RA synovial tissue are crucial in preclinical and translational research to evaluate novel diagnostic and/or therapeutic markers. Two-dimensional (2D) settings present very limited in vivo physiological proximity as they cannot recapitulate cell-cell and cell-matrix interactions occurring in the three-dimensional (3D) tissue compartment. Here, we present the engineering of a spheroid-based model of RA synovial tissue which mimics 3D interactions between cells and pro-inflammatory mediators present in the inflamed synovium. Methods: Spheroids were generated by culturing RA fibroblast-like-synoviocytes (RAFLS), human umbilical vein endothelial cells (ECs) and monocyte-derived macrophages in a collagen-based 3D scaffold. The spheroids were cultured in the presence or absence of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (bFGF) or RA synovial fluid (SF). Spheroid expansion and cell migration were quantified for all conditions using confocal microscopy and digital image analysis. Results: A novel approach using machine learning was developed to quantify spheroid outgrowth and used to reexamine the existing spheroid-based model of RA synovial angiogenesis consisting of ECs and RAFLS. A 2-fold increase in the spheroid outgrowth ratio was demonstrated upon VEGF/bFGF stimulation (p<0.05). The addition of macrophages within the spheroid structure (3.75x104 RAFLS, 7.5x104 ECs and 3.0x104 macrophages) resulted in good incorporation of the new cell type. The addition of VEGF/bFGF significantly induced spheroid outgrowth (p<0.05) in the new system. SF stimulation enhanced containment of macrophages within the spheroids. Conclusion: We present a novel spheroid based model consisting of RAFLS, ECs and macrophages that reflects the RA synovial tissue microenvironment. This model may be used to dissect the role of specific cell types in inflammatory responses in RA, to study specific signaling pathways involved in the disease pathogenesis and examine the effects of novel diagnostic (molecular imaging) and therapeutic compounds, including small molecule inhibitors and biologics.

Contribution of Type H Blood Vessels to Pathologic Osteogenesis and Inflammation in an Experimental Spondyloarthritis Model.

OBJECTIVE: Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. METHODS: We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)-deficient or TNF receptor type II (TNFRII)-deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. RESULTS: Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. CONCLUSION: The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention.

TNF blockade requires 1,25(OH)2D3 to control human Th17-mediated synovial inflammation.

OBJECTIVES: T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) controls Th17-mediated synovial inflammation. METHODS: Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17-RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)(2)D(3) and/or TNFα blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA. RESULTS: The authors show that the 1,25(OH)(2)D(3), but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17-RASF and synovial biopsy cultures. Combining 1,25(OH)(2)D(3) and TNFα blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17-RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue. CONCLUSIONS: These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)(2)D(3). The combination of neutralising TNF activity and 1,25(OH)(2)D(3) controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptor signalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

Malignant transformation of Slp65-deficient pre-B cells involves disruption of the Arf-Mdm2-p53 tumor suppressor pathway.

The adapter protein Slp65 is a key component of the precursor-B (pre-B) cell receptor. Slp65-deficient mice spontaneously develop pre-B cell leukemia, but the mechanism by which Slp65(-/-) pre-B cells become malignant is unknown. Loss of Btk, a Tec-family kinase that cooperates with Slp65 as a tumor suppressor, synergizes with deregulation of the c-Myc oncogene during lymphoma formation. Here, we report that the presence of the immunoglobulin heavy chain transgene V(H)81X prevented tumor development in Btk(-/-)Slp65(-/-) mice. This finding paralleled the reported effect of a human immunoglobulin heavy chain transgene on lymphoma development in Emu-myc mice, expressing transgenic c-Myc. Because activation of c-Myc strongly selects for spontaneous inactivation of the p19(Arf)-Mdm2-p53 tumor suppressor pathway, we investigated whether disruption of this pathway is a common alteration in Slp65(-/-) pre-B cell tumors. We found that combined loss of Slp65 and p53 in mice transformed pre-B cells very efficiently. Aberrations in p19(Arf), Mdm2, or p53 expression were found in all Slp65(-/-) (n = 17) and Btk(-/-)Slp65(-/-) (n = 32) pre-B cell leukemias analyzed. In addition, 9 of 10 p53(-/-)Slp65(-/-) pre-B cell leukemias manifested significant Mdm2 protein expression. These data indicate that malignant transformation of Slp65(-/-) pre-B cells involves disruption of the p19(Arf)-Mdm2-p53 tumor suppressor pathway.

Extrathymic AIRE-expressing cells: Friends or foes in autoimmunity and cancer?

Auto-immune regulator (AIRE) is a transcription factor that is mainly known for its crucial role in the thymus. Here, AIRE ensures central tolerance by promoting the expression of peripheral tissue antigens in thymic epithelial cells, which is essential for the negative selection of autoreactive T cells. Intriguingly, AIRE expressing cells have recently been identified in other tissues outside the thymus as well. However, the exact function of these extrathymic AIRE expressing cells (eTACs) remains largely enigmatic. Human eTACs are mainly found in secondary lymphoid tissues under homeostatic conditions, but are also found in pathologies such as the inflamed tissues of patients with autoimmune diseases and in various cancer tissues. eTACs have been demonstrated to express dendritic cell (DC)-like markers, such as MHCII, CD40 and CD127, but also CCR7, IDO and PD-L1. Interestingly, eTACs lack high expression of co-stimulatory molecules, such as CD80 or CD86. In mice, different types of peripheral AIRE expressing cells have been described, including cells with an innate lymphoid cell-like phenotype and antigen presenting cell (APC) function. These findings suggest that eTACs are APCs with the possibility to modulate or inhibit immune responses, which is confirmed by functional murine studies demonstrating the ability of eTACs to induce tolerance in autoreactive T cells. The potential immunomodulatory function of eTACs makes them promising targets to restore tolerance in autoimmunity or improve immunotherapy in cancer settings. Yet, this requires a better understanding of these cells and the molecular mechanisms involved. In this review we aim to summarize the current knowledge and understanding of eTACs, including their putative roles in health and disease.

Differential Contribution of NF-κB Signaling Pathways to CD4+ Memory T Cell Induced Activation of Endothelial Cells.

Endothelial cells (ECs) are important contributors to inflammation in immune-mediated inflammatory diseases (IMIDs). In this study, we examined whether CD4+ memory T (Tm) cells can drive EC inflammatory responses. Human Tm cells produced ligands that induced inflammatory responses in human umbilical vein EC as exemplified by increased expression of inflammatory mediators including chemokines and adhesion molecules. NF-κB, a key regulator of EC activation, was induced by Tm cell ligands. We dissected the relative contribution of canonical and non-canonical NF-κB signaling to Tm induced EC responses using pharmacological small molecule inhibitors of IKKß (iIKKß) or NF-κB inducing kinase (iNIK). RNA sequencing revealed substantial overlap in IKKß and NIK regulated genes (n=549) that were involved in inflammatory and immune responses, including cytokines (IL-1ß, IL-6, GM-CSF) and chemokines (CXCL5, CXCL1). NIK regulated genes were more restricted, as 332 genes were uniquely affected by iNIK versus 749 genes by iIKKß, the latter including genes involved in metabolism, proliferation and leukocyte adhesion (VCAM-1, ICAM-1). The functional importance of NIK and IKKß in EC activation was confirmed by transendothelial migration assays with neutrophils, demonstrating stronger inhibitory effects of iIKKß compared to iNIK. Importantly, iIKKß - and to some extent iNIK - potentiated the effects of currently employed therapies for IMIDs, like JAK inhibitors and anti-IL-17 antibodies, on EC inflammatory responses. These data demonstrate that inhibition of NF-κB signaling results in modulation of Tm cell-induced EC responses and highlight the potential of small molecule NF-κB inhibitors as a novel treatment strategy to target EC inflammatory responses in IMIDs.