Objectives SSc-interstitial lung disease (ILD) is one of the leading causes of mortality in SSc. Data from randomized controlled trials (RCTs) support rituximab and tocilizumab monotherapy but there are limited data regarding their use for those who fail standard immunomodulatory therapies.Methods SSc patients treated with rituximab or tocilizumab were retrospectively identified in a single centre cohort. Linear mixed effect models were used to analyse before and after treatment lung function trajectory, and identify patient characteristics associated with treatment response.Results A total of 127 patients were included for analysis. Fifty-one of 94 (54.2%) and 13 of 33 (39.4%) of the rituximab and tocilizumab cohorts, respectively, were receiving concurrent MMF. Pre-treatment decline in absolute change % forced vital capacity (%FVC)/year and % diffusion capacity for carbon monoxide (%DLCO)/year, respectively, was similar in both cohorts (-3.2% and -4.0% rituximab, and -3.2% and -3.6% tocilizumab). Both treatments resulted in lung function stabilization (%FVC/year and %DLCO/year: 1.2% and +0.2% rituximab cohort, 1.0% and 1.0% tocilizumab cohort). Anti-topoisomerase antibody (ATA)-positive patients had a significant response on %FVC/year to tocilizumab compared with ATA-negative patients. Gender had a significant impact on %FVC/year response to rituximab, with males responding to a greater degree than females. Age, ILD extent and skin subset had no impact on treatment response.Conclusion Combination rituximab or tocilizumab with background immunosuppressive therapy is associated with stabilization in lung function trajectory among those who remain refractory to standard immunosuppressives. Specific patient characteristics have an impact on lung function response. Improved FVC response among ATA patients receiving tocilizumab validate data from RCTs.

The mechanisms of endotoxin tolerance (ET), which down-regulate inflammation, are well described in response to exogenous toll-like receptor ligands, but few studies have focused on ET-associated mechanisms in inflammatory disease. As blocking TNF can attenuate the development of ET, the effect of anti-TNF on the expression of key ET-associated molecules in inflammatory auto-immune disease was measured; changes in inflammatory gene expression were confirmed using an ET bioassay. The expression of immunomodulatory molecules was measured in a murine model of arthritis treated with anti-TNF and the expression of ET-associated molecules was measured in whole blood in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients, before and after therapy. The expression of ET-associated genes was also measured in RA patient monocytes before and after therapy, in anti-TNF responders and non-responders. Tnfaip3, Ptpn6 and Irak3 were differentially expressed in affected paws, spleens, lymph nodes and circulating leucocytes in experimental murine arthritis treated with anti-TNF. Prior to therapy, the expression of TNFAIP3, INPP5D, PTPN6, CD38 and SIGIRR in whole blood differed between human healthy controls and RA or AS patients. In blood monocytes from RA patients, the expression of TNFAIP3 was significantly reduced by anti-TNF therapy in non-responders. Prior to therapy, anti-TNF non-responders had higher expression of TNFAIP3 and SLPI, compared to responders. Although the expression of TNFAIP3 was significantly higher in RA non-responders prior to treatment, the post-treatment reduction to a level similar to responders did not coincide with a clinical response to therapy.

Background/Aims Transitional B cells comprise a distinct population of B cells that have recently migrated to the periphery from the bone marrow. In systemic sclerosis (SSc), there are differences in the number and function of transitional B cells compared with healthy controls (HCs). In addition, we have previously shown that transitional B cells evade peripheral tolerance from SSc patients who are seropositive for anti-topoisomerase I autoantibodies (ATA). To investigate mechanisms that underpin this defect, we performed a paired bulk and single-cell RNA sequencing study of transitional B cell subsets in treatment-naive ATA-positive patients with diffuse cutaneous SSc (dcSSc) and HCs. Methods CD19(+)CD24(hi)CD38(hi) transitional B cells (5000) were sorted from four HCs and four treatment-naive ATA-positive dcSSc patients. Single-cell RNA-sequencing was performed on the sorted B cells and the data was analysed using Seurat in RStudio. Paired bulk RNA-sequencing of the sorted transitional T1 (CD19(+)CD24(hi)CD38(hi) CD27(-)IgM(hi)IgD(med)) and T2 (CD19(+)CD24(hi)CD38(hi)CD27(-)IgM(hi)IgD(hi)) subsets was undertaken in a subset of this cohort. Results From the sorted CD19(+)CD24(hi)CD38(hi) lymphocytes single cell transcriptomics identified six B cell clusters in SSc patients and HCs with differential expression of key genes such as CD19 and TCL1A. This included a cluster of marginal zone precursor cells expressing PLD4 and MZB1 which is expanded in SSc. We also identified an interesting memory-like B cell cluster which was diminished in the SSc group and expresses genes such as TNFRSF13B (BAFF), AICDA (AID) and S100A10. In the HCs, this cluster also expresses genes such as CD27 and IGHA1 which are absent in SSc indicating that B cell development is impaired in the disease. Subsequent pathway analyses provided evidence for the molecular bases of defective tolerance in SSc patients. This appears to be related to dysregulated NF-kappa B signalling and aberrant extrafollicular B cell development as evidenced through pseudotime trajectory analysis. Differential gene expression analyses using bulk RNA-sequencing of sorted T1 and T2 transitional B cells further identified five candidate gene signatures such as MYC-targets and DNA repair which are significantly enriched in the T2 subset. This latter finding is in congruence with the single cell data and provides further evidence to support the existence of a peripheral tolerance/selection checkpoint at the transitional B cell stage. Conclusion We have identified four transitional B cell subsets and have characterised their transcriptomic profile in SSc. Additionally, we provide new evidence for a potential breach in peripheral tolerance of SSc patients. Further studies are underway to identify key pathways that underpin B cell dysregulation in SSc and explore the relevance of such findings for better targeted therapies. Disclosure C. Beesley: Grants/research support; C.B. is supported through Versus Arthritis (grant number 558800). N. Goldman: None. D. Abraham: None. C. Denton: None. R. Mageed: None. V.H. Ong: None.

Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-β, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.

The immune system protects from infections and cancer through complex cellular networks. For this purpose, immune cells require well-developed mechanisms of energy generation. However, the immune system itself can also cause diseases when defective regulation results in the emergence of autoreactive lymphocytes. Recent studies provide insights into how differential patterns of immune cell responses are associated with selective metabolic pathways. This review will examine the changing metabolic requirements of Th17 cells and of B cells at different stages of their development and activation. Both cells provide protection but can also mediate diseases through the production of autoantibodies and the production of proinflammatory mediators. In health, B cells produce antibodies and cytokines and present antigens to T cells to mount specific immunity. Th17 cells, on the other hand, provide protection against extra cellular pathogens at mucosal surfaces but can also drive chronic inflammation. The latter cells can also promote the differentiation of B cells to plasma cells to produce more autoantibodies. Metabolism-regulated checkpoints at different stages of their development ensure the that self-reactive B cells clones and needless production of interleukin (IL-)17 are limited. The metabolic regulation of the two cell types has some similarities, e.g. the utility of hypoxia induced factor (HIF)1α during low oxygen tension, to prevent autoimmunity and regulate inflammation. There are also clear differences, as Th17 cells only are vulnerable to the lack of certain amino acids. B cells, unlike Th17 cells, are also dependent of mechanistic target of rapamycin 2 (mTORC2) to function. Significant knowledge has recently been gained, particularly on Th17 cells, on how metabolism regulates these cells through influencing their epigenome. Metabolic dysregulation of Th17 cells and B cells can lead to chronic inflammation. Disease associated alterations in the genome can, in addition, cause dysregulation to metabolism and, thereby, result in epigenetic alterations in these cells. Recent studies highlight how pathology can result from the cooperation between the two cell types but only few have so far addressed the key metabolic alterations in such settings. Knowledge of the impact of metabolic dysfunction on chronic inflammation and pathology can reveal novel therapeutic targets to treat such diseases.

Abstract Background/Aims Interstitial lung disease (ILD) is one of the leading causes of death in systemic sclerosis (SSc). Evidence from randomised controlled trials suggests a beneficial effect of tocilizumab and rituximab on preserving lung function. However, comparative data outside the arena of clinical trials remains limited. Deciding when and who to treat with specific treatments remains challenging. Methods We performed a retrospective analysis of all SSc patients attending a single specialist centre who had received rituximab or tocilizumab. Demographic, clinical and laboratory data was collected along with serial lung function. Patients were excluded if lung function pre- and/or post-therapy was not available. Data were analysed based on anti-topoisomerase I antibody (ATA) status and other clinical, laboratory and radiological features. Wilcoxon T test and Fisher’s exact test were used. Results 129 patients were included, of which 87 received rituximab, 32 tocilizumab and 10 both therapies. 8 of 10 (80%) patients who had received both therapies received rituximab prior to tocilizumab. 76 (58.9%) patients had diffuse SSc and 102 (79%) had ILD. Concurrent mycophenolate mofetil (MMF) was prescribed for 52 (53.6%) patients with rituximab and 17 (40.5%) patients with tocilizumab. Median pre-treatment lung function percentage forced vital capacity (%FVC) and percentage transfer factor (%DLCO) were 67.9% and 42.5% and 85.5% and 59.8% for rituximab and tocilizumab respectively. Median change %FVC and %DLCO pre- and post-treatment were +0.35% and -0.8% for rituximab and -0.9% and +0.2% for tocilizumab. Using minimally clinically important difference for change in %FVC in SSc, the majority of patients improved or remained stable (69.3% rituximab, 64.1% tocilizumab) with both treatments. A smaller percentage of patients on rituximab demonstrated FVC decline compared with tocilizumab. The effect from rituximab was not impacted by ATA status. In contrast, ATA positive patients were more likely to respond to tocilizumab (p = 0.0073) (median FVC change: tocilizumab, + 60ml ATA positive vs -110ml ATA negative; rituximab, 0ml ATA positive vs 0ml ATA negative). Disease duration and CRP had no effect on treatment response for either therapy. Conclusion Our retrospective cohort provides real-life data supporting the use of both rituximab and tocilizumab to stabilise ILD in SSc. Differential response based on autoantibody specificity and clinical parameters may help optimise patient selection for biological therapy in SSc-ILD. Further research to understand the exact mechanism of action driving the differential response in these patient groups is needed to greater understand the pathogenesis of SSc-ILD. Disclosure N.R. Goldman: Grants/research support; MRC/SRUK Clinical Research Training Fellowship [grant number MR/V030108/1]. A. Tynan: None. C. Beesley: None. R. Mageed: None. C. Denton: Consultancies; CD has been a consultant to Roche. V.H. Ong: None.

Abstract Background/Aims Aberrant complement activation is associated with autoimmune diseases including systemic sclerosis (SSc). As a leading cause of death in SSc, interstitial lung disease (ILD) can coexist with emphysema in non-smoking patients, worsening prognosis. It has been suggested that the airway destruction observed in SSc patients may be an exaggerated inflammatory response related to increased complement activation. Methods We analysed 10 complement proteins in plasma samples of 16 non-smoking SSc patients with emphysema (SSc-Emp), 8 SSc no-ILD, 8 SSc-ILD patients and 8 healthy controls (HC) (Table.1) selected from 1800 SSc patients under active follow up at our centre. The extent and distribution of emphysema was evaluated on high resolution computed tomography (HRCT) and enzyme-linked immunosorbent assays (ELISA) were performed for C1q, MASP-2, Factor B, Factor Bb, Factor H, C3, C3a, C4, C5, C5a and TCC (terminal complement complex). One-way ANOVA and post-hoc Tukey test were used for analysis. Results HRCT confirmed that amongst the emphysema cohort, there was a spectrum of mild to severe destruction including both paraseptal and centrilobular emphysema. All 16 SSc-Emp patients had ILD and 9 of the 16 patients demonstrated perivascular emphysema not previously described in SSc. The SSc-ILD group was characterised by extensive lung fibrosis on CT, mean FVC 71% predicted. Across the four groups, C1q was significantly reduced in the SSc-ILD cohort (SSc-Emp 950±404µg/mL, SSc-ILD 355±116µg/mL, SSc-no ILD 1087±206µg/mL, HC 858±210µg/mL, p= 0.0002). In contrast, the ratio of C3a/C3 was significantly increased in the SSc-ILD group compared with the SSc-Emp group (SSc-ILD 0.12±0.07 vs SSc-Emp 0.06±0.03, p= 0.0283). The ratio of Bb/B was significantly increased in the SSc no-ILD group compared with all other groups (SSc-Emp 1.57±0.80µg/mL, SSc-ILD 1.27±0.56µg/mL, SSc-no ILD 3.86±1.12µg/mL, HC 1.89±0.75µg/mL, p< 0.0001). There was a trend towards an increased ratio of C5a/C5 in SSc-ILD and SSc-Emp compared with HC and reduced MASP2 in the SSc groups compared with HC. Conclusion This data demonstrates dysregulated complement levels in three SSc subgroups. Notably, the perturbed C1q and C3a:C3 pathways in SSc-ILD are reversed in SSc-emphysema, supporting complement activation as part of the divergent tissue remodelling responses in these distinct SSc lung phenotypes. Disclosure C. Beesley: Grants/research support; Versus Arthritis [grant number 558800]. A. Cole: None. N. Goldman: None. J. Barnett: None. D. Abraham: None. C. Denton: None. R. Mageed: None. V. Ong: None.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

AbstractLymphocytes mount protective immunity from infectious pathogens and from cancer. Their engagement by target antigens prompt activation, differentiation to effector cells and proliferation. These responses require energy that is generated by cellular metabolic processes, such as glycolysis and oxidative phosphorylation. Glycolysis occurs in the cytoplasm while oxidative phosphorylation occurs in the mitochondria. Energy in the form of adenosine triphosphate is generated from the uptake of glucose, amino acids and free fatty acids. Important recent evidence indicates that naïve and activated lymphocytes and functionally distinct subsets preferentially use different metabolic pathways for their energy needs. Thus, effector Th17 cells primarily use glycolysis to generate energy required for their activities. Memory T and B cells and regulatory T cells, in contrast, rely on mitochondrial metabolism for their energy requirements. Naïve T and B are in a quiescent state with small mitochondria. Extrinsic factors such as oxygen tension and intrinsic substrates can also influence the choice of metabolic pathways and functional flexibility. Studies of lymphocytes in disease states reveal alterations to choices of metabolic pathways from those in lymphocytes in healthy individuals. This article provides an overview of metabolic pathways required for energy generation during homeostasis and those induced during cellular differentiation and responses. Furthermore, we explore available evidence for altered metabolic pathway induction in some autoimmune diseases. A focus of the overview will be on helper T lymphocytes involved in chronic inflammation and those that regulate the immune response. The report alludes to the potential that targeting metabolic pathways could provide a strategy for the treatment of chronic diseases.

OBJECTIVES:About half of RA patients treated with TNFα inhibitors either do not respond or lose their initial therapeutic response over time. The clinical response is measured by reduction in DAS28, which primarily reflects inflammation. However, other effects of TNFα inhibitors, such as impact on bone erosion, are not assessed by DAS28. We aimed to examine the effect of TNFα inhibitors on bone density, bone biomarkers and cytokine production in responder and non-responder patients and assessed mechanisms of action.METHODS:BMD in the lumbar spine and femur neck of 117 RA patients was measured by DEXA scan. Bone turnover biomarkers CTX, osteoprotegerin (OPG), osteocalcin and RANKL were measured by ELISA. Levels of 16 cytokines in plasma and in tissue culture supernatants of ex vivo T cells were measured by multiplex assays and ELISA. The effect of treatment with TNFα inhibitors on blood mononuclear cell (MNC) differentiation to osteoclast precursors (OCP) was measured flow cytometry and microscopy.RESULTS:TNFα inhibitors improved lumbar spine BMD but had modest effects on blood bone biomarkers, irrespective of patients' clinical response. Blood OCP numbers and the ability of monocytes to differentiate to OCP in vitro declined after treatment. Treatment also reduced RANK expression and IL-20 production. BMD improvement correlated with reduced levels of IL-20 in responder patients.CONCLUSION:This study reveals that TNFα inhibitors reduce lumbar spine bone loss in RA patients irrespective of changes in DAS28. The reduction in bone loss is associated with reduction in IL-20 levels in responder patients.

Background Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro-inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear. Design In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL-17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases. Results The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis. Conclusion This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases.

21 Background: CD24CD38 transitional B cells represent cells at a key stage in their 22 developmental pathway. During this stage, B cells undergo peripheral tolerance and functional 23 maturation. In addition, these B cells have been widely ascribed regulatory functions and 24 involvement in the control of chronic inflammatory diseases. However, the phenotypic and 25 functional overlap between these cells and regulatory B cells remain controversial. 26 Objective and Methods: In this study, we use multi-color flow cytometry in 27 combination with bioinformatics and functional studies to show that CD24CD38 B cells 28 can be distinguished into multiple subsets with different regulatory functions. 29 Results: The study reveals for the first time that human transitional B cells encompass 30 not only transitional type 1 (T1) and T2 B cells, as previously suggested, but also distinct anergic 31 T3 B cells as well as IL-10-producing CD27 transitional B cells. Interestingly, the latter two 32 subsets differentially regulate CD4 T cell proliferation and polarization towards Th1 effector 33 cells. Additional analyses reveal that the percentage of T3 B cells is reduced while the frequency 34 of CD27 transitional B cells is increased in patients with autoimmune diseases compared with 35 matched healthy individuals. 36 Conclusion: This study provides evidence for the existence of different transitional B cell 37 subsets each displaying unique phenotypic and regulatory functional profiles. Furthermore, the 38 study indicates that altered distribution of transitional B cells subsets highlights different 39 regulatory defects in different autoimmune diseases. 40

Objective Systemic sclerosis ( SS c) has the highest case‐specific mortality of any rheumatic disease, and no effective therapy is available. A clear manifestation of SS c is the presence of autoantibodies. However, the origin of autoantibody‐producing B lymphocytes, their mechanisms of activation and autoantibody production, and their role remain unclear. This study was undertaken to identify mechanisms that contribute to pathogenic B cell generation and involvement in SS c and to assess the altered distribution and function of B cells in SS c patients. Methods Multicolor flow cytometry was performed to determine B cell subset distribution, cytokine production, and tolerance induction in SS c patients and healthy controls. Cytokine production following stimulation of the cells ex vivo was determined by multiplex assay. Results A range of defects in B lymphocyte tolerance and cytokine production in SS c were noted. There was evidence of altered distribution of transitional B cell subsets, increased production of interleukin‐6 ( IL ‐6) and IL ‐8, and defective tolerance induction in SS c B cells. In addition, B cells from SS c patients had a reduced ability to produce IL ‐10 when stimulated through innate immune pathways. In contrast to healthy individuals, tolerance checkpoints in SS c patients failed to suppress the emergence of B cells that produce autoantibodies with specificity to the Scl‐70 antigen, which is strongly associated with SS c. These defects were paralleled by altered intracellular signaling and apoptosis following B cell receptor engagement. Conclusion Our findings provide new insights into mechanisms underlying defective B lymphocyte responses in patients with SS c and their contribution to disease.

B lymphocytes are critical for effective immunity; they produce antibodies and cytokines, present antigens to T lymphocytes and regulate immune responses. However, because of the inherent randomness in the process of generating their vast repertoire of antigen-specific receptors, B cells can also cause diseases through recognizing and reacting to self. Therefore, B lymphocyte selection and responses require tight regulation at multiple levels and at all stages of their development and activation to avoid diseases. Indeed, newly generated B lymphocytes undergo rigorous tolerance mechanisms in the bone marrow and, subsequently, in the periphery after their migration. Furthermore, activation of mature B cells is regulated through controlled expression of co-stimulatory receptors and intracellular signalling thresholds. All these regulatory events determine whether and how B lymphocytes respond to antigens, by undergoing apoptosis or proliferation. However, defects that alter regulated co-stimulatory receptor expression or intracellular signalling thresholds can lead to diseases. For example, autoimmune diseases can result from altered regulation of B cell responses leading to the emergence of high-affinity autoreactive B cells, autoantibody production and tissue damage. The exact cause(s) of defective B cell responses in autoimmune diseases remains unknown. However, there is evidence that defects or mutations in genes that encode individual intracellular signalling proteins lead to autoimmune diseases, thus confirming that defects in intracellular pathways mediate autoimmune diseases. This review provides a synopsis of current knowledge of signalling proteins and pathways that regulate B lymphocyte responses and how defects in these could promote autoimmune diseases. Most of the evidence comes from studies of mouse models of disease and from genetically engineered mice. Some, however, also come from studying B lymphocytes from patients and from genome-wide association studies. Defining proteins and signalling pathways that underpin atypical B cell response in diseases will help in understanding disease mechanisms and provide new therapeutic avenues for precision therapy.

Biologic TNFα inhibitors are a mainstay treatment option for patients with rheumatoid arthritis (RA) refractory to other treatment options. However, many patients either do not respond or relapse after initially responding to these agents. This study was carried out to identify biomarkers that can distinguish responder from non-responder patients before the initiation of treatment. The level of cytokines in plasma and those produced by ex vivo T cells, B cells and monocytes in 97 RA patients treated with biologic TNFα inhibitors was measured before treatment and after 1 and 3 months of treatment by multiplex analyses. The frequency of T cell subsets and intracellular cytokines were determined by flow cytometry. The results reveal that pre-treatment, T cells from patients who went on to respond to treatment with biologic anti-TNFα agents produced significantly more GM-CSF than non-responder patients. Furthermore, immune cells from responder patients produced higher levels of IL-1β, TNFα and IL-6. Cytokine profiling in the blood of patients confirmed the association between high levels of GM-CSF and responsiveness to biologic anti-TNFα agents. Thus, high blood levels of GM-CSF pre-treatment had a positive predictive value of 87.5% (61.6 to 98.5% at 95% CI) in treated RA patients. The study also shows that cells from most anti-TNFα responder patients in the current cohort produced higher levels of GM-CSF and TNFα pre-treatment than non-responder patients. Findings from the current study and our previous observations that non-responsiveness to anti-TNFα is associated with high IL-17 levels suggest that the disease in responder and non-responder RA patients is likely to be driven/sustained by different inflammatory pathways. The use of biomarker signatures of distinct pro-inflammatory pathways could lead to evidence-based prescription of the most appropriate biological therapies for different RA patients.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

TNFα is a principal pro-inflammatory cytokine vital for immunity to infections. However, its excessive production is involved in chronic inflammation and disease pathology in autoimmune diseases. Evidence for its pathogenic role is validated by the fact that its neutralisation by therapeutic agents in vivo is beneficial in ameliorating disease and controlling symptoms. Paradoxically, however, treatment with TNFα inhibitors can either have no clinical effects, or even exacerbate disease in some patients. The explanation for such contradictory outcomes may lay in how and which downstream signalling pathways are activated and drive disease. TNFα causes its effects by binding to either or both of two membrane-bound receptors, TNFR1 and TNFR2. Engagement of the receptors can induce cell death or cell proliferation. T cells both produce and respond to TNFα and depending on whether the cytokine is membrane-bound or soluble and the level of expression of its two receptors, the biological outcome can be distinct. In addition, polymorphisms in genes encoding TNFα and T cell signalling proteins can significantly impact the outcome of TNFα receptor engagement. Early studies revealed that effector T cells in patients with rheumatoid arthritis (RA) are hyporesponsive due to chronic exposure to TNFα. However, recent evidence indicates that the relationship between TNFα and T cell responses is complex and, at times, can be paradoxical. In addition, there is controversy as to the specific effects of TNFα on different T cell subsets. This review will summarise knowledge on how TNFα modulates T cell responses and the effect of engaging either of its two receptors. Furthermore, we discuss how such interactions can dictate the outcome of treatment with TNFα inhibitors.

Background: CD24(high)CD38(high) transitional B cells represent cells at a key stage in their developmental pathway. In addition, these B cells have been widely ascribed regulatory functions and involvement in the control of chronic inflammatory diseases. However, the phenotypic and functional overlap between these cells and regulatory B cells remains controversial.Objective: In this study we wanted to explore the regulatory properties of CD24(high)CD38(high) human B cells.Methods: We used multicolor flow cytometry in combination with bioinformatics and functional studies to show that CD24(high)CD38(high) B cells can be distinguished into multiple subsets with different regulatory functions.Results: For the first time, the study reveals that human transitional B cells encompass not only transitional type 1 and type 2 B cells, as previously suggested, but also distinct anergic type 3 B cells, as well as IL-10-producing CD27(+) transitional B cells. Interestingly, the latter 2 subsets differentially regulate CD4(+) T-cell proliferation and polarization toward T(H)1 effector cells. Additional analyses reveal that the percentage of type 3 B cells is reduced and the frequency of CD27(+) transitional B cells is increased in patients with autoimmune diseases compared with those in matched healthy subjects.Conclusion: This study provides evidence for the existence of different transitional B-cell subsets, each displaying unique phenotypic and regulatory functional profiles. Furthermore, the study indicates that altered distribution of transitional B-cell subsets highlights different regulatory defects in patients with different autoimmune diseases.