The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where antiviral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, inducing STAT1 and STAT4 signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment inhibited IL-15 induced NK cell proliferation in an IFN-I dependent manner and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. Coupled with the direct, OV-mediated killing of tumour cells, the activation of both CD56dim and CD56bright NK cells by antiviral pathways induces a spectrum of activity that includes the NK cell-mediated killing of tumour cells and modulation of adaptive responses via the trafficking of IFN-γ expressing CD56bright NK cells to lymph nodes.

Targeting human plasmacytoid dendritic cells through BDCA2 prevents skin inflammation and fibrosis in a novel xenotransplant mouse model of scleroderma.

OBJECTIVES: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. METHODS: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression. RESULTS: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses. CONCLUSIONS: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc.

Interleukin-7: a potential factor supporting B-cell maturation in the rheumatoid arthritis synovium.

OBJECTIVES: The exact function of interleukin-7 (IL-7) in autoimmune diseases remains unclear although it is a recognised therapeutic target for cytokine blockade. Our objective was to investigate the regulation and downstream effect of IL-7 in diseased tissue from rheumatoid arthritis (RA) patients notably with respect to its function as bone turnover regulator and tissue architecture (TA) organiser. METHODS: Synovial tissues (fresh, frozen or xed) were obtained from our tissue bank and distributed between experiments for live cell cultures, histology, immunohistochemistry or gene expression array by qPCR. RESULTS: IL-7 expression in synoviocyte cultures was up-regulated by pro-in ammatory cytokines, notably IL-6. Gene expression pro ling segregated synovial biopsies based on the presence of B/plasma cells and ectopic TA. IL-7 gene expression was associated with that of several genes whose function was to support B-cell maturation in tissue with distinct B-cell aggregates (despite the lack of IL-7-Receptor expression on B-cells) as well as with ectopic germinal-like centres. IL-7 was associated with bone turnover regulation in biopsies with diffuse in ltration. A novel relationship between the IL-7 and IL-6 axis was also highlighted in human tissue. CONCLUSIONS: Overall, IL-7 may contribute to the maintenance of the pro-in ammatory cycle perpetuating in ammation in RA synovium. We therefore propose a novel role for IL-7 as an orchestrator of TA with an impact on B-cell maturation in relation with IL-6.

Expanding Clinical Phenotype and Novel Insights into the Pathogenesis of ICOS Deficiency.

BACKGROUND: Inducible T cell co-stimulator (ICOS) deficiency has been categorized as a combined immunodeficiency often complicated by enteropathies, autoimmunity, lymphoproliferation, and malignancy. We report seven new patients and four novel ICOS mutations resulting in a common variable immunodeficiency (CVID)-like phenotype and show that dysregulated IL-12 release, reduced cytotoxic T lymphocyte-associated protein 4 (CTLA4) expression, and skewing towards a Th1-dominant phenotype are all associated with inflammatory complications in this condition. METHODS: A combination of whole exome and Sanger sequencing was used to identify novel mutations. Standard clinical and immunological evaluation was performed. FACS and ELISA-based assays were used to study cytokine responses and ICOS/ICOSL/CTLA4 expression following stimulation of whole blood and PBMCs with multiple TLR ligands, anti-CD3, and PHA. RESULTS: Four novel ICOS mutations included homozygous c.323_332del, homozygous c.451C>G, and compound heterozygous c.58+1G>A/c.356T>C. The predominant clinical phenotype was that of antibody deficiency associated with inflammatory complications in 4/7 patients. Six out of seven patients were treated with immunoglobulin replacement and one patient died from salmonella sepsis. All patients who were tested showed reduced IL-10 and IL-17 cytokine responses, normal IL-1ß, IL6, and TNF release following LPS stimulation and highly elevated IL-12 production in response to combined LPS/IFNγ stimulation. This was associated with skewing of CD4+ T cells towards Th1 phenotype and increased expression of ICOSL on monocytes. Lastly, reduced CTLA4 expression was found in 2 patients. One patient treated with ustekinumab for pancytopenia due to granulomatous bone marrow infiltration failed to respond to this targeted therapy. CONCLUSIONS: ICOS deficiency is associated with defective T cell activation, with simultaneously enhanced stimulation of monocytes. The latter is likely to result from a lack of ICOS/ICOSL interaction which might be necessary to provide negative feedback which limits monocytes activation.

Prediction of autoimmune connective tissue disease in an at-risk cohort: prognostic value of a novel two-score system for interferon status.

OBJECTIVE: To evaluate clinical, interferon and imaging predictors of progression from 'At Risk' to autoimmune connective tissue diseases (AI-CTDs). METHODS: A prospective observational study was conducted in At-Risk of AI-CTD (defined as antinuclear antibody (ANA) positive; ≤1 clinical systemic lupus erythematosus (SLE) criterion; symptom duration <12 months and treatment-naïve). Bloods and skin biopsy (non-lesional) were analysed for two interferon-stimulated gene expression scores previously described (IFN-Score-A and IFN-Score-B). Forty-nine healthy controls (HCs) and 114 SLE were used as negative and positive controls. Musculoskeletal ultrasound was performed. Progression was defined by meeting classification criteria for AI-CTDs at 12 months. RESULTS: 118 individuals with 12-month follow-up were included. Of these, 19/118 (16%) progressed to AI-CTD (SLE=14, primary Sjogren's=5). At baseline, both IFN scores differed among At-Risk, HCs and SLE groups (p<0.001) and both were elevated in At-Risk who progressed to AI-CTD at 12 months versus non-progressors, to a greater extent for IFN-Score-B (fold difference (95% CI) 3.22 (1.74 to 5.95), p<0.001) than IFN-Score-A (2.94 (1.14 to 7.54); p=0.018). Progressors did not have significantly greater baseline clinical characteristics or ultrasound findings. Fold difference between At-Risk and HCs for IFN-Score-A was markedly greater in skin than blood. In multivariable logistic regression, only family history of autoimmune rheumatic disease, OR 8.2 (95% CI 1.58 to 42.53) and IFN-Score-B, 3.79 (1.50-9.58) increased the odds of progression. CONCLUSION: A two-factor interferon score and family history predict progression from ANA positivity to AI-CTD. These interferon scores may allow stratification of individuals At-Risk of AI-CTD permitting early intervention for disease prevention and avoid irreversible organ damage.

Scleroderma fibroblasts suppress angiogenesis via TGF-ß/caveolin-1 dependent secretion of pigment epithelium-derived factor.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-ß) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-ß and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. METHODS: PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast-endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. RESULTS: PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-ß in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-ß receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. CONCLUSIONS: Our data reveal a new pathway by which TGF-ß suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

Network Analysis Identifies Proinflammatory Plasma Cell Polarization for Secretion of ISG15 in Human Autoimmunity.

Plasma cells (PCs) as effectors of humoral immunity produce Igs to match pathogenic insult. Emerging data suggest more diverse roles exist for PCs as regulators of immune and inflammatory responses via secretion of factors other than Igs. The extent to which such responses are preprogrammed in B-lineage cells or can be induced in PCs by the microenvironment is unknown. In this study, we dissect the impact of IFNs on the regulatory networks of human PCs. We show that core PC programs are unaffected, whereas PCs respond to IFNs with distinctive transcriptional responses. The IFN-stimulated gene 15 (ISG15) system emerges as a major transcriptional output induced in a sustained fashion by IFN-α in PCs and linked both to intracellular conjugation and ISG15 secretion. This leads to the identification of ISG15-secreting plasmablasts/PCs in patients with active systemic lupus erythematosus. Thus, ISG15-secreting PCs represent a distinct proinflammatory PC subset providing an Ig-independent mechanism of PC action in human autoimmunity.

Predicting and managing primary and secondary non-response to rituximab using B-cell biomarkers in systemic lupus erythematosus.

OBJECTIVE: To assess factors associated with primary and secondary non-response to rituximab in systemic lupus erythematosus (SLE) and evaluate management of secondary non-depletion non-response (2NDNR). METHODS: 125 patients with SLE treated with rituximab over 12 years were studied prospectively. A major clinical response was defined as improvement of all active British Isles Lupus Assessment Group (BILAG)-2004 domains to grade C/better and no A/B flare. Partial responders were defined by one persistent BILAG B. B-cell subsets were measured using highly sensitive flow cytometry. Patients with 2NDNR, defined by infusion reaction and defective depletion, were treated with ocrelizumab or ofatumumab. RESULTS: 117 patients had evaluable data. In cycle 1 (C1), 96/117 (82%) achieved BILAG response (major=50%, partial=32%). In multivariable analysis, younger age (OR 0.97, 95% CI 0.94 to 1.00) and B-cell depletion at 6 weeks (OR 3.22, 95% CI 1.24 to 8.33) increased the odds of major response. Complete depletion was predicted by normal complement and lower pre-rituximab plasmablasts and was not associated with increased serious infection post-rituximab. Seventy-seven (with data on 72) C1 responders were retreated on clinical relapse. Of these, 61/72 (85%) responded in cycle 2 (C2). Of the 11 C2 non-responders, nine met 2NDNR criteria (incidence=12%) and tested positive for anti-rituximab antibodies. Lack of concomitant immunosuppressant and higher pre-rituximab plasmablasts predicted 2NDNR. Five were switched to ocrelizumab/ofatumumab, and all depleted and responded. CONCLUSION: Treatment with anti-CD20 agents can be guided by B-cell monitoring and should aim to achieve complete depletion. 2NDNR is associated with anti-rituximab antibodies, and switching to humanised agents restores depletion and response. In SLE, alternative anti-CD20 antibodies may be more consistently effective.

Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone formation in spondyloarthropathies.

OBJECTIVES.: The SpAs are genetically and therapeutically linked to IL-23, which in turn regulates IL-22, a cytokine that has been implicated in the regulation of new bone formation in experimental models. We hypothesize that IL-22, a master regulator of stem cells in other niches, might also regulate human mesenchymal stem cell (MSC) osteogenesis. METHODS.: The effects of IL-22 on in vitro MSC proliferation, migration and osteogenic differentiation were evaluated in the presence or absence of IFN-γ and TNF (to ascertain IL-22 activity in pro-inflammatory environments). Colorimetric XTT assay, trans-well migration assays, quantitative real-time PCR (qRT-PCR) for MSC lineage markers and osteogenesis assays were used. RESULTS.: Combined treatment of MSC with IL-22, IFN-γ and TNF resulted in increased MSC proliferation ( P = 0.008) and migration ( P = 0.04), an effect that was not seen in cells treated with IL-22 alone and untreated cells. Osteogenic and adipogenic, but not chondrogenic, transcription factors were upregulated by IL-22 alone ( P < 0.05). MSC osteogenesis was enhanced following IL-22 exposure ( P = 0.03, measured by calcium production). The combination of IFN-γ and TNF with or without IL-22 suppressed MSC osteogenesis ( P = 0.03). CONCLUSION.: This work shows that IL-22 is involved in human MSC proliferation/migration in inflammatory environments, with MSC osteogenesis occurring only in the absence of IFN-γ/TNF. These effects of IL-22 on MSC function is a novel pathway for exploring pathological, post-inflammation osteogenesis in human SpA.

Altered natural killer cell subset homeostasis and defective chemotactic responses in paroxysmal nocturnal hemoglobinuria.

In paroxysmal nocturnal hemoglobinuria (PNH), hematopoietic cells lacking glycosylphosphatidylinositol (GPI)-linked proteins on their surface (GPI(neg)) exist alongside normal (GPI+) cells. Analysis of natural killer (NK) cell subsets in 47 PNH patients revealed that the ratio of CD56(bright):CD56(dim) NK cells differed in the GPI+ and GPI(neg) populations, with GPI(neg)CD56(bright) NK cells significantly more abundant in peripheral blood than their normal GPI+ counterparts. Indeed, GPI+CD56(bright) NK cells were not detected in the peripheral blood of some patients, suggesting their trafficking to a niche unavailable to the GPI(neg)CD56(bright) NK cell population. Defective cellular trafficking in this disease was supported by findings showing differential chemokine receptor expression between GPI+ and GPI(neg) NK cells and impaired stromal cell-derived factor 1 (SDF-1)-induced chemotaxis of GPI(neg) NK cells. Our results indicate a role for GPI-linked proteins in NK cell subset homeostasis and suggest that differential chemokine responses might contribute to the balance of GPI+ and GPI(neg) populations in this disease.

Abatacept or tocilizumab after rituximab in rheumatoid arthritis? An exploratory study suggests non-response to rituximab is associated with persistently high IL-6 and better clinical response to IL-6 blocking therapy.

OBJECTIVES: To evaluate the efficacy and safety of two different targeted approaches-abatacept or tocilizumab-after rituximab therapy in rheumatoid arthritis, and to explain observed difference in efficacy using blood and synovial studies of interleukin 6 (IL-6) and B cells in patients receiving rituximab therapy. METHODS: Consecutive series of patients who had discontinued rituximab therapy owing to inefficacy or toxicity were treated with abatacept (n=16) or tocilizumab (n=35). Clinical response and reasons for discontinuation were evaluated. Serial blood and synovial samples were obtained from a group of 57 and 25 rituximab-treated patients, respectively, and were analysed for B cells and IL-6 using flow cytometry, immunohistochemistry and quantitative real-time PCR. RESULTS: In the abatacept group, mean (SEM) Disease Activity Score in 28 joints calculated using the erythrocyte sedimentation rate (DAS28-ESR) reduced from 5.69 (0.42) at baseline to 4.94 (0.44) at 6 months (p=0.12). In the tocilizumab group: mean (SEM) DAS28- ESR reduced from 5.75 (0.21) at baseline to 3.28 (0.26) at 6 months (p<0.001). This was paralleled by a significant swollen joint count reduction in the tocilizumab (5.47 (0.70) to 2.70 (0.61), p=0.033), but not abatacept (6.23 (1.3) to 4.15 (1.2), p=0.26), group. In the synovium, despite complete depletion of B cells in 19/22 patients, IL-6 mRNA expression was not significantly reduced after rituximab. Blood B cell numbers remained low 12 months after rituximab. Serum IL-6 was raised at baseline and significantly higher in rituximab clinical non-responders (p=0.035) than responders. A significant reduction in serum IL-6 was seen in rituximab clinical responders (p=0.005) but not in non-responders (p=0.237). CONCLUSION: In patients with rheumatoid arthritis for whom rituximab therapy failed despite adequate B cell depletion, IL-6-directed therapy might be a more logical and effective treatment choice than T cell costimulation blockade. Further controlled studies investigating other possible mechanisms are needed to validate these initial findings.

An immunological biomarker to predict MTX response in early RA.

OBJECTIVES: The therapeutic goal for patients with rheumatoid arthritis (RA) is clinical remission. This is best achieved by early diagnosis and appropriate therapeutic intervention. RA is associated with dysregulation of T-cell subsets (naïve, regulatory (Treg) and inflammation-related cells (IRC)) early in the disease. Our aim was to test the hypothesis that T-cell subset quantification can predict the achievement of clinical remission with early treatment in RA. METHODS: T-cell subsets were quantified in 108 drug-naïve, early RA patients commencing methotrexate (MTX) or MTX+antitumor necrosis factor (anti-TNF) and in 105 healthy controls (HC). The primary outcome assessed was remission (DAS28<2.6). A pilot study used frozen cells (38 patients and 35 HCs, see online supplementary material) and was validated with fresh blood (70 patients and 70 HCs). RESULTS: Immune dysregulation in early RA was confirmed with an association between age and reduced naïve cells compared with HCs (p=0.006), a lower age-adjusted Treg and higher IRC frequency (p=0.001). Anticitrullinated peptide antibody (ACPA) positivity was associated with lower naïve (p=0.031) and Treg frequencies (p=0.039). In 50 patients treated with MTX, ACPA/age-adjusted analysis demonstrated that higher naïve cell frequency (relative to HC) was associated with remission (OR 5.90 (1.66 to 20.98), p=0.006, sensitivity/specificity 62%/79%, Positive Predictive Value (PPV)/Negative Predictive Value (NPV) 66%/76%). Remission with MTX+anti-TNF (n=20) was not found to be associated with naïve cell frequency, and for patients with reduced naïve cells the remission rate increased from 24% (MTX) to 42% (MTX+anti-TNF). CONCLUSIONS: Baseline T-cell subset analysis has a value in predicting early RA remission with first therapy with MTX. Immunological analysis could be used in conjunction with clinical/serological features to predict response to MTX and help select the most appropriate therapy at disease presentation.

A human NK cell activation/inhibition threshold allows small changes in the target cell surface phenotype to dramatically alter susceptibility to NK cells.

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing's sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing's sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.

Comprehensive genetic and functional analyses of Fc gamma receptors influence on response to rituximab therapy for autoimmunity.

BACKGROUND: Rituximab is widely used to treat autoimmunity but clinical response varies. Efficacy is determined by the efficiency of B-cell depletion, which may depend on various Fc gamma receptor (FcγR)-dependent mechanisms. Study of FcγR is challenging due to the complexity of the FCGR genetic locus. We sought to assess the effect of FCGR variants on clinical response, B-cell depletion and NK-cell-mediated killing in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). METHODS: A longitudinal cohort study was conducted in 835 patients [RA = 573; SLE = 262]. Clinical outcome measures were two-component disease activity score in 28-joints (2C-DAS28CRP) for RA and British Isles Lupus Assessment Group (BILAG)-2004 major clinical response (MCR) for SLE at 6 months. B-cells were evaluated by highly-sensitive flow cytometry. Single nucleotide polymorphism and copy number variation for genes encoding five FcγRs were measured using multiplex ligation-dependent probe amplification. Ex vivo studies assessed NK-cell antibody-dependent cellular cytotoxicity (ADCC) and FcγR expression. FINDINGS: In RA, carriage of FCGR3A-158V and increased FCGR3A-158V copies were associated with greater 2C-DAS28CRP response (adjusted for baseline 2C-DAS28CRP). In SLE, MCR was associated with increased FCGR3A-158V, OR 1.64 (95% CI 1.12-2.41) and FCGR2C-ORF OR 1.93 (95% CI 1.09-3.40) copies. 236/413 (57%) patients with B-cell data achieved complete depletion. Homozygosity for FCGR3A-158V and increased FCGR3A-158V copies were associated with complete depletion in combined analyses. FCGR3A genotype was associated with rituximab-induced ADCC, and increased NK-cell FcγRIIIa expression was associated with improved clinical response and depletion in vivo. Furthermore, disease status and concomitant therapies impacted both NK-cell FcγRIIIa expression and ADCC. INTERPRETATION: FcγRIIIa is the major low affinity FcγR associated with rituximab response. Increased copies of the FCGR3A-158V allele (higher affinity for IgG1), influences clinical and biological responses to rituximab in autoimmunity. Enhancing FcγR-effector functions could improve the next generation of CD20-depleting therapies and genotyping may stratify patients for optimal treatment protocols. FUNDING: Medical Research Council, National Institute for Health and Care Research, Versus Arthritis.

Head and neck squamous cell carcinoma: mismatch repair immunohistochemistry and promoter hypermethylation of hMLH1 gene.

UNLABELLED: Squamous cell carcinomas of the head and neck are the sixth most frequently occurring cancers and the seventh leading cause of cancer-related deaths worldwide. Epigenetic alteration, using promoter hypermethylation of hMLH1 gene, is important for the development of head and neck squamous cell carcinoma (HNSCC). AIM OF THIS WORK: The aim of the present study is to analyze the relationship between protein expression and promoter hypermethylation of the hMLH1 gene in HNSCC and correlating inactivation of this gene with clinical parameters. MATERIALS AND METHODS: Paired normal and tumor specimens from 49 patients with HNSCC were collected from Otolaryngology Department, Minia University Hospital, from 2006 to 2009. We analyzed hMLH1 protein expression and promoter hypermethylation by immunohistochemical and methylation-specific polymerase chain reaction (MSP). RESULTS: Decreased hMLH1 protein expression and hMLH1 promoter hypermethylation were shown in 15 (30.6%) and 14 (28.6%) cases, respectively. Eleven cases showed dysplasia and or carcinoma in situ in the surface squamous epithelia, and all were positively stained for the hMLH1 protein. hMLH1 promoter hypermethylation was detected in 10 (20.4%) cases of normal-appearing squamous mucosa adjacent to invasive carcinoma. Thirteen (86.7%) of 15 cases that were negative for the hMLH1 protein showed promoter hypermethylation, whereas 33 (97%) of 34 cases positive for the protein were negative of promoter methylation. Promoter hypermethylation was detected in 1 (7.1%) case in which invasive tumor cells were moderately positive for the hMLH1 protein. No significant correlation was observed between hMLH1 protein expression or hMLH1 promoter hypermethylation and any of clinicopathologic parameters. CONCLUSIONS: hMLH1 gene may be detected early in head and neck squamous carcinogenesis. Promoter hypermethylation is an important mechanism for hMLH1 gene inactivation in HNSCC.

Multipotent Mesenchymal Stromal Cells in Rheumatoid Arthritis and Systemic Lupus Erythematosus; From a Leading Role in Pathogenesis to Potential Therapeutic Saviors?

The pathogenesis of the autoimmune rheumatological diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is complex with the involvement of several immune cell populations spanning both innate and adaptive immunity including different T-lymphocyte subsets and monocyte/macrophage lineage cells. Despite therapeutic advances in RA and SLE, some patients have persistent and stubbornly refractory disease. Herein, we discuss stromal cells' dual role, including multipotent mesenchymal stromal cells (MSCs) also used to be known as mesenchymal stem cells as potential protagonists in RA and SLE pathology and as potential therapeutic vehicles. Joint MSCs from different niches may exhibit prominent pro-inflammatory effects in experimental RA models directly contributing to cartilage damage. These stromal cells may also be key regulators of the immune system in SLE. Despite these pro-inflammatory roles, MSCs may be immunomodulatory and have potential therapeutic value to modulate immune responses favorably in these autoimmune conditions. In this review, the complex role and interactions between MSCs and the haematopoietically derived immune cells in RA and SLE are discussed. The harnessing of MSC immunomodulatory effects by contact-dependent and independent mechanisms, including MSC secretome and extracellular vesicles, is discussed in relation to RA and SLE considering the stromal immune microenvironment in the diseased joints. Data from translational studies employing MSC infusion therapy against inflammation in other settings are contextualized relative to the rheumatological setting. Although safety and proof of concept studies exist in RA and SLE supporting experimental and laboratory data, robust phase 3 clinical trial data in therapy-resistant RA and SLE is still lacking.

SARS-CoV-2 Infection Induces Psoriatic Arthritis Flares and Enthesis Resident Plasmacytoid Dendritic Cell Type-1 Interferon Inhibition by JAK Antagonism Offer Novel Spondyloarthritis Pathogenesis Insights.

Objective: Bacterial and viral infectious triggers are linked to spondyloarthritis (SpA) including psoriatic arthritis (PsA) development, likely via dendritic cell activation. We investigated spinal entheseal plasmacytoid dendritic cells (pDCs) toll-like receptor (TLR)-7 and 9 activation and therapeutic modulation, including JAK inhibition. We also investigated if COVID-19 infection, a potent TLR-7 stimulator triggered PsA flares. Methods: Normal entheseal pDCs were characterized and stimulated with imiquimod and CpG oligodeoxynucleotides (ODN) to evaluate TNF and IFNα production. NanoString gene expression assay of total pDCs RNA was performed pre- and post- ODN stimulation. Pharmacological inhibition of induced IFNα protein was performed with Tofacitinib and PDE4 inhibition. The impact of SARS-CoV2 viral infection on PsA flares was evaluated. Results: CD45+HLA-DR+CD123+CD303+CD11c- entheseal pDCs were more numerous than blood pDCs (1.9 ± 0.8% vs 0.2 ± 0.07% of CD45+ cells, p=0.008) and showed inducible IFNα and TNF protein following ODN/imiquimod stimulation and were the sole entheseal IFNα producers. NanoString data identified 11 significantly upregulated differentially expressed genes (DEGs) including TNF in stimulated pDCs. Canonical pathway analysis revealed activation of dendritic cell maturation, NF-κB signaling, toll-like receptor signaling and JAK/STAT signaling pathways following ODN stimulation. Both tofacitinib and PDE4i strongly attenuated ODN induced IFNα. DAPSA scores elevations occurred in 18 PsA cases with SARS-CoV2 infection (9.7 ± 4 pre-infection and 35.3 ± 7.5 during infection). Conclusion: Entheseal pDCs link microbes to TNF/IFNα production. SARS-CoV-2 infection is associated with PsA Flares and JAK inhibition suppressed activated entheseal plasmacytoid dendritic Type-1 interferon responses as pointers towards a novel mechanism of PsA and SpA-related arthropathy.