OPTIMAL: An OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration.

Analysis of imaging mass cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single-cell segmentation and suboptimal approaches for data visualization and exploration. This can lead to inaccurate identification of cell phenotypes, states, or spatial relationships compared to reference data from single-cell suspension technologies. To this end we have developed the "OPTimized Imaging Mass cytometry AnaLysis (OPTIMAL)" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualization/clustering, and spatial neighborhood analysis. Using a panel of 27 metal-tagged antibodies recognizing well-characterized phenotypic and functional markers to stain the same Formalin-Fixed Paraffin Embedded (FFPE) human tonsil sample tissue microarray over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, 5 different dimensionality reduction algorithms, and 2 clustering methods. Finally, we assessed the optimal approach for performing neighborhood analysis. We found that single-cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bivariate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximized the statistical separation between negative and positive signal distributions and a simple Z-score normalization step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing phenograph in terms of cell type identification. We also found that neighborhood analysis was influenced by the method used for finding neighboring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly, OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output and allows for single-cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses.

Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24(+)CD64(-) DCs and contaminating CSF-1R-dependent CD24(-)CD64(+) macrophages. Functionally, loss of CD24(+)CD11b(+) DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24(+)CD11b(+) DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies.

Targeting of tolerogenic dendritic cells to heat-shock proteins in inflammatory arthritis.

BACKGROUND: Autologous tolerogenic dendritic cells (tolDC) are a promising therapeutic strategy for inflammatory arthritis (IA) as they can regulate autoantigen-specific T cell responses. Here, we investigated two outstanding priorities for clinical development: (i) the suitability of using heat-shock proteins (HSP), abundant in inflamed synovia, as surrogate autoantigens to be presented by tolDC and (ii) identification of functional biomarkers that confirm tolDC regulatory activity. METHODS: Cell proliferation dye-labelled human peripheral blood mononuclear cells of IA (rheumatoid arthritis (RA) and psoriatic arthritis (PsA)) patients or healthy donors were cultured with HSP40-, HSP60- and HSP70-derived peptides or recall antigens (e.g. tuberculin purified protein derivative (PPD)) in the presence or absence of tolDC or control DC for 9 days. Functional characteristics of proliferated antigen-specific T-cells were measured using flow cytometry, gene expression profiling and cytokine secretion immunoassays. Repeated measures analysis of variance (ANOVA) with Bonferroni correction for comparisons between multiple groups and paired Student t test for comparisons between two groups were used to determine significance. RESULTS: All groups showed robust CD4+ T-cell responses towards one or more HSP-derived peptide(s) as assessed by a stimulation index > 2 (healthy donors: 78%, RA: 73%, PsA: 90%) and production of the cytokines IFNγ, IL-17A and GM-CSF. Addition of tolDC but not control DC induced a type 1 regulatory (Tr1) phenotype in the antigen-specific CD4+ T-cell population, as identified by high expression of LAG3, CD49b and secretion of IL-10. Furthermore, tolDC inhibited bystander natural killer (NK) cell activation in a TGFß dependent manner. CONCLUSIONS: HSP-specific CD4+ T-cells are detectable in the majority of RA and PsA patients and can be converted into Tr1 cells by tolDC. HSP-loaded tolDC may therefore be suitable for directing T regulatory responses to antigens in inflamed synovia of IA patients. Tr1 markers LAG3, CD49b and IL-10 are suitable biomarkers for future tolDC clinical trials.

Targeting of tolerogenic dendritic cells towards heat-shock proteins: a novel therapeutic strategy for autoimmune diseases?

Tolerogenic dendritic cells (tolDCs) are a promising therapeutic tool to restore immune tolerance in autoimmune diseases. The rationale of using tolDCs is that they can specifically target the pathogenic T-cell response while leaving other, protective, T-cell responses intact. Several ways of generating therapeutic tolDCs have been described, but whether these tolDCs should be loaded with autoantigen(s), and if so, with which autoantigen(s), remains unclear. Autoimmune diseases, such as rheumatoid arthritis, are not commonly defined by a single, universal, autoantigen. A possible solution is to use surrogate autoantigens for loading of tolDCs. We propose that heat-shock proteins may be a relevant surrogate antigen, as they are evolutionarily conserved between species, ubiquitously expressed in inflamed tissues and have been shown to induce regulatory T cells, ameliorating disease in various arthritis mouse models. In this review, we provide an overview on how immune tolerance may be restored by tolDCs, the problem of selecting relevant autoantigens for loading of tolDCs, and why heat-shock proteins could be used as surrogate autoantigens.

Reduced TCR-dependent activation through citrullination of a T-cell epitope enhances Th17 development by disruption of the STAT3/5 balance.

Citrullination is a post-translational modification of arginine that commonly occurs in inflammatory tissues. Because T-cell receptor (TCR) signal quantity and quality can regulate T-cell differentiation, citrullination within a T-cell epitope has potential implications for T-cell effector function. Here, we investigated how citrullination of an immunedominant T-cell epitope affected Th17 development. Murine naïve CD4(+) T cells with a transgenic TCR recognising p89-103 of the G1 domain of aggrecan (agg) were co-cultured with syngeneic bone marrow-derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro-Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL-2, expressed lower levels of the IL-2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL-2 blockade in native p89-103-primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post-translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th-cell development in inflammatory disorders.

T-regulatory cells exhibit a biphasic response to prolonged endurance exercise in humans.

PURPOSE: T-regulatory cells (Tregs) are a sub-population of lymphocytes that act to suppress aberrant immune responses. We investigated changes in the numbers of naïve and terminally differentiated Tregs in the peripheral blood to establish their role in the immuno-suppressive response to prolonged exercise. METHODS: Blood was drawn from seventeen experienced runners (age 40 ± 12 years; height 1.75 ± 0.08 m; mass 71.4 ± 10.8 kg) before, ~1 h after (POST-1h), and on the day following the marathon (POST-1d). Tregs (CD3+CD4+Foxp3+CD25++CD127-) were analysed in peripheral blood mononuclear cells using flow cytometry. The markers CD45RA and HLA-DR were included to define naïve and terminally differentiated Tregs, respectively. RESULTS: The absolute number of Tregs decreased (27%) POST-1h marathon (P < 0.001) but increased (21%) at POST-1d (P < 0.01). Naïve CD45RA+ Tregs fell by 39% POST-1h (P < 0.01) but were unaffected POST-1d (P > 0.05). In contrast, an increased number of Tregs expressing HLA-DR was observed at POST-1d (P < 0.01). Interleukin (IL)-1ß, IL-6, IL-8 and IL-10 levels in the serum all increased POST-1h (P > 0.05) but returned to pre-exercise levels POST-1d. The suppressive cytokine, transforming growth factor-beta, was unaffected by the marathon (P > 0.05). CONCLUSIONS: These results suggest that Tregs do not play a major role in immune suppression in the early hours of recovery from a marathon. However, terminally differentiated HLA-DR+ Tregs are mobilized the following day, which could represent a compensatory attempt by the host to restore immune homeostasis and limit excessive cell damage.

Dendritic cell maturation and survival are differentially regulated by TNFR1 and TNFR2.

The capacity of dendritic cells (DC) to regulate adaptive immunity is controlled by their maturation state and lifespan. Although TNF is a well-known maturation and survival factor for DC, the role of the two TNFR, TNFR1 and TNFR2, in mediating these effects is poorly understood. By using unique TNF variants that selectively signal through TNFR1 and/or TNFR2, we demonstrate differential functions of TNFR in human monocyte-derived and blood CD1c(+) DC. Activation of TNFR1, but not TNFR2, efficiently induced DC maturation, as defined by enhanced expression of cell surface maturation markers (CD83, CD86, and HLA-DR) as well as enhanced T cell stimulatory capacity. In contrast, both TNFR1 and TNFR2 significantly protected DC against cell death, indicating that innate signals can promote DC survival in the absence of DC maturation. We further show differential activation of NF-κB signaling pathways by the TNFR: TNFR1 activated both the p65 and p52 pathways, whereas TNFR2 triggered p52, but not p65, activation. Accordingly, the p65 NF-κB pathway only played a role in the prosurvival effect of TNFR1. However, cell death protection through both TNFR was mediated through the Bcl-2/Bcl-xL pathway. Taken together, our data show that TNFR1, but not TNFR2, signaling induces DC maturation, whereas DC survival can be mediated independently through both TNFR. These data indicate differential but partly overlapping responses through TNFR1 and TNFR2 in both inflammatory and conventional DC, and they demonstrate that DC maturation and DC survival can be regulated through independent signaling pathways.

A negative feedback loop mediated by STAT3 limits human Th17 responses.

The transcription factor STAT3 is critically required for the differentiation of Th17 cells, a T cell subset involved in various chronic inflammatory diseases. In this article, we report that STAT3 also drives a negative-feedback loop that limits the formation of IL-17-producing T cells within a memory population. By activating human memory CD4(+)CD45RO(+) T cells at a high density (HiD) or a low density (LoD) in the presence of the pro-Th17 cytokines IL-1ß, IL-23, and TGF-ß, we observed that the numbers of Th17 cells were significantly higher under LoD conditions. Assessment of STAT3 phosphorylation revealed a more rapid and stronger STAT3 activation in HiD cells than in LoD cells. Transient inhibition of active STAT3 in HiD cultures significantly enhanced Th17 cell numbers. Expression of the STAT3-regulated ectonucleotidase CD39, which catalyzes ATP hydrolysis, was higher in HiD, than in LoD, cell cultures. Interestingly, inhibition of CD39 ectonucleotidase activity enhanced Th17 responses under HiD conditions. Conversely, blocking the ATP receptor P2X7 reduced Th17 responses in LoD cultures. These data suggest that STAT3 negatively regulates Th17 cells by limiting the availability of ATP. This negative-feedback loop may provide a safety mechanism to limit tissue damage by Th17 cells during chronic inflammation. Furthermore, our results have relevance for the design of novel immunotherapeutics that target the STAT3-signaling pathway, because inhibition of this pathway may enhance, rather than suppress, memory Th17 responses.

Clinical Use of Tolerogenic Dendritic Cells-Harmonization Approach in European Collaborative Effort.

The number of patients with autoimmune diseases and severe allergies and recipients of transplants increases worldwide. Currently, these patients require lifelong administration of immunomodulatory drugs. Often, these drugs are expensive and show immediate or late-occurring severe side effects. Treatment would be greatly improved by targeting the cause of autoimmunity, that is, loss of tolerance to self-antigens. Accumulating knowledge on immune mechanisms has led to the development of tolerogenic dendritic cells (tolDC), with the specific objective to restrain unwanted immune reactions in the long term. The first clinical trials with tolDC have recently been conducted and more tolDC trials are underway. Although the safety trials have been encouraging, many questions relating to tolDC, for example, cell-manufacturing protocols, administration route, amount and frequency, or mechanism of action, remain to be answered. Aiming to join efforts in translating tolDC and other tolerogenic cellular products (e.g., Tregs and macrophages) to the clinic, a European COST (European Cooperation in Science and Technology) network has been initiated-A FACTT (action to focus and accelerate cell-based tolerance-inducing therapies). A FACTT aims to minimize overlap and maximize comparison of tolDC approaches through establishment of minimum information models and consensus monitoring parameters, ensuring that progress will be in an efficient, safe, and cost-effective way.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19.

BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

The choice of adjuvant determines the cytokine profile of T cells in proteoglycan-induced arthritis but does not influence disease severity.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) -dependent disease that is induced with antigen in complete Freund's adjuvant, whereas PGIA is Th1-mediated and is induced using antigen in dimethyldioctadecyl-ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T-cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan-T-cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin-17 (IL-17) produced by T cells isolated from CFA-immunized mice after antigen-specific stimulation were not significantly different from those found in DDA-immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL-17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity.

Interplay between biomaterials and the immune system: Challenges and opportunities in regenerative medicine.

The use of biomaterials for tissue engineering and regenerative medicine applications has increased dramatically over recent years. However, the clinical uptake of a wide variety of biomaterials remains limited due to adverse effects commonly exhibited by patients, which are caused by the host immune response. Despite this, current in vitro evaluation standards (ISO-10993) for assessing the host response to biomaterials have limitations in predicting the likelihood of in vivo biomaterial acceptance. Furthermore, endotoxin contamination of biomaterials is rarely considered, despite its ability to significantly affect the performance of biomaterials and engineered tissues. This review highlights the importance of the immune response to biomaterials and discusses existing challenges and opportunities in the development and standardised assessment of the immune response to biomaterials, including the importance of endotoxin levels. In addition, the properties of biomaterials that impact the host immune response and the exploitation of immunomodulatory biomaterials in regenerative medicine are explored. Finally, a standardised in vitro pathway of evaluating the immune response to biomaterials (hydrogels) and their regenerative potential is proposed, aiming to ensure safety and consistency, while reducing costs and the use of animals in the biomaterials research for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: This review presents a critical analysis of the role of the interactions between the immune system and biomaterials in determining the therapeutic success of biomaterial-based approaches. No such review addressing the lack of understanding of biomaterial-immune system interactions during the developmental and pre-clinical stages of biomaterials, including the impact of the endotoxin levels of biomaterials on the immune response, is published. As there is a lack of in vitro regulations to evaluate the immune response to biomaterials, a standardised in vitro pathway to evaluate the immune response to biomaterials (hydrogels) and their immunomodulatory and regenerative potential for use in tissue engineering/regenerative medicine applications is presented. The aim of the proposed pathway of biomaterial evaluation is to ensure safety and consistency in the biomaterials research community, while reducing costs and animal use (through the concept of the 3R's - reduction, refinement, and replacement of animals).

In Vitro Generation of Human Tolerogenic Monocyte-Derived Dendritic Cells.

Human monocyte-derived dendritic cells (moDC) are commonly used as a research tool to investigate interactions between antigen-presenting cells and T cells. Generation of these cells involves the isolation of CD14 positive monocytes from peripheral blood and their in vitro differentiation into immature moDC by the cytokines GM-CSF and IL-4. Their functional characteristics can then be manipulated by maturing these cells with a cocktail of agents, which can be tailored to induce either immune activating or tolerogenic properties. Here, we describe a protocol for the generation of moDC with stable tolerogenic function, referred to as tolerogenic dendritic cells. These cells have been developed as an immunotherapeutic tool for the treatment of autoimmune disease but have also proven useful to dissect mechanisms of T cell tolerance induction in vitro.

Challenges in tolerogenic dendritic cell therapy for autoimmune diseases: the route of administration.

Tolerogenic dendritic cells (tolDCs) are a promising strategy to treat autoimmune diseases since they have the potential to re-educate and modulate pathological immune responses in an antigen-specific manner and, therefore, have minimal adverse effects on the immune system compared to conventional immunosuppressive treatments. TolDC therapy has demonstrated safety and efficacy in different experimental models of autoimmune disease, such as multiple sclerosis (MS), type 1 diabetes (T1D), and rheumatoid arthritis (RA). Moreover, data from phase I clinical trials have shown that therapy with tolDCs is safe and well tolerated by MS, T1D, and RA patients. Nevertheless, various parameters need to be optimized to increase tolDC efficacy. In this regard, one important parameter to be determined is the most appropriate route of administration. Several delivery routes, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranodal, and intraarticular routes, have been used in experimental models as well as in phase I clinical trials. This review summarizes data obtained from preclinical and clinical studies of tolDC therapy in the treatment of MS, T1D, and RA and their animal models, as well as data from the context of cancer immunotherapy using mature peptide-loaded DC, and data from in vivo cell tracking experiments, to define the most appropriate route of tolDC administration in relation to the most feasible, safest, and effective therapeutic use.

Tolerogenic dendritic cell reporting: Has a minimum information model made a difference?

Minimum information models are reporting frameworks that describe the essential information that needs to be provided in a publication, so that the work can be repeated or compared to other work. In 2016, Minimum Information about Tolerogenic Antigen-Presenting cells (MITAP) was created to standardize the reporting on tolerogenic antigen-presenting cells, including tolerogenic dendritic cells (tolDCs). tolDCs is a generic term for dendritic cells that have the ability to (re-)establish immune tolerance; they have been developed as a cell therapy for autoimmune diseases or for the prevention of transplant rejection. Because protocols to generate these therapeutic cells vary widely, MITAP was deemed to be a pivotal reporting tool by and for the tolDC community. In this paper, we explored the impact that MITAP has had on the tolDC field. We did this by examining a subset of the available literature on tolDCs. Our analysis shows that MITAP is used in only the minority of relevant papers (14%), but where it is used the amount of metadata available is slightly increased over where it is not. From this, we conclude that MITAP has been a partial success, but that much more needs to be done if standardized reporting is to become common within the discipline.

Effective Endotoxin Removal from Chitosan That Preserves Chemical Structure and Improves Compatibility with Immune Cells.

Chitosan is one of the most researched biopolymers for healthcare applications, however, being a naturally derived polymer, it is susceptible to endotoxin contamination, which elicits pro-inflammatory responses, skewing chitosan's performance and leading to inaccurate conclusions. It is therefore critical that endotoxins are quantified and removed for in vivo use. Here, heat and mild NaOH treatment are investigated as facile endotoxin removal methods from chitosan. Both treatments effectively removed endotoxin to below the FDA limit for medical devices (<0.5 EU/mL). However, in co-culture with peripheral blood mononuclear cells (PBMCs), only NaOH-treated chitosan prevented TNF-α production. While endotoxin removal is the principal task, the preservation of chitosan's structure is vital for the synthesis and lysozyme degradation of chitosan-based hydrogels. The chemical properties of NaOH-treated chitosan (by FTIR-ATR) were significantly similar to its native composition, whereas the heat-treated chitosan evidenced macroscopic chemical and physical changes associated with the Maillard reaction, deeming this treatment unsuitable for further applications. Degradation studies conducted with lysozyme demonstrated that the degradation rates of native and NaOH-treated chitosan-genipin hydrogels were similar. In vitro co-culture studies showed that NaOH hydrogels did not negatively affect the cell viability of monocyte-derived dendritic cells (moDCs), nor induce phenotypical maturation or pro-inflammatory cytokine release.

A CD4 T cell gene signature for early rheumatoid arthritis implicates interleukin 6-mediated STAT3 signalling, particularly in anti-citrullinated peptide antibody-negative disease.

OBJECTIVE: We sought clinically relevant predictive biomarkers present in CD4 T-cells, or in serum, that identified those patients with undifferentiated arthritis (UA) who subsequently develop rheumatoid arthritis (RA). METHODS: Total RNA was isolated from highly purified peripheral blood CD4 T cells of 173 early arthritis clinic patients. Paired serum samples were also stored. Microarray analysis of RNA samples was performed and differential transcript expression among 111 'training cohort' patients confirmed using real-time quantitative PCR. Machine learning approaches tested the utility of a classification model among an independent validation cohort presenting with UA (62 patients). Cytokine measurements were performed using a highly sensitive electrochemiluminescence detection system. RESULTS: A 12-gene transcriptional 'signature' identified RA patients in the training cohort and predicted the subsequent development of RA among UA patients in the validation cohort (sensitivity 68%, specificity 70%). STAT3-inducible genes were over-represented in the signature, particularly in anti-citrullinated peptide antibody-negative disease, providing a risk metric of similar predictive value to the Leiden score in seronegative UA (sensitivity 85%, specificity 75%). Baseline levels of serum interleukin 6 (IL-6) (which signals via STAT3) were highest in anti-citrullinated peptide antibodies-negative RA and distinguished this subgroup from non-RA inflammatory synovitis (corrected p<0.05).Paired serum IL-6 measurements correlated strongly with STAT3-inducible gene expression. CONCLUSION: The authors have identified IL-6-mediated STAT-3 signalling in CD4 T cells during the earliest clinical phase of RA, which is most prominent in seronegative disease. While highlighting potential biomarker(s) for early RA, the role of this pathway in disease pathogenesis awaits clarification.

Immunity 12 years after alemtuzumab in RA: CD5⁺ B-cell depletion, thymus-dependent T-cell reconstitution and normal vaccine responses.

OBJECTIVES: Lymphocyte depleting therapies have been used to treat refractory autoimmune disease, including RA, but treatment may be associated with long-term lymphopenia. It is unclear whether delayed reconstitution preferentially affects lymphocyte subsets, how this modulates immune challenges and whether thymic function influences the outcome. These questions are now addressed in a detailed analysis of RA patients 12 years after alemtuzumab (anti-CD52) treatment. METHODS: Blood was obtained from 20 RA patients 12 years after alemtuzumab treatment. Lymphocyte subsets were enumerated by flow cytometry. T-cell receptor excision circles (TRECs)/ml were determined to quantify thymic function, and serological responses to neoantigens and recall antigens were assessed. RESULTS: RA patients remained lymphopenic 12 years after their first dose of alemtuzumab. CD5(+) B cells, which may be associated with autoantibody production, were significantly reduced in alemtuzumab-treated patients compared with age-matched disease controls. In addition, naïve and memory CD4(+) T-cell subsets were present in altered proportions in patients who had received alemtuzumab, with increased effector memory CD4(+) T cells, and decreased naïve and central memory CD4(+) T cells. TRECs were detectable in alemtuzumab-treated patients and correlated with CD4(+) lymphocyte counts. Vaccine responses to neoantigens and recall antigens fell within the normal range for an ageing population. CONCLUSIONS: Alemtuzumab therapy resulted in long-term alterations in lymphocyte subsets. The significance of these changes remains uncertain but patients respond normally to antigenic challenges. Thymic function remains an important determinant of T-cell reconstitution even several years after lymphocytotoxic therapy.

Low-strength T-cell activation promotes Th17 responses.

We show that the strength of T-cell stimulation determines the capability of human CD4(+) T cells to become interleukin-17 (IL-17) producers. CD4(+) T cells received either high- (THi) or low (TLo)-strength stimulation via anti-CD3/CD28 beads or dendritic cells pulsed with superantigen in the presence of pro-Th17 cytokines IL-1ß, transforming growth factor ß, and IL-23. We found that TLo, but not THi, stimulation profoundly promoted Th17 responses by enhancing both the relative proportion and total number of Th17 cells. Titration of anti-CD3 revealed that low TCR signaling promoted Th17 cells, but only in the presence of anti-CD28. Impaired IL-17 production in THi cells could not be explained by high levels of Foxp3 or transforming growth factor ß-latency-associated peptide expressed by THi cells. Nuclear factor of activated T cells was translocated to the nucleus in both THi and TLo cells, but only bound to the proximal region of the IL-17 promoter in TLo cells. The addition of a Ca(2+) ionophore under TLo conditions reversed the pro-Th17 effect, suggesting that high Ca(2+) signaling impairs Th17 development. Although our data do not distinguish between priming of naive T cells versus expansion/differentiation of memory T cells, our results clearly establish an important role for the strength of T-cell activation in regulating Th17 responses.