The effect of abatacept on T-cell activation is not long-lived in vivo.

Abatacept, a co-stimulatory blocker comprising the extracellular portion of human CTLA-4 linked to the Fc region of IgG1, is approved for the treatment of rheumatoid arthritis. By impairing the interaction between CD28 on T cells and CD80/CD86 on APCs, its mechanisms of action include the suppression of follicular T helper cells (preventing the breach of self-tolerance in B cells), inhibition of cell cycle progression holding T cells in a state described as 'induced naïve' and reduction in DC conditioning. However, less is known about how long these inhibitory effects might last, which is a critical question for therapeutic use in patients. Herein, employing a murine model of OVA-induced DTH, we demonstrate that the effect of abatacept is short-lived in vivo and that the inhibitory effects diminish markedly when treatment is ceased.

Orally administered antigen can reduce or exacerbate pathology in an animal model of inflammatory arthritis dependent upon the timing of administration.

Currently, treatments for rheumatoid arthritis (RA) are focussed on management of disease symptoms rather than addressing the cause of disease, which could lead to remission and cure. Central to disease development is the induction of autoimmunity through a breach of self-tolerance. Developing approaches to re-establish antigen specific tolerance is therefore an important emerging area of RA research. A crucial step in this research is to employ appropriate animal models to test prospective antigen specific immunotherapies. In this short communication, we evaluate our previously developed model of antigen specific inflammatory arthritis in which ovalbumin-specific T cell receptor transgenic T cells drive breach of tolerance to endogenous antigens to determine the impact that the timing of therapy administration has upon disease progression. Using antigen feeding to induce tolerance we demonstrate that administration prior to articular challenge results in a reduced disease score as evidenced by pathology and serum antibody responses. By contrast, feeding antigen after initiation of disease had the opposite effect and resulted in the exacerbation of pathology. These preliminary data suggest that the timing of antigen administration may be key to the success of tolerogenic immunotherapies. This has important implications for the timing of potential tolerogenic therapies in patients.

Dissecting the molecular control of immune cell accumulation in the inflamed joint.

Mechanisms governing entry and exit of immune cells into and out of inflamed joints remain poorly understood. We sought herein to identify the key molecular pathways regulating such migration. Using murine models of inflammation in conjunction with mice expressing a photoconvertible fluorescent protein, we characterized the migration of cells from joints to draining lymph nodes and performed RNA-Seq analysis on isolated cells, identifying genes associated with migration and retention. We further refined the gene list to those specific for joint inflammation. RNA-Seq data revealed pathways and genes previously highlighted as characteristic of rheumatoid arthritis in patient studies, validating the methodology. Focusing on pathways associated with cell migration, adhesion, and movement, we identified genes involved in the retention of immune cells in the inflamed joint, namely junctional adhesion molecule A (JAM-A), and identified a role for such molecules in T cell differentiation in vivo. Thus, using a combination of cell-tracking approaches and murine models of inflammatory arthritis, we identified genes, pathways, and anatomically specific tissue signatures regulating cell migration in a variety of inflamed sites. This skin- and joint-specific data set will be an invaluable resource for the identification of therapeutic targets for arthritis and other inflammatory disorders.

Tofacitinib inhibits CD4 T cell polarisation to Th1 during priming thereby leading to clinical impact in a model of experimental arthritis.

OBJECTIVES: Janus kinases (JAK) are key cell membrane orientated tyrosine kinases that regulate inflammatory responses by transducing signals received by cytokine receptors that directly influence the polarisation and function of Th cells. Tofacitinib is a pan-JAK inhibitor approved for the treatment of RA. In this study, we explored the effects of tofacitinib in the outcomes of CD4+ T cell-dendritic cell (DC) interactions and their impact in autoimmune arthritis. METHODS: The impact of tofacitinib in CD4+ T cell outcomes during priming or re-activation were analysed using antigen-specific in vitro and/or in vivo systems. A breach of self-tolerance model of arthritis was used to investigate the effects of tofacitinib in the outcomes of newly primed and antigen experienced CD4+ T cells. RESULTS: Tofacitinib inhibited Th1 polarisation during priming both in vitro and in vivo. In vitro, impaired T-bet expression and IFN-y production persisted upon secondary antigen challenge. Tofacitinib treatment during re-activation in vitro did not impact differentiation of antigen experienced CD4+ T cell towards Th1 phenotype. Moreover, JAK inhibition limited adaptive immune responses mediated by recently activated T cells and subsequent breach of self-tolerance in experimental arthritis. CONCLUSIONS: Our findings provide a novel mode of action for tofacitinib, demonstrating a potential therapeutic utility via homeostatic immune restoration in very early autoimmune arthritis.

Revealing stromal and lymphoid sources of Col3a1-expression during inflammation using a novel reporter mouse.

One of the earliest signs of dysregulation of the homeostatic process of fibrosis, associated with pathology in chronic conditions such as rheumatoid arthritis, is the overexpression of collagen type III (COL-3). Critically, there is still relatively little known regarding the identity of the cell types expressing the gene encoding COL-3 (Col3a1). Identifying and characterizing Col3a1-expressing cells during the development of fibrosis could reveal new targets for the diagnosis and treatment of fibrosis-related pathologies. As such, a reporter mouse expressing concomitantly Col3a1 and mKate-2, a fluorescent protein, was generated. Using models of footpad inflammation, we demonstrated its effectiveness as a tool to measure the expression of COL-3 during the repair process and provided an initial characterization of some of the stromal and immune cells responsible for Col3a1 expression.

Nanoalum adjuvanted vaccines: small details make a big difference.

Purified vaccine antigens offer important safety and reactogenicity advantages compared with live attenuated or whole killed virus and bacterial vaccines. However, they require the addition of adjuvants to induce the magnitude, duration and quality of immune response required to achieve protective immunity. Aluminium salts have been used as adjuvants in vaccines for almost a century. In the literature, they are often referred to as aluminium-based adjuvants (ABAs), or aluminium salt-containing adjuvants or more simply "alum". All these terms are used to group aluminium suspensions that are very different in terms of atomic composition, size, and shape. They differ also in stability, antigen-adsorption, and antigen-release kinetics. Critically, these parameters also have a profound effect on the character and magnitude of the immune response elicited. Recent findings suggest that, by reducing the size of aluminium from micro to nanometers, a more effective adjuvant is obtained, together with the ability to sterile filter the vaccine product. However, the behaviour of aluminium nanoparticles in vaccine formulations is different from microparticles, requiring specific formulation strategies, as well as a more detailed understanding of how formulation influences the immune response generated. Here we review the current state of art of aluminium nanoparticles as adjuvants, with a focus on their immunobiology, preparation methods, formulation optimisation and stabilisation.

Corrigendum: To the Skin and Beyond: The Immune Response to African Trypanosomes as They Enter and Exit the Vertebrate Host.

[This corrects the article DOI: 10.3389/fimmu.2020.01250.].

Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Preclinical models of arthritis for studying immunotherapy and immune tolerance.

Increasingly earlier identification of individuals at high risk of rheumatoid arthritis (RA) (eg, with autoantibodies and mild symptoms) improves the feasibility of preventing or curing disease. The use of antigen-specific immunotherapies to reinstate immunological self-tolerance represent a highly attractive strategy due to their potential to induce disease resolution, in contrast to existing approaches that require long-term treatment of underlying symptoms.Preclinical animal models have been used to understand disease mechanisms and to evaluate novel immunotherapeutic approaches. However, models are required to understand critical processes supporting disease development such as the breach of self-tolerance that triggers autoimmunity and the progression from asymptomatic autoimmunity to joint pain and bone loss. These models would also be useful in evaluating the response to treatment in the pre-RA period.This review proposes that focusing on immune processes contributing to initial disease induction rather than end-stage pathological consequences is essential to allow development and evaluation of novel immunotherapies for early intervention. We will describe and critique existing models in arthritis and the broader field of autoimmunity that may fulfil these criteria. We will also identify key gaps in our ability to study these processes in animal models, to highlight where further research should be targeted.

Junctional adhesion molecule-A on dendritic cells regulates Th1 differentiation.

The junctional adhesion molecule-A (JAM-A) is an adhesion molecule present in the surface of several cell types, such as endothelial cells and leukocytes as well as Dendritic Cells (DC). Given the potential relevance of JAM-A in diverse pathological conditions such as inflammatory diseases and cancer, we investigated the role of JAM-A in CD4+ T cell priming. We demonstrate that JAM-A is present in the immunological synapse formed between T cells and DC during priming. Furthermore, an antagonistic anti-JAM-A mAb could disrupt the interaction between CD4+ T cell and DC. Antagonism of JAM-A also attenuated T cell activation and proliferation with a decrease in T-bet expression and increased IL-6 and IL-17 secretion. These findings demonstrate a functional role for JAM-A in interactions between CD4+ T cells and DCs during T cell priming as a positive regulator of Th1 differentiation.

TCRß Sequencing Reveals Spatial and Temporal Evolution of Clonal CD4 T Cell Responses in a Breach of Tolerance Model of Inflammatory Arthritis.

Effective tolerogenic intervention in Rheumatoid Arthritis (RA) will rely upon understanding the evolution of articular antigen specific CD4 T cell responses. TCR clonality of endogenous CD4 T cell infiltrates in early inflammatory arthritis was assessed to monitor evolution of the TCR repertoire in the inflamed joint and associated lymph node (LN). Mouse models of antigen-induced breach of self-tolerance and chronic polyarthritis were used to recapitulate early and late phases of RA. The infiltrating endogenous, antigen experienced CD4 T cells in inflamed joints and LNs were analysed using flow cytometry and TCRß sequencing. TCR repertoires from inflamed late phase LNs displayed increased clonality and diversity compared to early phase LNs, while inflamed joints remained similar with time. Repertoires from late phase LNs accumulated clones with a diverse range of TRBV genes, while inflamed joints at both phases contained clones expressing similar TRBV genes. Repertoires from LNs and joints at the late phase displayed reduced CDR3ß sequence overlap compared to the early disease phase, however the most abundant clones in LNs accumulate in the joint at the later phase. The results indicate CD4 T cell repertoire clonality and diversity broadens with progression of inflammatory arthritis and is first reflected in LNs before mirroring in the joint. These observations imply that antigen specific tolerogenic therapies could be more effective if targeted at earlier phases of disease when CD4 T cell clonality is least diverse.

Tolerance induction in memory CD4 T cells is partial and reversible.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.

Targeting Opposing Immunological Roles of the Junctional Adhesion Molecule-A in Autoimmunity and Cancer.

The junctional adhesion molecule-A (JAM-A) is a cell surface adhesion molecule expressed on platelets, epithelial cells, endothelial cells and leukocytes (e. g. monocytes and dendritic cells). JAM-A plays a relevant role in leukocyte trafficking and its therapeutic potential has been studied in several pathological conditions due to its capacity to induce leukocyte migration out of inflamed sites or infiltration into tumor sites. However, disruption of JAM-A pathways may worsen clinical pathology in some cases. As such, the effects of JAM-A manipulation on modulating immune responses in the context of different diseases must be better understood. In this mini-review, we discuss the potential of JAM-A as a therapeutic target, summarizing findings from studies manipulating JAM-A in the context of inflammatory diseases (e.g. autoimmune diseases) and cancer and highlighting described mechanisms.

The Impact of Malaria Parasites on Dendritic Cell-T Cell Interaction.

Malaria is caused by apicomplexan parasites of the genus Plasmodium. While infection continues to pose a risk for the majority of the global population, the burden of disease mainly resides in Sub-Saharan Africa. Although immunity develops against disease, this requires years of persistent exposure and is not associated with protection against infection. Repeat infections occur due to the parasite's ability to disrupt or evade the host immune responses. However, despite many years of study, the mechanisms of this disruption remain unclear. Previous studies have demonstrated a parasite-induced failure in dendritic cell (DCs) function affecting the generation of helper T cell responses. These T cells fail to help B cell responses, reducing the production of antibodies that are necessary to control malaria infection. This review focuses on our current understanding of the effect of Plasmodium parasite on DC function, DC-T cell interaction, and T cell activation. A better understanding of how parasites disrupt DC-T cell interactions will lead to new targets and approaches to reinstate adaptive immune responses and enhance parasite immunity.

To the Skin and Beyond: The Immune Response to African Trypanosomes as They Enter and Exit the Vertebrate Host.

African trypanosomes are single-celled extracellular protozoan parasites transmitted by tsetse fly vectors across sub-Saharan Africa, causing serious disease in both humans and animals. Mammalian infections begin when the tsetse fly penetrates the skin in order to take a blood meal, depositing trypanosomes into the dermal layer. Similarly, onward transmission occurs when differentiated and insect pre-adapted forms are ingested by the fly during a blood meal. Between these transmission steps, trypanosomes access the systemic circulation of the vertebrate host via the skin-draining lymph nodes, disseminating into multiple tissues and organs, and establishing chronic, and long-lasting infections. However, most studies of the immunobiology of African trypanosomes have been conducted under experimental conditions that bypass the skin as a route for systemic dissemination (typically via intraperitoneal or intravenous routes). Therefore, the importance of these initial interactions between trypanosomes and the skin at the site of initial infection, and the implications for these processes in infection establishment, have largely been overlooked. Recent studies have also demonstrated active and complex interactions between the mammalian host and trypanosomes in the skin during initial infection and revealed the skin as an overlooked anatomical reservoir for transmission. This highlights the importance of this organ when investigating the biology of trypanosome infections and the associated immune responses at the initial site of infection. Here, we review the mechanisms involved in establishing African trypanosome infections and potential of the skin as a reservoir, the role of innate immune cells in the skin during initial infection, and the subsequent immune interactions as the parasites migrate from the skin. We suggest that a thorough identification of the mechanisms involved in establishing African trypanosome infections in the skin and their progression through the host is essential for the development of novel approaches to interrupt disease transmission and control these important diseases.

Developing a xenograft model of human vasculature in the mouse ear pinna.

Humanised xenograft models allow for the analysis of human tissue within a physiological environment in vivo. However, current models often rely on the angiogenesis and ingrowth of recipient vasculature to perfuse tissues, preventing analysis of biological processes and diseases involving human blood vessels. This limits the effectiveness of xenografts in replicating human physiology and may lead to issues with translating findings into human research. We have designed a xenograft model of human vasculature to address this issue. Human subcutaneous fat was cultured in vitro to promote blood vessel outgrowth prior to implantation into immunocompromised mice. We demonstrate that implants survived, retained human vasculature and anastomosed with the circulatory system of the recipient mouse. Significantly, by performing transplants into the ear pinna, this system enabled intravital observation of xenografts by multiphoton microscopy, allowing us to visualise the steps leading to vascular cytoadherence of erythrocytes infected with the human parasite Plasmodium falciparum. This model represents a useful tool for imaging the interactions that occur within human tissues in vivo and permits visualization of blood flow and cellular recruitment in a system which is amenable to intervention for various studies in basic biology together with drug evaluation and mechanism of action studies.

Cretaceous gnetalean yields first preserved plant gum.

Some liquid plant exudates (e.g. resin) can be found preserved in the fossil record. However, due to their high solubility, gums have been assumed to dissolve before fossilisation. The visual appearance of gums (water-soluble polysaccharides) is so similar to other plant exudates, particularly resin, that chemical testing is essential to differentiate them. Remarkably, Welwitschiophyllum leaves from Early Cretaceous, Brazil provide the first chemical confirmation of a preserved gum. This is despite the leaves being exposed to water twice during formation and subsequent weathering of the Crato Formation. The Welwitschiophyllum plant shares the presence of gum ducts inside leaves with its presumed extant relative the gnetalean Welwitschia. This fossil gum presents a chemical signature remarkably similar to the gum in extant Welwitschia and is distinct from those of fossil resins. We show for the first time that a water-soluble plant exudate has been preserved in the fossil record, potentially allowing us to recognise further biomolecules thought to be lost during the fossilisation process.

The aorta can act as a site of naïve CD4+ T-cell priming.

AIMS: Aortic adaptive immunity plays a role in atherosclerosis; however, the precise mechanisms leading to T-cell activation in the arterial wall remain poorly understood. METHODS AND RESULTS: Here, we have identified naïve T cells in the aorta of wild-type and T-cell receptor transgenic mice and we demonstrate that naïve T cells can be primed directly in the vessel wall with both kinetics and frequency of T-cell activation found to be similar to splenic and lymphoid T cells. Aortic homing of naïve T cells is regulated at least in part by the P-selectin glycosylated ligand-1 receptor. In experimental atherosclerosis the aorta supports CD4+ T-cell activation selectively driving Th1 polarization. By contrast, secondary lymphoid organs display Treg expansion. CONCLUSION: Our results demonstrate that the aorta can support T-cell priming and that naïve T cells traffic between the circulation and vessel wall. These data underpin the paradigm that local priming of T cells specific for plaque antigens contributes to atherosclerosis progression.

A Novel Triple-Cell Two-Dimensional Model to Study Immune-Vascular Interplay in Atherosclerosis.

Atherosclerosis is a complex inflammatory pathology underpinning cardiovascular diseases (CVD), which are the leading cause of death worldwide. The interplay between vascular stromal cells and immune cells is fundamental to the progression and outcome of atherosclerotic disease, however, the majority of in vitro studies do not consider the implications of these interactions and predominantly use mono-culture approaches. Here we present a simple and robust methodology involving the co-culture of vascular endothelial (ECs) and smooth muscle cells (SMCs) alongside an inflammatory compartment, in our study containing THP-1 macrophages, for studying these complex interactions. Using this approach, we demonstrate that the interaction between vascular stromal and immune cells produces unique cellular phenotypes and soluble mediator profiles not observed in double-cell 2D cultures. Our results highlight the importance of cellular communication and support the growing idea that in vitro research must evolve from mono-culture systems to provide data more representative of the multi-cellular environment found in vivo. The methodology presented, in comparison with established approaches, has the advantage of being technically simple whilst enabling the isolation of pure populations of ECs, SMCs and immune cells directly from the co-culture without cell sorting. The approach described within would be applicable to those studying mechanisms of vascular inflammation, particularly in relation to understanding the impact cellular interaction has on the cumulative immune-vascular response to atherogenic or inflammatory stimuli.