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.

CD31 signaling promotes the detachment at the uropod of extravasating neutrophils allowing their migration to sites of inflammation.

Effective neutrophil migration to sites of inflammation is crucial for host immunity. A coordinated cascade of steps allows intravascular leukocytes to counteract the shear stress, transmigrate through the endothelial layer, and move toward the extravascular, static environment. Those events are tightly orchestrated by integrins, but, while the molecular mechanisms leading to their activation have been characterized, the regulatory pathways promoting their detachment remain elusive. In light of this, it has long been known that platelet-endothelial cell adhesion molecule (Pecam1, also known as CD31) deficiency blocks leukocyte transmigration at the level of the outer vessel wall, yet the associated cellular defects are controversial. In this study, we combined an unbiased proteomic study with in vitro and in vivo single-cell tracking in mice to study the dynamics and role of CD31 during neutrophil migration. We found that CD31 localizes to the uropod of migrating neutrophils along with closed ß2-integrin and is required for essential neutrophil actin/integrin polarization. Accordingly, the uropod of Pecam1-/- neutrophils is unable to detach from the extracellular matrix, while antagonizing integrin binding to extracellular matrix components rescues this in vivo migratory defect. Conversely, we showed that sustaining CD31 co-signaling actively favors uropod detachment and effective migration of extravasated neutrophils to sites of inflammation in vivo. Altogether, our results suggest that CD31 acts as a molecular rheostat controlling integrin-mediated adhesion at the uropod of egressed neutrophils, thereby triggering their detachment from the outer vessel wall to reach the inflammatory sites.

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.

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.].

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.

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.

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.

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.

Spatiotemporal Modeling of the Key Migratory Events During the Initiation of Adaptive Immunity.

Initiation of adaptive immunity involves distinct migratory cell populations coming together in a highly dynamic and spatially organized process. However, we lack a detailed spatiotemporal map of these events due to our inability to track the fate of cells between anatomically distinct locations or functionally identify cell populations as migratory. We used photo-convertible transgenic mice (Kaede) to spatiotemporally track the fate and composition of the cell populations that leave the site of priming and enter the draining lymph node to initiate immunity. We show that following skin priming, the lymph node migratory population is principally composed of cells recruited to the site of priming, with a minor contribution from tissue resident cells. In combination with the YAe/Eα system, we also show that the majority of cells presenting antigen are CD103+CD11b+ dendritic cells that were recruited to the site of priming during the inflammatory response. This population has previously only been described in relation to mucosal tissues. Comprehensive phenotypic profiling of the cells migrating from the skin to the draining lymph node by mass cytometry revealed that in addition to dendritic cells, the migratory population also included CD4+ and CD8+ T cells, B cells, and neutrophils. Taking our complex spatiotemporal data set, we then generated a model of cell migration that quantifies and describes the dynamics of arrival, departure, and residence times of cells at the site of priming and in the draining lymph node throughout the time-course of the initiation of adaptive immunity. In addition, we have identified the mean migration time of migratory dendritic cells as they travel from the site of priming to the draining lymph node. These findings represent an unprecedented, detailed and quantitative map of cell dynamics and phenotypes during immunization, identifying where, when and which cells to target for immunomodulation in autoimmunity and vaccination strategies.

Effects of host-derived chemokines on the motility and viability of Trypanosoma brucei.

African trypanosomes (Trypanosoma brucei spp.) are extracellular, hemoflagellate, protozoan parasites. Mammalian infection begins when the tsetse fly vector injects trypanosomes into the skin during blood feeding. The trypanosomes then reach the draining lymph nodes before disseminating systemically. Intravital imaging of the skin post-tsetse fly bite revealed that trypanosomes were observed both extravascularly and intravascularly in the lymphatic vessels. Whether host-derived cues play a role in the attraction of the trypanosomes towards the lymphatic vessels to aid their dissemination from the site of infection is not known. Since chemokines can mediate the attraction of leucocytes towards the lymphatics, in vitro chemotaxis assays were used to determine whether chemokines might also act as chemoattractants for trypanosomes. Although microarray data suggested that the chemokines CCL8, CCL19, CCL21, CCL27 and CXCL12 were highly expressed in mouse skin, they did not stimulate the chemotaxis of T brucei. Certain chemokines also possess potent antimicrobial properties. However, none of the chemokines tested exerted any parasiticidal effects on T brucei. Thus, our data suggest that host-derived chemokines do not act as chemoattractants for T brucei. Identification of the mechanisms used by trypanosomes to establish host infection will aid the development of novel approaches to block disease transmission.

A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo.

Dendritic cell activation of CD4 T cells in the lymph node draining a site of infection or vaccination is widely considered the central event in initiating adaptive immunity. The accepted dogma is that this occurs by stimulating local activation and antigen acquisition by dendritic cells, with subsequent lymph node migration, however the generalizability of this mechanism is unclear. Here we show that in some circumstances antigen can bypass the injection site inflammatory response, draining freely and rapidly to the lymph nodes where it interacts with subcapsular sinus (SCS) macrophages resulting in their death. Debris from these dying SCS macrophages is internalized by monocytes recruited from the circulation. This coordinated response leads to antigen presentation by monocytes and interactions with naïve CD4 T cells that can drive the initiation of T cell and B cell responses. These studies demonstrate an entirely novel pathway leading to initiation of adaptive immune responses in vivo.

Non-Invasive Multiphoton Imaging of Islets Transplanted Into the Pinna of the NOD Mouse Ear Reveals the Immediate Effect of Anti-CD3 Treatment in Autoimmune Diabetes.

We present a novel and readily accessible method facilitating cellular time-resolved imaging of transplanted pancreatic islets. Grafting of islets to the mouse ear pinna allows non-invasive, in vivo longitudinal imaging of events in the islets and enables improved acquisition of experimental data and use of fewer experimental animals than is possible using invasive techniques, as the same mouse can be assessed for the presence of islet infiltrating cells before and after immune intervention. We have applied this method to investigating therapeutic protection of beta cells through the well-established use of anti-CD3 injection, and have acquired unprecedented data on the nature and rapidity of the effect on the islet infiltrating T cells. We demonstrate that infusion of anti-CD3 antibody leads to immediate effects on islet infiltrating T cells in islet grafts in the pinna of the ear, and causes them to increase their speed and displacement within 20 min of infusion. This technique overcomes several technical challenges associated with intravital imaging of pancreatic immune responses and facilitates routine study of beta islet cell development, differentiation, and function in health and disease.

Visualising the interaction of CD4 T cells and DCs in the evolution of inflammatory arthritis.

OBJECTIVES: Successful early intervention in rheumatoid arthritis (RA) with the aim of resetting immunological tolerance requires a clearer understanding of how specificity, cellular kinetics and spatial behaviour shape the evolution of articular T cell responses. We aimed to define initial seeding of articular CD4+ T cell responses in early experimental arthritis, evaluating their dynamic behaviour and interactions with dendritic cells (DCs) in the inflamed articular environment. METHODS: Antigen-induced arthritis was used to model articular inflammation. Flow cytometry and PCR of T cell receptor (TCR) diversity genes allowed phenotypic analysis of infiltrating T cells. The dynamic interactions of T cells with joint residing DCs were visualised using intravital multiphoton microscopy. RESULTS: Initial recruitment of antigen-specific T cells into the joint was paralleled by accumulation of CD4+ T cells with diverse antigen-receptor expression and ability to produce tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) on mitogenic restimulation. A proportion of this infiltrate demonstrated slower motility speeds and engaged for longer periods with articular DCs in vivo. Abatacept treatment did not disrupt these interactions but did reduce T cell expression of inducible costimulatory (ICOS) molecule. We also demonstrated that non-specific CD4+ T cells could be recruited during these early articular events. CONCLUSIONS: We demonstrate that CD4+ T cells engage with articular DCs supporting antigen specific T cell reactivation. This cellular dialogue can be targeted therapeutically to reduce local T cell activation.

Model answers: Rational application of murine models in arthritis research.

Advances in targeted immune therapeutics have profoundly improved clinical outcomes for patients with inflammatory arthropathies particularly rheumatoid arthritis. The landscape of disease that is observed and the treatment outcomes desired for the future have also progressed. As such there is an increasing move away from traditional models of end-stage, chronic disease with recognition of the need to consider the earliest phases of pathogenesis as a target for treatment leading to resolution and/or cure. In order to continue the discovery process and enhance our understanding of disease and treatment, we therefore need to continuously revisit the animal models we employ and assess their relevance and utility in the light of contemporary therapeutic goals. In this review, we highlight the areas where we consider new developments in animal models and their application are most required. Thus, we have contextualised the relevant mouse models and their use within the current concepts of human inflammatory arthritis pathogenesis and highlight areas of need.

Sphingosine-1-Phosphate Promotes the Persistence of Activated CD4 T Cells in Inflamed Sites.

Inflammation can be protective or pathogenic depending on context and timeframe. Acute inflammation, including the accumulation of CD4 T cells, accompanies protective immune responses to pathogens, but the presence of activated CD4 T cells at sites of inflammation is associated with chronic inflammatory disease. While significant progress has been made in understanding the migration of CD4 T cells into inflamed sites, the signals that lead to their persistence are poorly characterized. Using a murine ear model of acute inflammation and intravital two-photon imaging, we have dissected the signals that mediate CD4 T cell persistence. We report the unexpected finding that the bioactive lipid, sphingosine-1-phosphate (S1P), is both necessary and sufficient for the persistence of activated CD4 T cells at peripheral tissues in acute inflammation. S1P mediated the enhanced motility of CD4 T cells at inflamed tissues but did not affect their migration to the downstream draining lymph node. We found that sphingosine kinase-1, which regulates S1P production is increased at inflamed sites in mice and in patients with the chronic inflammatory disease, rheumatoid arthritis. Together, these data suggest that S1P, or its regulators, may be key targets to promote or disrupt accumulation of CD4 T cells at inflamed tissues.

Visualizing and Tracking T Cell Motility In Vivo.

Advanced cellular tracking and imaging techniques allow the dynamic nature of immune responses to be studied in detail and in a physiological context. Here we describe two methods applying multiphoton laser scanning microscopy to the visualization and tracking of fluorescently labeled CD4+ T cells and dendritic cells (DCs) within the complex lymph node (LN) environment. Ex vivo imaging of LNs allows the study of cell populations without the need for skilled surgical techniques while providing comparable data. While more technically demanding, intravital imaging of the popliteal LN allows aspects of T cell/DC responses to be studied in the context of an intact lymph and blood supply. We also describe methods to aid the acquisition of time series data suitable for cellular tracking, providing a quantitative approach to real-time analysis of DC and T cell LN responses.

T Cell Response in the Lung Following Influenza Virus Infection.

Methods that enable the identification of virus-specific CD4 and CD8 T cells are key to our understanding of how the adaptive immune response controls viral infection. Here we describe two distinct methods to evaluate the T cell response to influenza A virus (IAV). The number and phenotype of T cells that respond to natural IAV epitopes can be assessed by flow cytometry using MHC class I and class II tetramers. Using this system, IAV-specific T cells can be tracked in various organs within the same animal, or, in different cohorts, the response can be evaluated at several time points following infection. While providing clear quantitative data, flow cytometry cannot provide any information about T cell location within the lung or interactions between responding T cells and other cell types. Here we also describe a method to examine activated CD4 T cells in the lungs of living animals using multiphoton intravital microscopy, thus providing real-time analysis of T cell behavior during an infection.