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.

Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin ß Receptors.

Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe(-/-) mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4(+) T cells, generated CD4(+), CD8(+), T regulatory (Treg) effector and central memory cells, converted naive CD4(+) T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin ß receptors (VSMC-LTßRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTßRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe(-/-)Ltbr(-/-) and to a similar extent in aged Apoe(-/-)Ltbr(fl/fl)Tagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTßRs participate in atherosclerosis protection via ATLOs.

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).

Resolving the apparent transmission paradox of African sleeping sickness.

Human African trypanosomiasis (HAT), or African sleeping sickness, is a fatal disease found throughout sub-Saharan Africa. The disease is close to elimination in many areas, although it was similarly close to elimination once before and subsequently reemerged, despite seemingly low rates of transmission. Determining how these foci persisted and overcame an apparent transmission paradox is key to finally eliminating HAT. By assessing clinical, laboratory, and mathematical data, we propose that asymptomatic infections contribute to transmission through the presence of an overlooked reservoir of skin-dwelling parasites. Our assessment suggests that a combination of asymptomatic and parasitaemic cases is sufficient to maintain transmission at foci without animal reservoirs, and we argue that the current policy not to treat asymptomatic HAT should be reconsidered.

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.

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.

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.

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.

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.

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.

Tolerance induction in memory CD4 T cells requires two rounds of antigen-specific activation.

A major goal for immunotherapy is to tolerize the immune cells that coordinate tissue damage in autoimmune and alloantigen responses. CD4 T cells play a central role in many of these conditions and improved antigen-specific regulation or removal of these cells could revolutionize current treatments. A confounding factor is that little is known about whether and how tolerance is induced in memory CD4 T cells. We used MHC class II tetramers to track and analyze a population of endogenous antigen-specific memory CD4 T cells exposed to soluble peptide in the absence of adjuvant. We found that such memory T cells proliferated and reentered the memory pool apparently unperturbed by the incomplete activation signals provided by the peptide. Upon further restimulation in vivo, CD4 memory T cells that had been previously exposed to peptide proliferated, provided help to primary responding B cells, and migrated to inflamed sites. However, these reactivated memory cells failed to survive. The reduction in T-cell number was marked by low expression of the antiapoptotic molecule B cell lymphoma 2 (Bcl2) and increased expression of activated caspase molecules. Consequently, these cells failed to sustain a delayed-type hypersensitivity response. Moreover, following two separate exposures to soluble antigen, no T-cell recall response and no helper activity for B cells could be detected. These results suggest that the induction of tolerance in memory CD4 T cells is possible but that deletion and permanent removal of the antigen-specific T cells requires reactivation following exposure to the tolerogenic antigen.

The thymus and rheumatology: should we care?

PURPOSE OF REVIEW: The purpose of this review is to discuss the mechanisms of central and peripheral tolerance in relation to T-cell mediated autoimmunity in rheumatoid arthritis (RA). RECENT FINDINGS: The well established association between major histocompatibility complex class II and RA has led us to understand that T cells, and the adaptive immune response, are important in the pathogenesis of disease. In order for autoimmune disease to develop, there is a breach of tolerance to self antigen and the mechanisms of both central and peripheral tolerance aim to prevent this. Here, we review evidence from mouse models indicating that alterations in T-cell receptor signalling thresholds during thymic selection may be linked to the escape of T cells that mediate autoimmune arthritis. In addition, we summarize the role of dendritic cells and Foxp3+ regulatory T cells in both peripheral and thymic tolerance, and highlight their relevance to what we know about the aetiology of RA. SUMMARY: Mechanisms of central tolerance in the thymus and peripheral tolerance are in place to control autoreactive T cells and to prevent the development of autoimmune disease. We anticipate that a better understanding of these mechanisms will lead to the development of better, antigen-specific therapeutics to restore tolerance.

Assessment of murine collagen-induced arthritis by longitudinal non-invasive duplexed molecular optical imaging.

OBJECTIVE: In the present study we evaluated the use of four commercially available fluorescent probes to monitor disease activity in murine CIA and its suppression during glucocorticoid therapy. METHODS: Arthritis was induced in male DBA/1 mice by immunization with type II collagen in Complete Freund's Adjuvant, followed by a boost of collagen in PBS. Four fluorescent probes from PerkinElmer in combination [ProSense 750 fluorescent activatable sensor technology (FAST) with Neutrophil Elastase 680 FAST and MMPSense 750 FAST with CatK 680 FAST] were used to monitor disease development from day 5 through to day 40 post-immunization. Fluorescence generated in vivo by the probes was correlated with clinical and histological score and paw measurements. RESULTS: The fluorescence intensity emitted by each probe was shown to correlate with the conventional measurements of disease. The highest degree of correlation was observed with ProSense 750 FAST in combination with Neutrophil Elastase 680 FAST; these probes were then used to successfully assess CIA suppression during dexamethasone treatment. CONCLUSION: We have demonstrated that longitudinal non-invasive duplexed optical fluorescence imaging provides a simple assessment of arthritic disease activity within the joints of mice following the induction of CIA and may represent a powerful tool to monitor the efficacy of drug treatments in preclinical studies.

The active metabolite of spleen tyrosine kinase inhibitor fostamatinib abrogates the CD4⁺ T cell-priming capacity of dendritic cells.

OBJECTIVES: Spleen tyrosine kinase (SYK) is a core signalling protein that drives inflammatory responses and is fundamental to the propagation of signals via numerous immune receptors, including the B cell receptor and Fc receptors (FcRs). Fostamatinib, a small molecule SYK inhibitor, has shown evidence of ameliorating inflammation in RA patients. We sought to understand how the active metabolite of fostamatinib, R406, affects the inflammatory response at the cellular level. METHODS: Antigen-specific in vivo systems and in vitro fluorescence microscopy were combined to investigate the effects of fostamatinib on antigen-specific interactions between dendritic cells (DCs) and CD4(+) T cells. RESULTS: Although it has previously been shown that R406 reduces the response of DCs to immune complexes (ICs), we found that fostamatinib failed to reduce specific CD4(+) T cell proliferation in mice after immunization with ICs. However, we observed in vitro that R406 reduces both the area and duration of cellular interactions between IC-activated DCs and specific CD4(+) T cells during the initial phase of cellular crosstalk. This led to diminished proliferation of antigen-specific CD4(+) T cells after R406 treatment compared with vehicle controls. This decreased proliferative capacity of CD4(+) T cells was accompanied by reduced expression of the co-stimulatory molecules, inducible T cell co-stimulator (ICOS) and PD-1, and abrogation of the production of inflammatory cytokines such as IFN-γ and IL-17. CONCLUSION: Our findings indicate a potential mechanism by which this compound may be effective in inhibiting FcR-driven CD4(+) T cell responses.

Applying refinement to the use of mice and rats in rheumatoid arthritis research.

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat 'models' of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research.

A novel method to allow noninvasive, longitudinal imaging of the murine immune system in vivo.

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior.

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.

D6 facilitates cellular migration and fluid flow to lymph nodes by suppressing lymphatic congestion.

Lymphatic endothelial cells are important for efficient flow of antigen-bearing fluid and antigen-presenting cells (APCs) from peripheral sites to lymph nodes (LNs). APC movement to LNs is dependent on the constitutive chemokine receptor CCR7, although how conflicting inflammatory and constitutive chemokine cues are integrated at lymphatic surfaces during this process is not understood. Here we reveal a previously unrecognized aspect of the regulation of this process. The D6 chemokine-scavenging receptor, which is expressed on lymphatic endothelial cells (LECs), maintains lymphatic surfaces free of inflammatory CC-chemokines and minimizes interaction of inflammatory leukocytes with these surfaces. D6 does not alter the level of CCR7 ligands on LECs, thus ensuring selective presentation of homeostatic chemokines for interaction with CCR7(+) APCs. Accordingly, in D6-deficient mice, inflammatory CC-chemokine adherence to LECs results in inappropriate perilymphatic accumulation of inflammatory leukocytes at peripheral inflamed sites and draining LNs. This results in lymphatic congestion and impaired movement of APCs, and fluid, from inflamed sites to LNs. We propose that D6, by suppressing inflammatory chemokine binding to lymphatic surfaces, and thereby preventing inappropriate inflammatory leukocyte adherence, is a key regulator of lymphatic function and a novel, and indispensable, contributor to the integration of innate and adaptive immune responses.

Antigen depot is not required for alum adjuvanticity.

Alum adjuvants have been in continuous clinical use for more than 80 yr. While the prevailing theory has been that depot formation and the associated slow release of antigen and/or inflammation are responsible for alum enhancement of antigen presentation and subsequent T- and B-cell responses, this has never been formally proven. To examine antigen persistence, we used the chimeric fluorescent protein EαGFP, which allows assessment of antigen presentation in situ, using the Y-Ae antibody. We demonstrate that alum and/or CpG adjuvants induced similar uptake of antigen, and in all cases, GFP signal did not persist beyond 24 h in draining lymph node antigen-presenting cells. Antigen presentation was first detectable on B cells within 6-12 h of antigen administration, followed by conventional dendritic cells (DCs) at 12-24 h, then finally plasmacytoid DCs at 48 h or later. Again, alum and/or CpG adjuvants did not have an effect on the magnitude or sequence of this response; furthermore, they induced similar antigen-specific T-cell activation in vivo. Notably, removal of the injection site and associated alum depot, as early as 2 h after administration, had no appreciable effect on antigen-specific T- and B-cell responses. This study clearly rules out a role for depot formation in alum adjuvant activity.