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Summary: CD8+ T cells have evolved as one of the most motile mammalian cell types, designed to continuously scan peptide–major histocompatibility complexes class I on the surfaces of other cells. Chemoattractants and adhesion molecules direct CD8+ T‐cell homing to and migration within secondary lymphoid organs, where these cells colocalize with antigen‐presenting dendritic cells in confined tissue volumes. CD8+ T‐cell activation induces a switch to infiltration of non‐lymphoid tissue (NLT), which differ in their topology and biophysical properties from lymphoid tissue. Here, we provide a short overview on regulation of organism‐wide trafficking patterns during naive T‐cell recirculation and their switch to non‐lymphoid tissue homing during activation. The migratory lifestyle of CD8+ T cells is regulated by their actomyosin cytoskeleton, which translates chemical signals from surface receptors into mechanical work. We explore how properties of the actomyosin cytoskeleton and its regulators affect CD8+ T cell function in lymphoid and non‐lymphoid tissue, combining recent findings in the field of cell migration and actin network regulation with tissue anatomy. Finally, we hypothesize that under certain conditions, intrinsic regulation of actomyosin dynamics may render NLT CD8+ T‐cell populations less dependent on input from extrinsic signals during tissue scanning. [ABSTRACT FROM AUTHOR]

Background: Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis—because of injury, disease, or aging—initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration. Methods: In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration. Results: We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood–retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1+ cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8+ T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response. Conclusions: Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely. [ABSTRACT FROM AUTHOR]

Background: Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis—because of injury, disease, or aging—initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration. Methods: In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration. Results: We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood–retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1+ cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8+ T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response. Conclusions: Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely. [ABSTRACT FROM AUTHOR]

A prominent activity of the chemokine system is the regulation of leukocyte trafficking. Here we summarize recent findings on the initial steps in chemokine receptor–induced signal transduction in leukocytes. In particular, we discuss the potential influences of the formation of oligomers of ligand and receptor and of coupling between chemokine signals and regulators of the cytoskeleton, such as small GTPases. [ABSTRACT FROM AUTHOR]

Chemokines are a large family of small, generally secreted polypeptides which guide lymphocyte movement throughout the body by controlling integrin avidity and inducing migration. Here, we look at recent, exciting findings on chemokine function throughout lymphocyte development and co-ordinated T and B cell migration during immune responses. Finally, we will review data on the regional control of immunity by tissue-specific chemokine receptors on effector/memory lymphocytes. [ABSTRACT FROM AUTHOR]

Presents a study that investigated ligands that modulate B and T cell immunity. Role of tumor necrosis factor family in inducing various biological responses such as cell proliferation; Methodology and results of the study.

The transcription factor KLF2 directs expression of receptors involved in trafficking of naive T cells. In this issue of Immunity, demonstrate that KLF2 additionally represses IL-4 production, which otherwise induces CXCR3 expression. [Copyright &y& Elsevier]

Background: Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury. Methods: Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina. Results: Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer. Conclusions: Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration. [ABSTRACT FROM AUTHOR]

Simple Summary: Bruton's tyrosine kinase inhibitor ibrutinib restores T-cell immunity and increases circulating T-cell numbers in chronic lymphocytic leukemia (CLL) patients. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, this physiological change might reflect, as well, a forced retention in PB derived of impaired migration to SLO. With the aim to investigate the impact of ibrutinib on CCR7-mediated homeostasis in CLL T-cells, we have documented receptor expression in a large cohort of ibrutinib-treated patients, and performed different in vivo and in vitro migration models. We show that ibrutinib has no effect on CCR7 expression or receptor-mediated T-cell chemotaxis, homing to SLO, and interstitial migration. Together, these results indicate that ibrutinib T-lymphocytosis is not caused by accumulation in the blood stream, and therefore back the T-cell expansion as the most plausible cause. Bruton's tyrosine kinase inhibitor ibrutinib has significantly changed treatment landscape in chronic lymphocytic leukemia (CLL). Growing evidence supports ibrutinib to work beyond the effect on tumor cells by means of, for example, restoring functionality of the T-cell compartment and increasing circulating T-cell numbers. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, whether the latter physiological change is also a consequence of a forced retention in blood remains undisclosed. Since CCR7 is the main chemokine receptor taking over the homing of T-cells from peripheral compartments to lymph nodes and other SLO, we aimed to investigate the impact of ibrutinib on CCR7 functionality in T-cells. To this end, we documented receptor expression in T-cells from a large cohort of ibrutinib-treated CLL patients, and performed different in vivo and in vitro migration models. Overall, our data confirm that CCR7 expression or receptor-mediated migration in CLL T-cells is not affected by ibrutinib. Furthermore, it does not modulate CCR7-driven homing nor nodal interstitial migration. Together, our results support that ibrutinib-induced CLL T-cell accumulation in the blood stream is not derived from an impairment of CCR7-driven recirculation between the SLO and bloodstream, and therefore T-cell expansion is the most plausible cause. [ABSTRACT FROM AUTHOR]

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine-scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC-chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro-lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2-deficient mice and reduced in CCR2-deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro-lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels. [ABSTRACT FROM AUTHOR]

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTβ) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTβR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTβR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTβR and RANK with implications for the immune response. [ABSTRACT FROM AUTHOR]

Alzheimer disease (AD) is one of the main causes of age-related dementia and neurodegeneration. However, the onset of the disease and the mechanisms causing cognitive defects are not well understood. Aggregation of amyloidogenic peptides is a pathological hallmark of AD and is assumed to be a central component of the molecular disease pathways. Pan-neuronal expression of Aβ42Arctic peptides in Drosophila melanogaster results in learning and memory defects. Surprisingly, targeted expression to the mushroom bodies, a center for olfactory memories in the fly brain, does not interfere with learning but accelerates forgetting. We show here that reducing neuronal excitability either by feeding Levetiracetam or silencing of neurons in the involved circuitry ameliorates the phenotype. Furthermore, inhibition of the Rac-regulated forgetting pathway could rescue the Aβ42Arctic-mediated accelerated forgetting phenotype. Similar effects are achieved by increasing sleep, a critical regulator of neuronal homeostasis. Our results provide a functional framework connecting forgetting signaling and sleep, which are critical for regulating neuronal excitability and homeostasis and are therefore a promising mechanism to modulate forgetting caused by toxic Aβ peptides. One of the early hallmarks in Alzheimer's disease is increased forgetting. This study shows that restricted expression of amyloid beta in the memory center of fruit flies causes enhanced forgetting without affecting the ability to learn; forgetting is caused by increased excitability and can be restored with drugs, increased sleep or modulation of dopamine signalling. [ABSTRACT FROM AUTHOR]

The article discusses understanding the fate of dendritic cells (DCs) productive immune synapses (postsynaptic DCs) with T cells for antigen presentation has neglected in favor of deciphering the nuances of T cell activation and memory generation. Topics include cells display epigenomic and metabolic changes, sustaining the expression of host defense–related genes; and an active exchange of information ensues, with DCs communicating antigenic information to T cells.

Malaria is a life-threatening disease, caused by Apicomplexan parasites of the Plasmodium genus. The Anopheles mosquito is necessary for the sexual replication of these parasites and for their transmission to vertebrate hosts, including humans. Imaging of the parasite within the insect vector has been attempted using multiple microscopy methods, most of which are hampered by the presence of the light scattering opaque cuticle of the mosquito. So far, most imaging of the Plasmodium mosquito stages depended on either sectioning or surgical dissection of important anatomical sites, such as the midgut and the salivary glands. Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) enable imaging fields of view in the centimeter scale whilst providing micrometer resolution. In this paper, we compare different optical clearing protocols and present reconstructions of the whole body of Plasmodium-infected, optically cleared Anopheles stephensi mosquitoes and their midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy and enable overall localization of parasites in midguts. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in extracted midguts. This work was submitted as a pre-print to bioRxiv, available at https://www.biorxiv.org/content/10.1101/682054v2. [ABSTRACT FROM AUTHOR]

Cell migration involves dynamic changes in cell shape. Intricate patterns of cell shape can be analyzed and classified using advanced shape descriptors, including spherical harmonics (SPHARM). Though SPHARM have been used to analyze and classify migrating cells, such classification did not exploit SPHARM spectra in their dynamics. Here, we examine whether additional information from dynamic SPHARM improves classification of cell migration patterns. We combine the static and dynamic SPHARM approach with a support-vector-machine classifier and compare their classification accuracies. We demonstrate that the dynamic SPHARM analysis classifies cell migration patterns more accurately than the static one for both synthetic and experimental data. Furthermore, by comparing the computed accuracies with that of a naive classifier, we can identify the experimental conditions and model parameters that significantly affect cell shape. This capability should – in the future – help to pinpoint factors that play an essential role in cell migration. [ABSTRACT FROM AUTHOR]

Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMVTT-VLPs) incorporating a universal Tetanus toxoid epitope TT830-843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMVTT-VLPs using bio-orthogonal Cu-free click chemistry. The CuMVTT-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Results: Our results showed that CuMVTT-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMVTT-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8+, specific p33 T cell response or tumour protection were assessed. Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic. [ABSTRACT FROM AUTHOR]

Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1- deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study, we analyzed morphology and migration parameters of murine wild-type and Flot1-/- CD8+ T cells using in vitro assays and intravital two-photon microscopy of lymphoid and nonlymphoid tissues. Flot1-/- CD8+ T cells displayed significant alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and intact in vitro migration to chemokines. Furthermore, their clonal expansion and infiltration into nonlymphoid tissues during primary and secondary antiviral immune responses was comparable to wild-type CD8+ T cells. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8+ T cell behavior under physiological conditions. [ABSTRACT FROM AUTHOR]

Melanoma is the most aggressive skin cancer in humans. One severe complication is the formation of brain metastasis, which requires extravasation of melanoma cells across the tight blood–brain barrier (BBB). Previously, VLA-4 has been assigned a role for the adhesive interaction of melanoma cells with non-BBB endothelial cells. However, the role of melanoma VLA-4 for breaching the BBB remained unknown. In this study, we used a mouse in vitro BBB model and imaged the shear resistant arrest of melanoma cells on the BBB. Similar to effector T cells, inflammatory conditions of the BBB increased the arrest of melanoma cells followed by a unique post-arrest behavior lacking immediate crawling. However, over time, melanoma cells intercalated into the BBB and compromised its barrier properties. Most importantly, antibody ablation of VLA-4 abrogated melanoma shear resistant arrest on and intercalation into the BBB and protected the BBB from barrier breakdown. A tissue microarray established from human brain metastasis revealed that indeed a majority of 92% of all human melanoma brain metastases stained VLA-4 positive. We propose VLA-4 as a target for the inhibition of brain metastasis formation in the context of personalized medicine identifying metastasizing VLA-4 positive melanoma. [ABSTRACT FROM AUTHOR]

Animal models have for long been pivotal for parasitology research. Over the last few years, techniques such as intravital, optoacoustic and magnetic resonance imaging, optical projection tomography, and selective plane illumination microscopy developed promising potential for gaining insights into host–pathogen interactions by allowing different visualization forms in vivo and ex vivo. Advances including increased resolution, penetration depth, and acquisition speed, together with more complex image analysis methods, facilitate tackling biological problems previously impossible to study and/or quantify. Here we discuss advances and challenges in the in vivo imaging toolbox, which hold promising potential for the field of parasitology. Highlights Technological advances in IVM include ultrafast tunable infrared lasers, super-resolution imaging in vivo , and better access to all organs by improved optics or motion-correction mechanisms. Optical clearance methods for use in OPT and SPIM allow imaging entire organs and organisms (such as mice or insect vectors), while achieving cellular and subcellular resolution. OAI combines optics and acoustics via the thermoelastic effect to investigate molecular mechanisms. Key advances in MRI include improved contrast agents, multimodal mesoscopic imaging, and functional MRI to visualize the metabolic function of tissues. Developments in bioluminescence imaging include red-shifted probes with high sensitivity; a larger diversity of probes with emission peaks at various wavelengths; ultrabright probes, and the use of BRET in vivo. X-ray-based and gamma-ray-based imaging platforms are noninvasive and enable the study of anatomical structures, metabolism, biochemical composition of tissues, and dynamics. [ABSTRACT FROM AUTHOR]

HIV-1 Nef is a multifunctional protein that optimizes virus spread and promotes immune evasion of infected cells to accelerate disease progression in AIDS patients. As one of its activities, Nef reduces the motility of infected CD4+ T lymphocytes in confined space. In vivo, Nef restricts T lymphocyte homing to lymph nodes as it reduces the ability for extravasation at the diapedesis step. Effects of Nef on T lymphocyte motility are typically mediated by its ability to reduce actin remodeling. However, interference with diapedesis does not depend on residues in Nef required for inhibition of host cell actin dynamics. In search for an alternative mechanism by which Nef could alter T lymphocyte extravasation, we noted that the viral protein interferes with the polarization of primary human CD4+ T lymphocytes upon infection with HIV-1. Expression of Nef alone is sufficient to disrupt T cell polarization, and this effect is conserved among lentiviral Nef proteins. Nef acts by arresting the oscillation of CD4+ T cells between polarized and nonpolarized morphologies. Mapping studies identified the binding site for the Nef-associated kinase complex (NAKC) as critical determinant of this Nef activity and a NAKC-binding-deficient Nef variant fails to impair CD4+ T lymphocyte extravasation and homing to lymph nodes. These results thus imply the disruption of T lymphocyte polarity via its NAKC binding site as a novel mechanism by which lentiviral Nef proteins alter T lymphocyte migration in vivo. [ABSTRACT FROM AUTHOR]

Bone protective effects of TNFα inhibition in rheumatoid arthritis are thought to be mediated by inhibiting synovial osteoclast differentiation and activity. However, it has not been addressed, if TNFα inhibitors alter the pool of peripheral osteoclast precursor cells (OPCs). Here, we blocked TNFα function in C57BL/6 mice with antigen induced arthritis (AIA) using the soluble TNFα receptor etanercept. Synovial bone lesions and osteoclasts were markedly reduced upon Etanercept in the early chronic phase of AIA. Unexpectedly this was not associated with a reduced recruitment of circulating OPCs to the arthritic joint nor to reduced synovial inflammation. In contrast we found that OPC numbers in bone marrow and blood were significantly reduced. Overall our study suggests that arrest of osteoclast mediated bone lesions upon inhibition of TNFα is, at least initially, based on reduced OPC availability in the periphery, and not on OPC recruitment or local anti-inflammatory effects in the arthritic joint. [ABSTRACT FROM AUTHOR]

Parenchymal migration of naive CD4+ T cells in lymph nodes (LNs) is mediated by the Rac activator DOCK2 and PI3Kg and is widely assumed to facilitate efficient screening of dendritic cells (DCs) presenting peptide-MHCs (pMHCs). Yet how CD4+ T cell motility, DC density, and pMHC levels interdependently regulate such interactions has not been comprehensively examined. Using intravital imaging of reactive LNs in DC-immunized mice, we show that pMHC levels determined the occurrence and timing of stable CD4+ T cell-DC interactions. Despite the variability in interaction parameters, ensuing CD4+ T cell proliferation was comparable over a wide range of pMHC levels. Unexpectedly, decreased intrinsic motility of DOCK2-/- CD4+ T cells did not impair encounters with DCs in dense paracortical networks and, instead, increased interaction stability, whereas PI3Kγ deficiency had no effect on interaction parameters. In contrast, intravital and whole-organ imaging showed that DOCK2-driven T cell motility was required to detach from pMHClow DCs and to find rare pMHChigh DCs. In sum, our data uncover flexible signal integration by scanning CD4+ T cells, suggesting a search strategy evolved to detect low-frequency DCs presenting high cognate pMHC levels. [ABSTRACT FROM AUTHOR]

While autoimmune T cells are present in most individuals, only a minority of the population suffers from an autoimmune disease. To better appreciate the limits of T cell tolerance, we carried out experiments to determine how many autoimmune T cells are required to initiate an experimental autoimmune disease. Variable numbers of autoimmune OT-I T cells were transferred into RIP-OVA mice, which were injected with antigen-loaded DCs in a single footpad; this restricted T cell priming to a few OT-I T cells that are present in the draining popliteal lymph node. Using selective plane illumination microscopy (SPIM) we counted the number of OT-I T cells present in the popliteal lymph node at the time of priming. Analysis of our data suggests that a single autoimmune T cell cannot induce an experimental autoimmune disease, but a “quorum” of 2–5 autoimmune T cells clearly has this capacity. [ABSTRACT FROM AUTHOR]

DNA rich in unmethylated CG motifs (CpGs) engage Toll-Like Receptor 9 (TLR-9) in endosomes and are well described stimulators of the innate and adaptive immune system. CpGs therefore can efficiently improve vaccines' immunogenicity. Packaging CpGs into nanoparticles, in particular into virus-like particles (VLPs), improves the pharmacological characteristics of CpGs as the protein shell protects them from DNAse activity and delivers the oligomers to the endosomal compartments of professional antigen presenting cells (APCs). The current consensus in packaging and delivering CpGs in VLP-based vaccines is that both adjuvants and antigens should be kept in close proximity (i.e. physically linked) to ensure delivery of antigens and adjuvants to the same APCs. In the current study, we harness the draining properties of the lymphatic system and show that also non-linked VLPs are efficiently co-delivered to the same APCs in lymph nodes. Specifically, we have shown that CpGs can be packaged in one VLP and mixed with another VLP displaying the antigen prior to administration in vivo . Both VLPs efficiently reached the same draining lymph node where they were taken up and processed by the same APCs, namely dendritic cells and macrophages. This resulted in induction of specific CTLs producing cytokines and killing target cells in vivo at levels seen when using VLPs containing both CpGs and chemically conjugated antigen. Thus, delivery of antigens and adjuvants in separate nanoparticles eliminates the need of physical conjugation and thus can be beneficial when designing precision medicine VLP-based vaccines or help to re-formulate existing VLP vaccines not naturally carrying immunostimulatory sequences. [ABSTRACT FROM AUTHOR]

The development of multi-photon intravital microscopy, in particular two-photon microscopy (2PM), has been a breakthrough technique for deep-tissue imaging of dynamic cell behavior inside live organisms and has substantially advanced the field of immunology. However, intravital time-lapse imaging over prolonged time periods is complicated by slow tissue drifts caused by vital activity, leading to shifting fields of views and making the acquired image sequence partially or completely unanalyzable. To solve this issue, we have established a system that performs continuous drift offset correction in real time using fine pattern matching during 2PM acquisition. We incorporated an extensive use of graphical processing unit (GPU) for high-speed computing required for real time correction during data acquisition. This allowed us to perform prolonged acquisitions and increase the proportion of analyzable datasets to nearly 100% in lymphoid and non-lymphoid tissues. Considering the straightforward implementation of our newly developed system, we anticipate that it will be applicable for other users interested in improving the quality of live imaging data acquisition. [ABSTRACT FROM AUTHOR]

Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses. [ABSTRACT FROM AUTHOR]

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5–10 mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues. [ABSTRACT FROM AUTHOR]

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4+ T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell--dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4+ Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings. [ABSTRACT FROM AUTHOR]

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4+ T cells. We propose that activated LNSCs acquire enhanced Agpresentation capacity, serving as an extrinsic brake system for CD4+ T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease. [ABSTRACT FROM AUTHOR]

Summary: The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-β receptor (LTβR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTβR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTβR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection. [Copyright &y& Elsevier]

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute. [ABSTRACT FROM AUTHOR]

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef's influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef's ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor. [ABSTRACT FROM AUTHOR]

To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42. yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially [ABSTRACT FROM AUTHOR]

Abstract: New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin−CD105+) were harvested from GFP+-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP+-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration. [Copyright &y& Elsevier]

Deep tissue imaging has become state of the art in biology, but now the problem is to quantify spatial information in a global, organ-wide context. Although access to the raw data is no longer a limitation, the computational tools to extract biologically useful information out of these large data sets is still catching up. In many cases, to understand the mechanism behind a biological process, where molecules or cells interact with each other, it is mandatory to know their mutual positions. We illustrate this principle here with the immune system. Although the general functions of lymph nodes as immune sentinels are well described, many cellular and molecular details governing the interactions of lymphocytes and dendritic cells remain unclear to date and prevent an in-depth mechanistic understanding of the immune system. We imaged ex vivo lymph nodes isolated from both wildtype and transgenic mice lacking key factors for dendritic cell positioning and used software written in MATLAB to determine the spatial distances between the dendritic cells and the internal high endothelial vascular network. This allowed us to quantify the spatial localization of the dendritic cells in the lymph node, which is a critical parameter determining the effectiveness of an adaptive immune response. [ABSTRACT FROM AUTHOR]

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p 160 coiled-coil kinase (ROCK)-myosin H-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonacti- vated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine- coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated I cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod con- traction during lymphocyte recirculation. [ABSTRACT FROM AUTHOR]

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P1) by CD69-/- DCs, whereas S1P3 expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P1 function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery. [ABSTRACT FROM AUTHOR]

T lymphocytes lacking the lymph node–homing receptors L-selectin and CCR7 do not migrate to lymph nodes in the steady state. Instead, we found here that lymph nodes draining sites of mature dendritic cells or adjuvant inoculation recruited L-selectin-negative CCR7− effector and memory CD8+ T cells. This recruitment required CXCR3 expression on T cells and occurred through high endothelial venules in concert with lumenal expression of the CXCR3 ligand CXCL9. In reactive lymph nodes, recruited T cells established stable interactions with and killed antigen-bearing dendritic cells, limiting the ability of these dendritic cells to activate naive CD4+ and CD8+ T cells. The inducible recruitment of blood-borne effector and memory T cells to lymph nodes may represent a mechanism for terminating primary and limiting secondary immune responses. [ABSTRACT FROM AUTHOR]

Chemokines guide lymphocytes from blood to secondary lymphoid organs by triggering integrin-dependent firm adhesion under vascular flow and directed migration of T and B lymphocytes within lymphoid tissue. Here, we analyze the roles of DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, and phosphoinositide-3-kinase (PI3K) during lymphocyte recirculation. DOCK2 mediated efficient lymphocyte migration in a largely PI3K-independent manner, although a minor, PI3K-dependent pathway for migration was observed in wild-type and DOCK2-deficient lymphocytes. In T cells, this residual migration depended mainly on PI3Kγ, whereas other PI3K isoforms were implicated in B cells. In vitro adhesion assays and intravital microscopy of lymphoid organ vasculature uncovered an unexpected defect in integrin activation in DOCK2-/- B cells, whereas lack of DOCK2 did not affect chemokine-triggered integrin activation in T cells. DOCK2 and PI3Kγ thus play distinct roles during T and B cell integrin activation and migration. [Copyright &y& Elsevier]

Following publication of the original article [1], the author reported an author's family name has been misspelled. Paul Engroff should be replace Paul Engeroff. Furthermore, there are two mistake in two affiliations: 9) Department of dermatology, University of Zurich, Bern, Switzerland and 10) Department of Oncology, University of Lausanne, Bern,Switzerland should be replace with 9) Department of dermatology, University of Zurich, Zurich, Switzerland. 10) Department of Oncology, University of Lausanne, Lausanne, Switzerland. [ABSTRACT FROM AUTHOR]

Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMVTT-VLPs) incorporating a universal Tetanus toxoid epitope TT830–843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMVTT-VLPs using bio-orthogonal Cu-free click chemistry. The CuMVTT-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Results: Our results showed that CuMVTT-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMVTT-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8+, specific p33 T cell response or tumour protection were assessed. Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic. [ABSTRACT FROM AUTHOR]

Background: T lymphocytes are highly dynamic elements of the immune system with a tightly regulated migration. T cell-based transfer therapies are promising therapeutic approaches which in vivo efficacy is often limited by the small proportion of administered cells that reaches the region of interest. Manipulating T cell localisation to improve specific targeting will increase the effectiveness of these therapies. Nanotechnology has been successfully used for localized release of drugs and biomolecules. In particular, magnetic nanoparticles (MNPs) loaded with biomolecules can be specifically targeted to a location by an external magnetic field (EMF). The present work studies whether MNP-loaded T cells could be targeted and retained in vitro and in vivo at a site of interest with an EMF. Results: T cells were unable to internalize the different MNPs used in this study, which remained in close association with the cell membrane. T cells loaded with an appropriate MNP concentration were attracted to an EMF and retained in an in vitro capillary flow-system. MNP-loaded T cells were also magnetically retained in the lymph nodes after adoptive transfer in in vivo models. This enhanced in vivo retention was in part due to the EMF application and to a reduced circulating cell speed within the organ. This combined use of MNPs and EMFs did not alter T cell viability or function. Conclusions: These studies reveal a promising approach to favour cell retention that could be implemented to improve cell-based therapy. [ABSTRACT FROM AUTHOR]