Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

Interferon-γ couples CD8+ T cell avidity and differentiation during infection.

Effective responses to intracellular pathogens are characterized by T cell clones with a broad affinity range for their cognate peptide and diverse functional phenotypes. How T cell clones are selected throughout the response to retain a breadth of avidities remains unclear. Here, we demonstrate that direct sensing of the cytokine IFN-γ by CD8+ T cells coordinates avidity and differentiation during infection. IFN-γ promotes the expansion of low-avidity T cells, allowing them to overcome the selective advantage of high-avidity T cells, whilst reinforcing high-avidity T cell entry into the memory pool, thus reducing the average avidity of the primary response and increasing that of the memory response. IFN-γ in this context is mainly provided by virtual memory T cells, an antigen-inexperienced subset with memory features. Overall, we propose that IFN-γ and virtual memory T cells fulfil a critical immunoregulatory role by enabling the coordination of T cell avidity and fate.

IFNγ signaling in cytotoxic T cells restricts anti-tumor responses by inhibiting the maintenance and diversity of intra-tumoral stem-like T cells.

IFNγ is an immune mediator with concomitant pro- and anti-tumor functions. Here, we provide evidence that IFNγ directly acts on intra-tumoral CD8 T cells to restrict anti-tumor responses. We report that expression of the IFNγ receptor ß chain (IFNγR2) in CD8 T cells negatively correlates with clinical responsiveness to checkpoint blockade in metastatic melanoma patients, suggesting that the loss of sensitivity to IFNγ contributes to successful antitumor immunity. Indeed, specific deletion of IFNγR in CD8 T cells promotes tumor control in a mouse model of melanoma. Chronic IFNγ inhibits the maintenance, clonal diversity and proliferation of stem-like T cells. This leads to decreased generation of T cells with intermediate expression of exhaustion markers, previously associated with beneficial anti-tumor responses. This study provides evidence of a negative feedback loop whereby IFNγ depletes stem-like T cells to restrict anti-tumor immunity. Targeting this pathway might represent an alternative strategy to enhance T cell-based therapies.

Spatiotemporal behavior of T cells in vaccination.

Vaccines are the most cost-effective resource to contain and eliminate infectious diseases. Despite decades of research in the field, several pathogens have eluded the effect of conventional vaccines mostly due their failure in inducing strong T cell responses. There is a need for new vaccine technologies that can surpass this problem. Recent advances in imaging techniques have allowed the study of T cell dynamics within their surrounding cellular niches. This information is invaluable to elucidate the main cellular mechanisms to target in order to optimize vaccine efficiency. In this review, we summarize the most recent key discoveries in T cell behavior in the context of vaccination and immunization.

LFA-1 in T cell priming, differentiation, and effector functions.

The integrin LFA-1 is crucial for T cell entry into mammalian lymph nodes and tissues, and for promoting interactions with antigen-presenting cells (APCs). However, it is increasingly evident that LFA-1 has additional key roles beyond the mere support of adhesion between T cells, the endothelium, and/or APCs. These include roles in homotypic T cell-T cell (T-T) communication, the induction of intracellular complement activity underlying Th1 effector cell polarization, and the support of long-lasting T cell memory. Here, we briefly summarize current knowledge of LFA-1 biology, discuss novel cytoskeletal regulators of LFA-1 functions, and review new aspects of LFA-1 mechanobiology that are relevant to its function in immunological synapses and in specific pathologies arising from LFA-1 dysregulation.

Th1 responses in vivo require cell-specific provision of OX40L dictated by environmental cues.

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.

Modes of Communication between T Cells and Relevance for Immune Responses.

T cells are essential mediators of the adaptive immune system, which constantly patrol the body in search for invading pathogens. During an infection, T cells that recognise the pathogen are recruited, expand and differentiate into subtypes tailored to the infection. In addition, they differentiate into subsets required for short and long-term control of the pathogen, i.e., effector or memory. T cells have a remarkable degree of plasticity and heterogeneity in their response, however, their overall response to a given infection is consistent and robust. Much research has focused on how individual T cells are activated and programmed. However, in order to achieve a critical level of population-wide reproducibility and robustness, neighbouring cells and surrounding tissues have to provide or amplify relevant signals to tune the overall response accordingly. The characteristics of the immune response-stochastic on the individual cell level, robust on the global level-necessitate coordinated responses on a system-wide level, which facilitates the control of pathogens, while maintaining self-tolerance. This global coordination can only be achieved by constant cellular communication between responding cells, and faults in this intercellular crosstalk can potentially lead to immunopathology or autoimmunity. In this review, we will discuss how T cells mount a global, collective response, by describing the modes of T cell-T cell (T-T) communication they use and highlighting their physiological relevance in programming and controlling the T cell response.

Quorum Regulation via Nested Antagonistic Feedback Circuits Mediated by the Receptors CD28 and CTLA-4 Confers Robustness to T Cell Population Dynamics.

T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from other cell types or if activated T cells themselves contribute to the orchestration of these population dynamics-akin to bacterial quorum regulation-is unclear. Here, we examined this question using time-lapse microscopy, genetic perturbation, bioinformatic predictions, and mathematical modeling. We found that ICAM-1-mediated cell clustering enabled CD8+ T cells to collectively regulate the balance between proliferation and apoptosis. Mechanistically, T cell expressed CD80 and CD86 interacted with the receptors CD28 and CTLA-4 on neighboring T cells; these interactions fed two nested antagonistic feedback circuits that regulated interleukin 2 production in a manner dependent on T cell density as confirmed by in vivo modulation of this network. Thus, CD8+ T cell-population-intrinsic mechanisms regulate cellular behavior, thereby promoting robustness of population dynamics.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Paracrine costimulation of IFN-γ signaling by integrins modulates CD8 T cell differentiation.

The cytokine IFN-γ is a critical regulator of immune system development and function. Almost all leukocytes express the receptor for IFN-γ, yet each cell type elicits a different response to this cytokine. Cell type-specific effects of IFN-γ make it difficult to predict the outcomes of the systemic IFN-γ blockade and limit its clinical application, despite many years of research. To better understand the cell-cell interactions and cofactors that specify IFN-γ functions, we focused on the function of IFN-γ on CD8 T cell differentiation. We demonstrated that during bacterial infection, IFN-γ is a dominant paracrine trigger that skews CD8 T cell differentiation toward memory. This skewing is preferentially driven by contact-dependent T cell-T cell (T-T) interactions and the localized IFN-γ secretion among activated CD8 T cells in a unique splenic microenvironment, and is less sensitive to concurrent IFN-γ production by other immune cell populations such as natural killer (NK) cells. Modulation of CD8 T cell differentiation by IFN-γ relies on a nonconventional IFN-γ outcome that occurs specifically within 24 hours following infection. This is driven by IFN-γ costimulation by integrins at T-T synapses, and leads to synergistic phosphorylation of the proximal STAT1 molecule and accelerated IL-2 receptor down-regulation. This study provides evidence of the importance of context-dependent cytokine signaling and gives another example of how cell clusters and the microenvironment drive unique biology.

Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

Visualization of immediate immune responses to pioneer metastatic cells in the lung.

Lung metastasis is the lethal determinant in many cancers and a number of lines of evidence point to monocytes and macrophages having key roles in its development. Yet little is known about the immediate fate of incoming tumour cells as they colonize this tissue, and even less known about how they make first contact with the immune system. Primary tumours liberate circulating tumour cells (CTCs) into the blood and we have developed a stable intravital two-photon lung imaging model in mice for direct observation of the arrival of CTCs and subsequent host interaction. Here we show dynamic generation of tumour microparticles in shear flow in the capillaries within minutes of CTC entry. Rather than dispersing under flow, many of these microparticles remain attached to the lung vasculature or independently migrate along the inner walls of vessels. Using fluorescent lineage reporters and flow cytometry, we observed 'waves' of distinct myeloid cell subsets that load differentially and sequentially with this CTC-derived material. Many of these tumour-ingesting myeloid cells collectively accumulated in the lung interstitium along with the successful metastatic cells and, as previously understood, promote the development of successful metastases from surviving tumour cells. Although the numbers of these cells rise globally in the lung with metastatic exposure and ingesting myeloid cells undergo phenotypic changes associated with microparticle ingestion, a consistently sparse population of resident conventional dendritic cells, among the last cells to interact with CTCs, confer anti-metastatic protection. This work reveals that CTC fragmentation generates immune-interacting intermediates, and defines a competitive relationship between phagocyte populations for tumour loading during metastatic cell seeding.

T cell migration, search strategies and mechanisms.

T cell migration is essential for T cell responses; it allows for the detection of cognate antigen at the surface of antigen-presenting cells and for interactions with other cells involved in the immune response. Although appearing random, growing evidence suggests that T cell motility patterns are strategic and governed by mechanisms that are optimized for both the activation stage of the cell and for environment-specific cues. In this Opinion article, we discuss how the combined effects of T cell-intrinsic and -extrinsic forces influence T cell motility patterns in the context of highly complex tissues that are filled with other cells involved in parallel motility. In particular, we examine how insights from 'search theory' can be used to describe T cell movement across an 'exploitation-exploration trade-off' in the context of activation versus effector function and lymph nodes versus peripheral tissues.

A septin requirement differentiates autonomous and contact-facilitated T cell proliferation.

T cell proliferation is initiated by T cell antigen receptor (TCR) triggering, soluble growth factors or both. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and septin-independent pathways. Septin-deficient T cells failed to complete cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was not dependent on septins when cell-cell contacts, such as those with antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and costimulatory signals. We were able to differentiate between cytokine- and antigen-driven expansion in vivo and thus show that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation.

Detection of rare antigen-presenting cells through T cell-intrinsic meandering motility, mediated by Myo1g.

To mount an immune response, T lymphocytes must successfully search for foreign material bound to the surface of antigen-presenting cells. How T cells optimize their chances of encountering and responding to these antigens is unknown. T cell motility in tissues resembles a random or Levy walk and is regulated in part by external factors including chemokines and lymph-node topology, but motility parameters such as speed and propensity to turn may also be cell intrinsic. Here we found that the unconventional myosin 1g (Myo1g) motor generates membrane tension, enforces cell-intrinsic meandering search, and enhances T-DC interactions during lymph-node surveillance. Increased turning and meandering motility, as opposed to ballistic motility, is enhanced by Myo1g. Myo1g acts as a "turning motor" and generates a form of cellular "flânerie." Modeling and antigen challenges show that these intrinsically programmed elements of motility search are critical for the detection of rare cognate antigen-presenting cells.

Secondary T cell-T cell synaptic interactions drive the differentiation of protective CD8+ T cells.

Immunization results in the differentiation of CD8+ T cells, such that they acquire effector abilities and convert into a memory pool. Priming of T cells takes place via an immunological synapse formed with an antigen-presenting cell (APC). By disrupting synaptic stability at different times, we found that the differentiation of CD8+ T cells required cell interactions beyond those made with APCs. We identified a critical differentiation period that required interactions between primed T cells. We found that T cell-T cell synapses had a major role in the generation of protective CD8+ T cell memory. T cell-T cell synapses allowed T cells to polarize critical secretion of interferon-γ (IFN-γ) toward each other. Collective activation and homotypic clustering drove cytokine sharing and acted as regulatory stimuli for T cell differentiation.

Evolving immune circuits are generated by flexible, motile, and sequential immunological synapses.

The immune system is made up of a diverse collection of cells, each of which has distinct sets of triggers that elicit unique and overlapping responses. It is correctly described as a 'system' because its overall properties (e.g. 'tolerance', 'allergy') emerge from multiple interactions of its components cells. To mobilize a response where needed, the majority of the cells of the system are obligatorily highly motile and so must communicate with one another over both time and space. Here, we discuss the flexibility of the primary immunological synapse (IS) with respect to motility. We then consider the primary IS as an initiating module that licenses 'immunological circuits': the latter consisting of two or more cell-cell synaptic interactions. We discuss how two or three component immunological circuits interact might with one another in sequence and how the timing, stoichiometry, milieu, and duration of assembly of immunological circuits are likely to be key determinants in the emergent outcome and thus the system-wide immune response. An evolving consideration of immunological circuits, with an emphasis on the cell-cell modules that complement T-antigen-presenting cell interaction, provides a fundamental starting point for systems analysis of the immune response.

Regulation of T cell priming by lymphoid stroma.

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues.

Evidence for a positive role of PtdIns5P in T-cell signal transduction pathways.

Phosphatidylinositol 5-phosphate (PtdIns5P) is emerging as a potential lipid messenger involved in several cell types, from plants to mammals. Expression of IpgD, a PtdIns(4,5)P(2) 4-phosphatase induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T-cells. Expression of a new PtdIns5P interacting domain blocks IpgD-induced T-cell activation and selective signaling molecules downstream of TCR triggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting the threshold of T-cell activation and contributing to maintain T-cell homeostasis.