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Climate change presents a grave and growing threat to the productivity and resilience of smallholder farming systems, impacts on which are already being felt in Pacific Island nations. Climate information plays a critical role in adaptation to these threats. Yet its accessibility and use may differ dramatically between groups of farmers, highlighting the need to understand the different paths of information exchange within communities. In this study, we examined weather and climate information sharing networks of both male and female farmers, and their perceived barriers to information access and use through a survey in three provinces in Papua New Guinea. The results provided insights about the most prominent actors as well as information sharing relationships, highlighting key gender differences. Women demonstrated stronger information connectivity with family and friends, capitalizing on close-knit networks. Conversely, the men’s network showed the potential to bridge between external information sources like media, and community leaders, church groups, and friends and family. To ensure equitable communication and use of information for climate adaptation, it is important to strike a balance between the strong bonding and open exchange aspects of the network, and consider the central sources and perceived hurdles affecting access and use of this information.

Social-ecological networks (SENs) represent the complex relationships between ecological and social systems and are a useful tool for analyzing and managing ecosystem services. However, mainstreaming the application of SENs in ecosystem service research has been hindered by a lack of clarity about how to match research questions to ecosystem service conceptualizations in SEN (i.e., as nodes, links, attributes, or emergent properties). Building from different disciplines, we propose a typology to represent ecosystem service in SENs and identify opportunities and challenges of using SENs in ecosystem service research. Our typology provides guidance for this growing field to improve research design and increase the breadth of questions that can be addressed with SEN to understand human-nature interdependencies in a changing world.

Food security, in the context of a rapidly changing climate, is one of the most prominent global challenges facing human societies today. Pacific Island Countries and Territories (PICTs) are particularly vulnerable to the adverse effects of climate change, while facing additional stress from globalisation and increased supply chain disruption. This review aims to document our understanding of food system vulnerability to climate change in the region, and identify the existing studies that could inform policy and decision-making. The review also serves to discern the dominant focal areas of research, as well as where gaps exist for emerging research.Using keyword searches on the web, scholarly databases, and targeted organisational websites, we identified 104 studies published from 2010 to 2022 that looked at the impacts of climate change on some aspect of the food system in at least one PICT. We found that the majority of empirical research on climate impacts on food systems in PICTs focused on the biophysical components of food production rather than the complex interactions between the socio-economic and biophysical factors that make up food systems. An incomplete understanding of the impacts from climate change could result in maladaptation and an undermining of food system resilience.

Autoimmunity arises when mechanisms of immune tolerance fail. Here we discuss mechanisms of T cell activation and tolerance and the dynamics of the autoimmune response at the site of disease. Live imaging of autoimmunity provides the ability to analyze immune cell dynamics at the single-cell level within the complex intact environment where disease occurs. These analyses have revealed mechanisms of T cell activation and tolerance in the lymph nodes, mechanisms of T cell entry into sites of autoimmune disease, and mechanisms leading to pathogenesis or protection in the autoimmune lesions. The overarching conclusions point to stable versus transient T cell antigen presenting cell interactions dictating the balance between T cell activation and tolerance, and T cell restimulation as a driver of pathogenesis at the site of autoimmunity. Findings from models of multiple sclerosis and type 1 diabetes are highlighted, however, the results have implications for basic mechanisms of T cell regulation during immune responses, tumor immunity, and autoimmunity.

Summary Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1D) has therapeutic potential, since targeting of T cell and B cell trafficking has been clinically effective in other autoimmune diseases. Trafficking to the islets is characterized by redundancy in adhesion molecule and chemokine usage, which has not enabled effective targeting to date. Additionally, cognate antigen is not consistently required for T cell entry into the islets throughout the progression of disease. However, myeloid cells are required to enable T cell and B cell entry into the islets, and may serve as a convergence point in the pathways controlling this process. In this review we describe current knowledge of the factors that mediate immune cell trafficking to pancreatic islets during T1D progression.

Naive T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigens presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Ena/VASP proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell activation and expansion in vivo. Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of antigen-specific T cell impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by 2-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also found that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling downstream of T-APC interactions required for the initiation of stable T cell conjugates during APC scanning and for efficient activation and expansion of T cells in vivo. Supported by NIH (R01AI125553; T32AI007405)

Lymphocyte migration is essential for the function of the adaptive immune system, and regulation of T cell entry into tissues is an effective therapy in autoimmune diseases. Little is known about the specific role of cytoskeletal effectors that mediate mechanical forces and morphological changes essential for migration in complex environments. We developed a new Formin-like-1 (FMNL1) knock-out mouse model and determined that the cytoskeletal effector FMNL1 is selectively required for effector T cell trafficking to inflamed tissues, without affecting naïve T cell entry into secondary lymphoid organs. Here, we identify a FMNL1-dependent mechanism of actin polymerization at the back of the cell that enables migration of the rigid lymphocyte nucleus through restrictive barriers. Furthermore, FMNL1-deficiency impairs the ability of self-reactive effector T cells to induce autoimmune disease. Overall, our data suggest that FMNL1 may be a potential therapeutic target to specifically modulate T cell trafficking to inflammatory sites.

Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets, promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in pre-diabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in the islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects the islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen.

To perform their functions, T cells migrate from the blood vasculature into tissues by traversing the vascular wall through a process known as transendothelial migration (TEM). While many of the extracellular cues that guide this process have been identified, the role of downstream cytoskeletal effectors that mediate force generation and morphological changes required for TEM remains an area of active research. Formin-like-1 (FMNL1) is a cytoskeletal effector that mediates cytoskeletal remodeling. However, the role of FMNL1 in T cell functions, and migration in particular, is mostly unknown. The goal of our study was to examine the role of FMNL1 in T cell extravasation and trafficking. We generated a new FMNL1 knock-out (KO) mouse and identified a novel role for FMNL1 in promoting T cell migration through restrictive endothelial barriers during TEM. In vitro, FMNL1-deficient T cells had reduced actin polymerization in response to chemokine signaling. Furthermore, FMNL1 KO T cells had a selective impairment in chemotaxis through restrictive 3μm pores vs permissive 5μm pores. Using time-lapse microscopy, we determined that FMNL1-deficient T cells are severely impaired in their capacity to complete TEM through endothelial barriers. In vivo, we found that FMNL1 was dispensable for T cell extravasation through permissive high endothelial venules and homeostatic trafficking to lymphoid organs. Instead, FMNL1 regulated the ability of activated T cells to extravasate into inflamed peripheral tissues and enabled self-reactive T cells to induce autoimmune disease. Overall, our data identify FMNL1 as a key regulator of lymphocyte migration through restrictive barriers and as a potential target for modulating effector T cell trafficking.

We have identified T cells and B cells in the islets of autoimmune resistant mice, yet the role of these lymphocytes is unknown. In the islets of autoimmune resistant C57BL/6 mice, local antigen receptor signaling was assessed using Nur77-GFP mice. Nur77-GFP expression was increased in islet CD8+ T cells and B cells suggesting that these lymphocyte subsets receive local antigenic stimulus. Islet T cell and B cell populations expressed the regulatory cytokine IL-10, assessed using the B6.TIGER reporter mouse. T cell populations lacked expression of the pro-inflammatory cytokine IFN-γ. These findings indicate that islet T cell populations are likely playing a regulatory role. In addition, cultured whole islets produced IL-10 protein, but not IFN-γ protein. To test the stability of the islet lymphocyte populations under inflammatory conditions, mice were exposed to metabolic stress through a diet-induced obesity (DIO) model. Obesity and type 2 diabetes (T2D) are associated with circulating inflammatory cytokines. In T2D patients, islet lymphocytes have been identified, though the function of these populations is largely unknown. C57BL/6 mice were fed a high fat diet (DIO) or a matched control diet (Control) starting at 5 weeks of age, and then analyzed at 16–25 weeks of age. The number and phenotype of lymphocytes in the islets were similar in DIO and control mice. Notably, under the conditions of obesity-associated systemic inflammation, islet lymphocytes in the DIO mice maintained IL-10 production. From these data, we propose that islet lymphocytes play a regulatory role to protect the islets from systemic inflammation through IL-10 production.

We recently established that hybrid insulin peptides (HIPs), formed in islet β-cells by fusion of insulin C-peptide fragments to peptides of chromogranin A or islet amyloid polypeptide, are ligands for diabetogenic CD4 T-cell clones. The goal of this study was to investigate whether HIP-reactive T cells were indicative of ongoing autoimmunity. MHC class II tetramers were used to investigate the presence, phenotype, and function of HIP-reactive and insulin-reactive T cells in NOD mice. Insulin-reactive T cells encounter their antigen early in disease, but they express FoxP3 and therefore may contribute to immune regulation. In contrast, HIP-reactive T cells are proinflammatory and highly diabetogenic in an adoptive transfer model. Because the frequency of antigen-experienced HIP-reactive T cells increases over progression of disease, they may serve as biomarkers of autoimmune diabetes.

The generation of class-switched, high-affinity, antibody-producing B cells plays a critical role in the establishment of type 2 immunity to intestinal helminths as well as in the pathogenesis of allergy and asthma. The generation of these high-affinity, antibody-producing B cells occurs in germinal centers (GC) and relies on interactions with follicular dendritic cells (FDCs) and T follicular helper (Tfh) cells. One critical mediator produced by Tfh cells in GCs is interleukin-4 (IL-4). Tfh-derived IL-4 drives class switching to type 2 antibody isotypes IgE and IgG1 and is required for high-affinity IgG1 production. In vivo detection of IL-4-expressing Tfh cells is required to better understand the role of these cells during the GC response. Detection of IL-4-expressing cells has been greatly improved by the generation of the IL-4

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the β cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA−/− mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA+/+ mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c+ B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c+ B cells form significantly more stable interactions with T cells than do FO cells. Together, these data identify a previously undescribed role for ABCs as potent APCs and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity.

The generation of class-switched, high-affinity, antibody-producing B cells plays a critical role in the establishment of type 2 immunity to intestinal helminths as well as in the pathogenesis of allergy and asthma. The generation of these high-affinity, antibody-producing B cells occurs in germinal centers (GC) and relies on interactions with follicular dendritic cells (FDCs) and T follicular helper (Tfh) cells. One critical mediator produced by Tfh cells in GCs is interleukin-4 (IL-4). Tfh-derived IL-4 drives class switching to type 2 antibody isotypes IgE and IgG1 and is required for high-affinity IgG1 production. In vivo detection of IL-4-expressing Tfh cells is required to better understand the role of these cells during the GC response. Detection of IL-4-expressing cells has been greatly improved by the generation of the IL-44get reporter mice, which read out IL-4 expression as green fluorescent protein (GFP). Much has been learned from these mice with regard to type 2 immunity using flow cytometry and immunohistochemistry. However, these methods do not allow the study of cellular behavior and interactions in real time. In contrast, multi-photon microscopy allows for deep tissue imaging and tracking of multiple cell types in intact tissues over time. Here, we describe a protocol for in vivo detection of IL-4-expressing Tfh cells in an explanted popliteal lymph node by multi-photon microscopy. The dynamics of Tfh cell motility and their interactions with FDC networks in the GCs were analyzed.

Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages., Author summary Interferon (IFN)γ promotes host resistance to invasive bacterial infections, but type I IFNs suppress macrophage activation and increase susceptibility to L. monocytogenes and other pathogenic bacteria. IFNγ promotes resistance by potently activating macrophages to become pro-inflammatory and antimicrobial. Type I IFNs have the opposite effect on resistance during many bacterial infections. They can also suppress macrophage responsiveness to IFNγ through the down regulation of its signaling receptor (IFNGR). Here, we developed an experimental system to investigate the significance of IFNGR1 down regulation as a mechanism contributing to pro-bacterial effects of type I IFNs. A transgenic mouse (fGR1) was generated in which macrophages resist type I IFN-induced down regulation of IFNGR1. We report that macrophages from fGR1 mice retain responsiveness to IFNγ when exposed to type I IFNs. fGR1 mice thus have increase resistance to systemic infection by L. monocytogenes. This resistance is dependent on IFNγ and associates with increased evidence of macrophage activation and anti-bacterial activity. Our findings thus suggest modulation of IFNGR1 is an important mechanism by which type I IFNs increase host susceptibility to bacterial infections.

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naive T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.

T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subsequently, they must navigate to effector sites, which they enter through the process of trans-endothelial migration (TEM). During interstitial migration, T cells migrate according to one of two modes that are distinguished by the strength and sequence of adhesions and the requirement for Myosin-IIA. In contrast during TEM, T cells require Myosin-IIA for the final process of pushing their nucleus through the endothelium. A generalized model emerges with dual roles for Myosin-IIA: This motor protein acts like a tensioning or expansion spring, transmitting force across the cell cortex to sites of surface contact and also optimizing the frictional coupling with substrata by modulating the surface area of the contact. The phosphorylation and deactivation of this motor following TCR engagement can allow T cells to rapidly alter the degree to which they adhere to surfaces and to switch to a mode of interaction with surfaces that is more conducive to forming a synapse with an antigen-presenting cell.

B cells have been strongly implicated in the development of human type 1 diabetes and are required for disease in the NOD mouse model. These functions are dependent on B cell antigen receptor (BCR) specificity and expression of MHC, implicating linked autoantigen recognition and presentation to effector T cells. BCR-antigen affinity requirements for participation in disease are unclear. We hypothesized that BCR affinity for the autoantigen insulin differentially affects lymphocyte functionality, including tolerance modality and the ability to acquire and become activated in the diabetogenic environment. Using combined transgenic and retrogenic heavy and light chain to create multiple insulin-binding BCRs, we demonstrate that affinity for insulin is a critical determinant of the function of these autoreactive cells. We show that both BCR affinity for insulin and genetic background affect tolerance induction in immature B cells. We also find new evidence that may explain the enigmatic ability of B cells expressing 125 anti-insulin BCR to support development of TID in NOD mice despite a reported affinity beneath requirements for binding insulin at in vivo concentrations. We report that when expressed as an antigen receptor the affinity of 125 is much higher than determined by measurements of the soluble form. Finally, we show that in vivo acquisition of insulin requires both sufficient BCR affinity and permissive host/tissue environment. We propose that a confluence of BCR affinity, pancreas environment, and B cell tolerance-regulating genes in the NOD animal allows acquisition of insulin and autoimmunity.

Multi-photon microscopy has taken hold as a widely used technique in immunology, allowing for imaging of the kinetics of immune cell motility and cell-cell interactions, but what have we learned from this technique about the processes involved in peripheral tolerance and autoimmunity? Various studies have now looked at the dynamics of several mechanisms of peripheral T cell tolerance and efforts to examine the dynamics of the autoimmune response at the disease site are also under way. Here, we will discuss the findings of studies that use multi-photon microscopy to examine the dynamics of T cell tolerance in the lymph nodes and of the autoimmune processes involved in models of type 1 diabetes and multiple sclerosis. An emerging theme from these studies is that short T cell-antigen presenting cell interactions can lead to tolerance, and that autoreactive T cell restimulation at the disease site can play an important role in autoimmune disease exacerbation.

Real-time imaging defines the dynamics of TCR and T cell motility during early T cell activation in lymph nodes., The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation.

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4 + T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell–antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell–APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell–APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.

During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts versus the need to traverse an entire organ. Here we show that in vivo, myosin IIA-deficient T cells had a triad of defects, including overadherence to high-endothelial venules, less interstitial migration and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of three-dimensional motility in microchannels showed that the degree of confinement and myosin IIA function, rather than integrin adhesion (as proposed by the haptokinetic model), optimized motility rate. This motility occurred via a myosin IIA-dependent rapid 'walking' mode with multiple small and simultaneous adhesions to the substrate, which prevented spurious and prolonged adhesions. Adhesion discrimination provided by myosin IIA is thus necessary for the optimization of motility through complex tissues.

Type 1 diabetes (T1D) is a largely T cell mediated autoimmune disease that destroys the beta cells of the pancreatic islets. Inhibition of cell trafficking to an active disease site has been an effective therapy for multiple autoimmune diseases, but the requirements of lymphocyte trafficking to the islets are not fully understood. We show that lymphocyte entry into previously infiltrated islets is dependent on the presence of CD11c+ cells in the islets. T cells and B cells transferred prior to CD11c+ cell depletion were able to enter the islets; whereas, short-term CD11c+ cell depletion rapidly prevented further entry. CD11c+ cells include cells that are highly efficient antigen presenters; however, we show entry of both activated or naïve T cells into previously infiltrated islets was not reliant on antigen. An alternative role for CD11c+ cells in lymphocyte entry into the islets is the production of chemokines and cytokines. These molecules can then either directly recruit lymphocytes through chemoattraction or increased adhesion to the vasculature. Initial experiments show that there are no changes in adhesion to the vasculature after CD11c depletion, suggesting that CD11c+ cells act through recruitment by chemokines following lymphocyte arrest on the vascular endothelium. CD11c+ cells in the islets express high levels of CXCL9, a chemoattractant for T and B lymphocytes. CXCR3, the receptor for CXCL9, is present on both T and B cells in the islets. We hypothesize that CD11c+ cells facilitate the process of extravasation into the islets through chemokine production. Targeting CD11c+ produced chemokine production may be useful therapeutically for the treatment of T1D by preventing T cell entry into remaining or transplanted islets.

Dendritic cells (DCs) are essential in T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans in type 1 diabetes. In this study, we investigated T cell induction of intra-islet DC maturation during the progression of the disease in both autoimmune-prone NOD and resistant C57BL/6 mice. We demonstrated steady-state capture and retention of unprocessed β cell-derived proteins by semimature intra-islet DCs in both mouse strains. T cell-mediated intra-islet inflammation induced an increase in CD40 and CD80 expression and processing of captured Ag by resident DCs without inducing the expression of the p40 subunit of IL-12/23. Some of the CD40high intra-islet DCs up-regulated CCR7, and a small number of CD40high DCs bearing unprocessed islet Ags were detected in the pancreatic lymph nodes in mice with acute intra-islet inflammation, demonstrating that T cell-mediated tissue inflammation augments migration of mature resident DCs to draining lymph nodes. Our results identify an amplification loop during the progression of autoimmune diabetes, in which initial T cell infiltration leads to rapid maturation of intra-islet DCs, their migration to lymph nodes, and expanded priming of more autoreactive T cells. Therapeutic interventions that intercept this process may be effective at halting the progression of type 1 diabetes.

SummaryT cells slow their motility, increase adherence, and arrest after encounters with antigen-presenting cells (APCs) bearing peptide-MHC complexes. Here, we analyzed the cell-cell communication among activating T cells. In vivo and in vitro, activating T cells associated in large clusters that collectively persisted for >30 min, but they also engaged in more transient interactions, apparently distal to APCs. Homotypic aggregation was driven by LFA-1 integrin interactions. Ultrastructural analysis revealed that cell-cell contacts between activating T cells were organized as multifocal synapses, and T cells oriented both the microtubule-organizing complex and interleukin-2 (IL-2) secretion toward this synapse. T cells engaged in homotypic interactions more effectively captured IL-2 relative to free cells. T cells receiving paracrine synaptic IL-2 polarized their IL-2 signaling subunits into the synaptic region and more efficiently phosphorylated the transcription factor STAT5, likely through a synapse-associated signaling complex. Thus, synapse-mediated cytokine delivery accelerates responses in activating T cells.

T lymphocytes are capable of rapid motility in vitro and in vivo. Upon antigen recognition, they may stop crawling and form a stable cell–cell contact called the ‘immunological synapse’ (IS). However, it is becoming clear that this outcome may not occur with the reliability that was once presumed. T cells, particularly naive cells, are apparently triggered partly ‘on the fly’ during short contacts with peptide–MHC (pMHC) bearing antigen-presenting cells (APCs) and are also influenced in both activity and synapse duration by a multitude of external cues. Underlying the emerging issues is a paucity of data concerning the cell biology of T lymphocytes. Here, we review the molecular mechanisms of crawling and adhesion versus the various potential modes of ‘stopping’ in T lymphocytes. Both motility and arrest involve similar processes: adhesion, actin elongation and internal tension control, but with different coordination. We will attempt to integrate this with the known and potential external cues that signal for T cell motility versus stopping to form a synapse in vivo. Finally, we discuss how this interplay may give rise to unexpectedly complex motile and morphological behavior.

Prostein is a prostate tissue-specific protein that is uniquely and abundantly expressed in normal and cancerous prostate tissues. Due to this expression profile, we examined the immunogenicity of prostein as a potential vaccine candidate for prostate cancer. To determine the presence of CD8 T cells specific for naturally processed prostein-derived epitopes in healthy individuals, we developed and applied an in vitro stimulation protocol. Using this protocol, we identified CD8 T cells specific for prostein in the peripheral blood of a male and a female donor. Prostein-specific CD8 T cell clones specifically recognized prostein-expressing targets, including prostate tumor cell lines expressing the relevant HLA alleles. CD8 T cell clones isolated from the male donor were significantly less effective in recognizing target cells compared to cells isolated from the female donor and appeared to recognize subdominant epitopes. The identification of a prostein-specific CD8 T cell repertoire supports the development of prostein in vaccination strategies against prostate cancer. Furthermore, the naturally processed peptide epitopes identified provide tools for the development of peptide-based vaccination strategies against prostate cancer and for monitoring of prostein-specific responses in vaccinated patients.

Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8(+) T cells. During early-islet infiltration, T-cell interactions with CD11c(+) antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell-APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling in vitro. Using timed introduction of effector T cells, we demonstrate that elements of the autoimmune-tissue microenvironment control the dynamics of autoantigen recognition by T cells and their resulting pathogenic effector functions.

Down-modulation of CD3ζ expression on CD8 T lymphocytes occurs, independently of other T-cell receptor (TCR)-CD3 components, in tumor-infiltrating lymphocytes, human immunodeficiency virus infection, and autoimmune disease. These associations suggest that it might be related to chronic antigenic stimulation. CD3ζ down-modulation was found, however, in CD8 T cells that proliferate in response to acute viral infections. In 3 otherwise healthy donors with acute gastroenteritis, infectious mononucleosis, and Epstein–Barr virus/cytomegalovirus/mononucleosis, 30% to 60% of circulating CD8 T cells had down-modulated CD3ζ to below the level of detection. The CD3ζ-T cells were also CD28− but expressed the activation markers HLA-DR and CD57. CD3ζ–CD28– T cells are effector CTL because they express perforin and produce IFN-γ, but not IL-2, on activation and contain the viral-specific cytotoxic T lymphocyte (CTL). However, CD3ζ–CD28–T cells generally do not express CD25 after anti-CD3 and anti-CD28 stimulation and are not cytotoxic until they are cultured with IL-2 overnight. Cytotoxicity coincides with the re-expression of CD3ζ but not CD28. Down-modulation of CD3ζ and CD28 on effector CTL may control CTL triggering and proliferation to prevent immunopathogenesis.

CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell death. Either granzyme A or B can act with perforin to trigger apoptosis. Granzyme B activates a ubiquitous apoptotic cascade induced by caspase cleavage, but the granzyme A pathway is largely unknown. Using affinity chromatography with recombinant mutant inactive granzyme A, we previously isolated two granzyme A-binding proteins, PHAP (putative HLA-associated protein) I and II. PHAP II, a substrate of granzyme A, is degraded within minutes of CTL attack. Two additional cytoplasmic proteins of 27 and 53 kDa bind strongly to the mutant granzyme A column, requiring 6 M urea to elute. Sequencing identified these as the monomer and dimer of hsp27, a small heat shock protein up-regulated by stress and cellular activation. Hsp27 coprecipitates with granzyme A from cytoplasmic lysates and is not a substrate of the enzyme. Hsp27 translocates to the detergent-insoluble fraction of target cells and relocalizes from diffuse cytoplasmic staining to long filamentous fibers, especially concentrated in a perinuclear region, within minutes of CTL attack. Hsp27 may participate in morphologic changes during granule-mediated lysis. Low or absent levels of hsp27 expression in T lymphocytes, even after heat shock, may play a role in CTL resistance to granule-mediated lysis.

Many vaccines include aluminum salts (alum) as adjuvants despite little knowledge of alum’s functions. Host DNA rapidly coats injected alum. Here, we further investigated the mechanism of alum and DNA’s adjuvant function. Our data show that DNase coinjection reduces CD4 T-cell priming by i.m. injected antigen + alum. This effect is partially replicated in mice lacking stimulator of IFN genes, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNase treatment impairs dendritic cell (DC) migration from the peritoneal cavity to the draining lymph node in mice immunized i.p. with alum. However, our data show that DNase does not affect accumulation of, or expression of costimulatory proteins on, antigen-loaded DCs in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNase does inhibit prolonged T-cell–DC conjugate formation and antigen presentation between antigen-positive DCs and antigen-specific CD4 T cells following i.m. injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DCs, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC class II presentation and better DC–T-cell interactions.

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.

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins that have been implicated in T cell actin cytoskeleton remodeling in response to T cell receptor signaling. However, the role of these proteins in T cell motility, transendothelial migration, and in vivo trafficking remained unknown. We investigated the role of EVL and VASP in T cell trafficking and found that these cytoskeletal effectors are selectively required for activated but not naïve T cell trafficking to the lymph nodes and spleen. Moreover, in a model of Multiple Sclerosis, EVL/VASP deficiency severely impaired activated T cell trafficking to the inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE). Deletion of EVL and VASP resulted in impaired Very Late Antigen-4 (VLA-4) expression and function, but unexpectedly did not cause alterations in shear-resistant adhesion and motility of T cells on primary brain microvascular endothelial cells in vitro. Instead, deletion of EVL and VASP caused a significant impairment in the ability of T cells to initiate and complete the diapedesis step of transendothelial migration. Furthermore, upon blockade of VLA-4, T cell diapedesis became equivalent between control and EVL/VASP-deficient T cells. Together, these data suggest that that EVL and VASP selectively regulate activated T cell trafficking by specifically mediating the diapedesis step of transendothelial migration in a VLA-4-dependent manner.

Modern imaging approaches are proving important for addressing contemporary issues in the immune system. These approaches are especially useful for characterizing the complex orchestration of immune responses in vivo. Multicolor, two-photon imaging has been proven to be especially enabling for such studies because of its superior tissue penetration, reduced image degradation by light scattering leading to better resolution, and its high image quality deep inside tissues. Here, we examine the functional requirements of two-photon imaging instruments necessary for such immune studies. These requirements include frame rate, spatial resolution and the number of emission channels. We use this discussion as a starting point to compare commercial systems and to introduce a custom technology platform that meets those requirements. This platform is noteworthy because it is very cost-effective, flexible and experimentally useful. Representative data collected with this instrument is used to demonstrate the utility of this platform. Finally, as the field is rapidly evolving, consideration is given to some of the cutting-edge developments in multiphoton microscopy that will likely improve signal strength, depth penetration and/or the experimental usefulness of this approach.

Modern imaging approaches are proving important for addressing contemporary issues in the immune system. These approaches are particularly useful for characterizing the complex orchestration of immune responses in vivo. Multicolor, two-photon imaging has been proven to be especially enabling for such studies because of its superior tissue penetration, reduced image degradation by light scattering leading to better resolution and its high image quality deep inside tissues. Here, we examine the functional requirements of two-photon imaging instruments necessary for such immune studies. These requirements include frame rate, spatial resolution and the number of emission channels. We use this discussion as a starting point to compare commercial systems and to introduce a custom technology platform that meets these requirements. This platform is noteworthy because it is very cost-effective, flexible and experimentally useful. Representative data collected with this instrument is used to demonstrate the utility of this platform. Finally, as the field is rapidly evolving, consideration is given to some of the cutting-edge developments in multiphoton microscopy that will likely improve signal strength, depth penetration and/or the experimental usefulness of this approach.

The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator (Aire) gene. Here we report the identification of extrathymicAire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen–expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

T cell activation in vivo occurs in a lymphoid milieu that presents chemotactic and T cell receptor signals concurrently. Here we demonstrate that T cell zone chemokines such as CCL21 are bound to the surface of lymph node dendritic cells. Contact with antigen-presenting cells bearing chemokines costimulated T cells by a previously unknown two-step contact mechanism. T cells initially formed an antigen-independent 'tethered' adhesion on chemokine-bearing antigen-presenting cells. The formation of those tethers superseded T cell receptor signaling and immunological synapse formation. However, chemokine-tethered T cells were hyper-responsive to subsequent contacts with antigen-presenting cells. Thus, T cells are costimulated 'in trans' and sequentially after initial engagement with their chemokine-rich environment.

The developmental transcription factor SOX-4 has been shown to be highly and differentially overexpressed in primary small cell lung carcinomas (SCLC). To examine the potential of SOX-4 for broad use as a lung cancer vaccine, we have evaluated the expression of SOX-4 in a panel of primary adenocarcinoma, squamous, and large cell tumor samples as well as in a panel of established small cell and non-small cell lung carcinoma tumor cell lines. SOX-4 mRNA is shown to be overexpressed in a substantial fraction of each of these lung tumor types. To examine the immunological potential of SOX-4, we have evaluated the presence of SOX-4-specific CD4 and CD8 T cells in PBMC of healthy donors and the presence of SOX4-specific Abs in sera from SCLC patients. We demonstrate the presence of both CD4 and CD8 T cells that recognize naturally processed epitopes derived from SOX-4 as well as the presence of SOX-4-specific Abs in sera from SCLC patients, but not in sera from healthy donors. The lung tumor-specific overexpression and demonstration of a comprehensive Ag-specific immune response specific for SOX-4 support the use of this molecule in the development of whole gene-, peptide-, or protein-based vaccination strategies against lung cancer. Furthermore, the identification of naturally processed T cell and Ab epitopes from SOX-4 provides valuable tools for the development of peptide-based vaccination strategies against lung cancer as well as to monitor SOX-4-specific responses in vaccinated patients.