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Abstract: Advancements in rheumatoid-arthritis-(RA) therapies have shown considerable progresses in the comprehension of disease. However, the development of new potential agents with relative safety and efficacy continues and natural compounds have been considered as alternatives to identify new entities. Since previous in-vivo data and our in-vitro findings showed that torilin has a strong anti-inflammatory property, we further investigated its effect against collagen-induced-arthritis-(CIA) in mice. CIA-induced DBA/1J mice were treated with torilin or methotrexate (MTX) for 5-weeks. Arthritis severity was evaluated by arthritic score and joint histopathology. Draining lymph node (dLN), joint and peripheral-blood mononuclear-cell (PBMC) counts, and activation/localization of T-/B-lymphocytes, dendritic cells (DCs) and neutrophils were examined by FACS analysis. Serum anti-type-II-collagen-(CII) antibody levels and cultured-splenocyte and serum cytokines were also evaluated. Torilin markedly reduced CIA-induced arthritic score, histopathology and leukocyte counts. Besides, torilin suppressed CIA-activated T-cells including CD3+, CD3+/CD69+, CD8+, CD4+ and CD4+/CD25+ in dLNs or joints. It also modified CD19+ or CD20+/CD23+ (B-cells), MHCII+/CD11c+ (DCs) and Gr-1+/CD11b+ (neutrophil) subpopulations. It further depressed total anti-CII-IgG, anti-CII-IgG1 and anti-CII-IgG2a antibody productions. Moreover, while IFN-γ and IL-10 were not affected, torilin suppressed CIA-induced serum TNF-α, IL-1β and IL-6 levels. Interestingly, torilin also blocked IFN-γ, IL-17 and IL-6 cytokines while it did not affect IL-10 but enhanced IL-4 in splenocytes. These results show that torilin attenuated arthritis severity, modified leukocyte activations in dLNs or joints, and restored serum and splenocyte cytokine imbalances. Torilin may have immunomodulatory and anti-inflammatory properties with the capacity to ameliorate the inflammatory response in CIA-mice. [Copyright &y& Elsevier]

Antigen processing and recognition is a key feature of antibacterial immune responses to intracellular bacteria. In contrast to viruses, which are primarily controlled by conventional MHC II- and MHC I-restricted CD4+ or CD8+ T cells, respectively, unconventional T cells participate additionally in antibacterial protection. These unconventional T cells include glycolipid-specific CD1-restricted T cells and phospholigand-specific γδ T cells. We are just beginning to understand the broad spectrum of antigen recognition and stimulation of distinct T-cell populations by bacterial pathogens. From the host perspective, a broad spectrum of different T-cell populations that recognize proteins, lipids and carbohydrates strengthens protective immunity. From the perspective of the pathogen, antigen presentation represents a bottleneck that should be exploited for evasion from, or devastation of, acquired immunity. Although several such mechanisms have been described in viral systems, few have thus far been elucidated in bacterial infections. [Copyright &y& Elsevier]

Advances in immune visualization have enabled the physical tracking of immune responses in vivo. The adaptation of such technology to models of infectious disease holds the promise of a more detailed analysis of host–pathogen interactions in a natural setting. However, the visualization of pathogen-specific immune responses in vivo confronts challenges that are inherent to the study of infectious disease systems. Recent attempts to track pathogen-specific immune responses in vivo validate the usefulness and underline the complexity of this experimental strategy. [Copyright &y& Elsevier]

Advances in imaging technology have been essential to our understanding of T-cell activation and effector functions. Much of the progress stems from the use of fluorescent fusion proteins combined with high resolution imaging techniques, including confocal and multiphoton microscopy. However, these techniques have limitations, and other modes of imaging, including new developments on the horizon, might add promising new tools for the visualization of cytoskeleton-dependent processes in living cells. [Copyright &y& Elsevier]

Over the past few years, the crucial role of Notch signaling in multiple stages of T-cell development has become apparent. Recent studies have helped to define more precisely the functions and components of the Notch signaling pathway in T-cell development, including during the T versus B fate decision and in early CD4-CD8-double-negative thymocytes. In addition, new evidence points to a requirement for Notch2 in the development of marginal zone B cells. Finally, recent studies have provided our first glimpse into the complex and paradoxical roles of Notch signaling in the activation and differentiation of mature T cells. [Copyright &y& Elsevier]

The interaction of T cells with antigen-presenting cells results in the formation of a contact face, termed the immunological synapse. The prototypical dynamics of this process are well established and involve cessation of crawling, a highly fluid ‘immature’ synapse phase during which signaling is initiated, and ultimately the formation of a ‘mature’ synapse characterized by centralized and peripheral supramolecular activating complexes. Ongoing research is directed towards defining how these supramolecular assemblies are formed and, more importantly, to what end. With regard to the former, progress has been made in defining the order in which various molecules are recruited to signaling centers in prototypical settings. With regard to the latter, however, the issue now appears more complex, as both developmental changes in T cells and variations in the environment appear to modulate features of mature synapse development. Although many details of the immunological synapse have been established, emerging evidence suggests a great variability in the ultimate form of these contacts and their effects on T-cell functions. [Copyright &y& Elsevier]

By mediating non-covalent protein–protein interactions, adaptors organize and assemble the multimolecular signalling complexes that coordinate intracellular programs leading to the activation and differentiation of lymphocytes. The co-ordinated interaction between adaptor and effector molecules is required for the propagation and dynamic modification of externally applied signals. Recent advances have been made regarding our understanding of how adaptors regulate signalling within lymphocytes. An unexpected function has been revealed for the well-known adaptor protein LAT in pre-B-cell receptor signalling. In addition, the adaptors BCAP, Bcl10, CARMA1 and Malt1 seem to regulate the development of particular B-cell subsets. In contrast to Shc, c-Cbl and LAT, which are involved in early signalling events, BCAP, Bcl10, CARMA1 and Malt1 seem to act more distally, by controlling NF-κB activation. Additional transmembrane adaptors, such as NTAL/LAB and LIME, have been identified and partially characterized. Finally, an involvement of the cytosolic adaptors ADAP, SKAP-55 and Cbl-b in the regulation of lymphocyte adhesion and migration has been demonstrated. [Copyright &y& Elsevier]

The analysis of recent data reveals that T-cell co-stimulation is a hierarchical process with elements of mutual interdependence between individual co-stimulators. The expression and function of co-stimulatory molecules is biased on various T-cell subsets and is dependent on the T-cell differentiation state. The classical paradigm of T-cell co-stimulation by professional antigen-presenting cells has to incorporate the newly recognized concept of T-cell co-stimulation in the interaction with peripheral tissues, such as endothelial or epithelial cells. The two signal paradigm of T-cell co-stimulation is being replaced by a multisignal integration concept of central and peripheral co-stimulation. [Copyright &y& Elsevier]

The essential role played by CD25+CD4+ regulatory T cells (TR cells) in the control of physiological as well as pathological immunity is now well established, but many aspects of their biology still remain unclear. One of the unresolved issues regards their development: where does this occur, what signals are required, and how do TR cells fit into the larger taxonomy of the T-cell family? Recent data has begun to shed light on the development and function of these important cells. [Copyright &y& Elsevier]

Clonal deletion is arguably the most important mechanism of eliminating self-reactive thymocytes from the T-cell repertoire. Recent work has identified new players in this process. On the thymocyte side, several molecules have been newly implicated in the pathway from initial T-cell receptor signaling through to the final result: gene transcription and thymocyte apoptosis. In addition, several proapoptotic molecules have been found to be necessary for the death of self-reactive thymocytes. On the antigen-presenting cell side, the expression of peripheral self-antigens, regulated at least in part by the autoimmune regulator (AIRE) protein, is crucial for complete elimination of autoreactive thymocytes. The importance of thymic peripheral antigen expression and clonal deletion to self-tolerance is demonstrated in the autoimmune diseases autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy and type-1 diabetes mellitus. [Copyright &y& Elsevier]

The Notch family of evolutionarily conserved proteins regulates a broad spectrum of cell-fate decisions and differentiation processes during fetal and post-natal development. The best characterized role of Notch signaling during mammalian hematopoiesis and lymphopoiesis is the essential function of the Notch1 receptor in T-cell lineage commitment. More recent studies have addressed the roles of other Notch receptors and ligands, as well as their downstream targets, revealing additional novel functions of Notch signaling in intra-thymic T-cell development, B-cell development and peripheral T-cell function. [Copyright &y& Elsevier]

The development of new imaging techniques has made it possible to investigate the dynamic movements of molecules involved in T-cell signalling. Fluorescence resonance energy transfer (FRET) imaging allows the investigation of protein–protein interactions in live cells, and has demonstrated that T-cell receptors (TCRs) and CD4 are brought together in the immunological synapse during antigen recognition. This interaction is inhibited by antagonist ligands. Antagonism works through competition between agonist and antagonist ligands for TCR binding, as well as through feedback via the SHP-1 tyrosine phosphatase and extracellular signal-related kinase. Early signalling events result in the clustering of co-receptors and TCRs at the synapse, and the activation of various signalling molecules. Recent data show that some T-cell signalling precedes the formation of the mature form of the immunological synapse, but that full T-cell activation depends on sustained signalling, which in turn requires the synapse. [Copyright &y& Elsevier]

The MHC class II molecules have been recognized as signaling receptors for more than a decade, and recent work has revealed the importance of their signaling for the immune response. Today, we know that the function of MHC class II molecules on antigen-presenting cells (APCs) is not limited to their role as antigen-presenting structures; they are flexible receptors that, by triggering a variety of signaling pathways, can regulate APC activities from proliferation and maturation to apoptosis. Recent advances have provided insights into how these molecules might accommodate such regulation. [Copyright &y& Elsevier]

Monoclonal antibodies specific for defined peptide–MHC complexes are now being used to physically detect T-cell receptor ligands. These reagents have resulted in the identification of the cells that present antigen in lymphoid and non-lymphoid tissues after various forms of antigen administration. In addition, recent advances in real-time imaging technology have begun to measure the rate and directionality of T-cell movement relative to antigen-presenting cells in lymph nodes, shedding light on the earliest events in T-cell activation in a physiological setting. [Copyright &y& Elsevier]

Activation of CD4+ T cells requires the interaction of multiple T-cell receptors with MHC class II–peptide complexes on the surface of antigen-presenting cells (APCs). Recent studies have shown that MHC class II complexes are clustered in APC plasma membrane microdomains, thereby providing a mechanism for localized concentration of MHC class II–peptide complexes. The integrity of one type of APC membrane microdomain, the lipid raft, is important for antigen presentation to T cells. We propose a model in which the coordinated processes of MHC class II peptide loading and intracellular trafficking enhance T-cell activation by loading specific MHC class II–peptide complexes in discrete lipid raft microdomains. [Copyright &y& Elsevier]

Recent advances in the field of Toll-like receptors (TLRs) have established their importance for acute innate immune responses and their fundamental requirement for initiation of CD4+ T-cell responses. Current directions in the field have been towards understanding the role of cytokines secreted by professional antigen-presenting cells in response to TLR ligands and also pathological manifestations in terms of increased production of rheumatoid factors due to TLR ligation on B cells. Thus, it is becoming clear that TLRs are important for induction of adaptive immune responses, but misguided responses can lead to autoimmune pathology. [Copyright &y& Elsevier]

MHC class I tetramer staining, intracellular cytokine staining and ELISPOT assays have made it possible to quantify CD8+ T-cell responses precisely during and following viral and bacterial infection. Although these quantitative methods are by now familiar and trusted components of the immunologist’s toolbox, their application to models of microbial infection continues to provide surprising insights into mammalian adaptive immunity. In the past year there have been many exciting new findings on CD8+ T-cell priming, expansion and memory formation in response to microbial infection. [Copyright &y& Elsevier]

The development of immunity depends upon the capacity of responding T cells to become mobilized from lymphoid tissues where they are primed to sites of antigen exposure, wherever they occur in the body. Activation-induced alterations in the ability of T cells to migrate signify a fundamental change in biological function. Considerable attention is now focused on identifying mechanisms that regulate the migration and persistence of T cells that disseminate to non-lymphoid compartments as effector cells, and those that are retained in the lymphoid compartment. There are many unanswered questions about the developmental relationships and roles in protective immunity of antigen-experienced T cells that partition in different tissues. [Copyright &y& Elsevier]

Antigen-dependent T-cell activation is initiated by the organized interactions of a host of cell-surface receptors in the interface between the T cell and an antigen-presenting cell. Recent structural studies of many of the receptors that comprise this ‘immunological synapse’, including integrins, cadherins and co-stimulatory molecules, reveal in detail the molecular interactions that bridge the synapse. Additionally, structural analyses of the cytoplasmic tails of integrins and co-stimulatory receptors in complex with intracellular signaling proteins are providing insight into the mechanisms that couple receptor clustering on the cell surface with the initiation of cytoplasmic signaling. [Copyright &y& Elsevier]

The formerly distinct fields of lymphocyte signal transduction and cytoskeletal remodeling have recently become linked, as proteins involved in transducing signals downstream of lymphocyte antigen receptors have also been implicated in actin cytoskeleton remodeling, microtubule dynamics and regulation of cell polarity. These discoveries have fuelled interest in understanding both the role of the actin cytoskeleton as an integral component of lymphocyte activation and the interplay between lymphoid cell–cell contact sites (immunological synapse), retractile pole structures (uropod, distal pole complex), and Rho-family GTPases (Rac, Rho, Cdc42), their upstream activators (Dbl-family guanine nucleotide exchange factors) and their downstream effectors (WASp, Arp2/3, ADAP). To understand how these complex regulatory networks are wired, a new breed of computational biologists uses mathematical language to reproduce and simulate signaling circuits ‘in silico’. [Copyright &y& Elsevier]

One of the most widely used methods for the study of T-helper (Th) cell activity is the interleukin 2 (IL-2) assay. Typically, this assay is a two-step process involving (a) the activation of Th cells in vitro and (b) the testing of IL-2-dependent indicator cells for growth in the presence of Th cell supernatants. The assay has served to quantify and characterize Th responses to a variety of unique pathogens and immunization regimens. A one-step, single-cell assay is also available for the testing of mouse Th cell hybridomas. In this assay, cells fused with the BWZ.36 parent line are scored for positive responses based on their expression of a β-galactosidase gene linked to an IL-2 enhancer element. The experiments described in this report were designed to examine the conventional and single-cell assays in a side-by-side comparison. Results revealed a striking difference between the two assays. The single-cell assay proved to be extremely sensitive and identified Th responses that were altogether missed by the conventional test. Based on these results, we suggest that (i) the conventional and single-cell assays should not be used interchangeably, and (ii) negative results in the conventional IL-2 assay should be considered preliminary until the more sensitive, single-cell assay can be performed. [Copyright &y& Elsevier]

Mycosis fungoides (MF) and Sézary syndrome (SS) comprise approximately 53% of cutaneous lymphomas. Both MF and SS may clinically and histologically mimic benign skin conditions, posing a diagnostic challenge to the dermatologist. Precise clinicopathologic correlation is necessary to support a diagnosis, especially in the early stages of disease. In addition to the identification of histopathologic criteria, ancillary studies, including the identification of CD4+ T cells with aberrant immunophenotypes and T-cell receptor gene rearrangements within skin lesions and peripheral blood are used to support the diagnosis. Recent studies evaluating the pathogenesis of MF have found that the skin microenvironment, including immune cells, such as dendritic cells and reactive cytotoxic and regulatory T cells, plays a crucial supporting role in MF. The skin-homing ability of malignant T cells is the result of chemokines, cytokines, adhesion molecules, and defective apoptosis, and is believed to play a role in disease pathogenesis and progression. In addition, recent studies have also suggested that MF and SS arise from distinct memory T cell subsets and advanced/erythrodermic MF and SS may be distinguished by identification of certain molecules, including Programmed-Death-1. [Copyright &y& Elsevier]

Background: Asthma is a predominantly TH2 cell–dominated inflammatory disease characterized by airway inflammation and a major public health concern affecting millions of persons. The Tec family tyrosine kinase IL-2–inducible T-cell kinase (Itk) is primarily expressed in T cells and critical for the function and differentiation of TH cells. Itk−/− mice have a defective TH2 response and are not susceptible to allergic asthma. Objective: We sought to better understand the role of Itk signaling in TH differentiation programs and in the development and molecular pathology of allergic asthma. Methods: Using a murine model of allergic airway inflammation, we dissected the role of Itk in regulating TH cell differentiation through genetic ablation of critical genes, chromatin immunoprecipitation assays, and house dust mite–driven allergic airway inflammation. Results: Peripheral naive Itk−/− CD4+ T cells have substantially increased transcripts and expression of the prototypic TH1 genes Eomesodermin, IFN-γ, T-box transcription factor (T-bet), and IL-12Rβ1. Removal of IFN-γ on the Itk−/− background rescues expression of TH2-related genes in TH cells and allergic airway inflammation in Itk−/− mice. Furthermore, small hairpin RNA–mediated knockdown of Itk in human peripheral blood T cells results in increased expression of mRNA for IFN-γ and T-bet and reduction in expression of IL-4. Conclusion: Our results indicate that Itk signals suppress the expression of IFN-γ in naive CD4+ T cells, which in a positive feed-forward loop regulates the expression of TH1 factors, such as T-bet and Eomesodermin, and suppress development of TH2 cells and allergic airway inflammation. [Copyright &y& Elsevier]

Background: Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. Objective: We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. Methods: Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2–Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2–Conj–sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. Results: nDer p 2–Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce TH2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG2a levels in sera. The conjugate exhibited reduced ability to activate human FcεRI+ cells without inducing TH17 cells or autoantibodies. Conclusions: The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects. [Copyright &y& Elsevier]