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Immune recognition occurs at specialized cell‐cell junctions when immune cells and target cells physically touch. In this junction, groups of receptor‐ligand complexes assemble and experience molecular forces that are ultimately generated by the cellular cytoskeleton. These forces are in the range of piconewton (pN) but play crucial roles in immune cell activation and subsequent effector responses. In this minireview, we will review the development of DNA based molecular tension sensors and their applications in mapping and quantifying mechanical forces experienced by immunoreceptors including T‐cell receptor (TCR), Lymphocyte function‐associated antigen (LFA‐1), and the B‐cell receptor (BCR) among others. In addition, we will highlight the use of DNA as a mechanical gate to manipulate mechanotransduction and decipher how mechanical forces regulate antigen discrimination and receptor signaling. [ABSTRACT FROM AUTHOR]

Aim: The regulation of osteoclasts (OCs) by inhibitory immunoreceptors maintains bone homeostasis and is considered an important determinant of the extent of periodontal pathology. The aim of this study was to investigate the role of the inhibitory immunoreceptor CD300lf and its ligand ceramide in osteoclastogenesis in periodontitis. Materials and Methods: The expression of CD300lf was measured in vitro and in a ligature‐induced periodontitis model. The effect of CD300lf ablation on osteoclastogenesis was examined in ligature‐retained and ligature removal periodontitis models. The effect of ceramide, the ligand of CD300lf, was examined in osteoclastogenesis in vitro and in vivo by smearing 20 μg of ceramide dissolved in carboxymethylcellulose on teeth and gingiva every other day in an experimental periodontitis model and ligature removal model. Results: CD300lf expression was downregulated during osteoclastogenesis. Ablation of CD300lf in the ligature‐induced periodontitis model increased the number of OCs and exacerbated bone damage. Bone resorption caused by CD300lf ablation was reversible following ligature removal. CD300lf‐ceramide binding suppressed osteoclastogenesis in vitro and inhibited alveolar bone loss in a mouse periodontitis model. Conclusions: Our findings reveal that CD300lf‐ceramide binding plays a critical negative role in alveolar bone loss in periodontitis by inhibiting OCs differentiation. [ABSTRACT FROM AUTHOR]

Measuring immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), can rely on antibodies, reactive T cells and other factors, with T-cell-mediated responses appearing to have greater sensitivity and longevity. Because each T cell carries an essentially unique nucleic acid sequence for its T-cell receptor (TCR), we can interrogate sequence data derived from DNA or RNA to assess aspects of the immune response. This review deals with the utility of bulk, rather than single-cell, sequencing of TCR repertoires, considering the importance of study design, in terms of cohort selection, laboratory methods and analysis. The advances in understanding SARS-CoV-2 immunity that have resulted from bulk TCR repertoire sequencing are also be discussed. The complexity of sequencing data obtained by bulk repertoire sequencing makes analysis challenging, but simple descriptive analyses, clonal analysis, searches for specific sequences associated with immune responses to SARS-CoV-2, motif-based analyses, and machine learning approaches have all been applied. TCR repertoire sequencing has demonstrated early expansion followed by contraction of SARS-CoV-2-specific clonotypes, during active infection. Maintenance of TCR repertoire diversity, including the maintenance of diversity of anti-SARS-CoV-2 response, predicts a favourable outcome. TCR repertoire narrowing in severe COVID-19 is most likely a consequence of COVID-19-associated lymphopenia. It has been possible to follow clonotypic sequences longitudinally, which has been particularly valuable for clonotypes known to be associated with SARS-CoV-2 peptide/MHC tetramer binding or with SARS-CoV-2 peptide-induced cytokine responses. Closely related clonotypes to these previously identified sequences have been shown to respond with similar kinetics during infection. A possible superantigen-like effect of the SARS-CoV-2 spike protein has been identified, by means of observing V-segment skewing in patients with severe COVID-19, together with structural modelling. Such a superantigen-like activity, which is apparently absent from other coronaviruses, may be the basis of multisystem inflammatory syndrome and cytokine storms in COVID-19. Bulk TCR repertoire sequencing has proven to be a useful and cost-effective approach to understanding interactions between SARS-CoV-2 and the human host, with the potential to inform the design of therapeutics and vaccines, as well as to provide invaluable pathogenetic and epidemiological insights. [ABSTRACT FROM AUTHOR]

Osteoclasts require coordinated co-stimulation by several signaling pathways to initiate and regulate their cellular differentiation. Receptor activator for NF-κB ligand (RANKL or TNFSF11), a tumor necrosis factor (TNF) superfamily member, is the master cytokine required for osteoclastogenesis with essential co-stimulatory signals mediated by immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptors, DNAX-associated protein 12 kDa size (DAP12) and FcεRI gamma chain (FcRγ). The ITAM-signaling adaptors do not have an extracellular ligand-binding domain and, therefore, must pair with ligand-binding immunoreceptors to interact with their extracellular environment. DAP12 pairs with a number of different immunoreceptors including triggering receptor expressed on myeloid cells 2 (TREM2), myeloid DAP12-associated lectin (MDL-1), and sialic acid-binding immunoglobulin-type lectin 15 (Siglec-15); while FcRγ pairs with a different set of receptors including osteoclast-specific activating receptor (OSCAR), paired immunoglobulin receptor A (PIR-A), and Fc receptors. The ligands for many of these receptors in the bone microenvironment remain unknown. Here, we will review immunoreceptors known to pair with either DAP12 or FcRγ that have been shown to regulate osteoclastogenesis. Co-stimulation and the effects of ITAM-signaling have turned out to be complex, and now include paradoxical findings that ITAM-signaling adaptor-associated receptors can inhibit osteoclastogenesis and immunoreceptor tyrosine-based inhibitory motif (ITIM) receptors can promote osteoclastogenesis. Thus, co-stimulation of osteoclastogenesis continues to reveal additional complexities that are important in the regulatory mechanisms that seek to maintain bone homeostasis.

Advancement in our understanding of immune cell recognition and emerging cellular engineering technologies during the last decades made active manipulation of the T cell response possible. Synthetic immunology is providing us with an expanding set of composite receptor molecules capable to reprogram immune cell function in a predefined fashion. Since the first prototypes in the late 1980s, the design of chimeric antigen receptors (CARs; T-bodies, immunoreceptors), has followed a clear line of stepwise improvements from antigen-redirected targeting to designed "living factories" delivering transgenic products on demand. Building on basic research and creative clinical exploration, CAR T cell therapy has been achieving spectacular success in the treatment of hematologic malignancies, now beginning to improve the outcome of cancer patients. In this study, we briefly review the history of CARs and outline how the progress in the basic understanding of T cell recognition and of cell engineering technologies made novel therapies possible. [ABSTRACT FROM AUTHOR]

Although tumor-infiltrating regulatory T (Treg) cells play a pivotal role in tumor immunity, how Treg cell activation are regulated in tumor microenvironments remains unclear. Here, we found that mice deficient in the inhibitory immunoreceptor CD300a on their dendritic cells (DCs) have increased numbers of Treg cells in tumors and greater tumor growth compared with wild-type mice after transplantation of B16 melanoma. Pharmacological impairment of extracellular vesicle (EV) release decreased Treg cell numbers in CD300a-deficient mice. Coculture of DCs with tumor-derived EV (TEV) induced the internalization of CD300a and the incorporation of EVs into endosomes, in which CD300a inhibited TEV-mediated TLR3–TRIF signaling for activation of the IFN-β-Treg cells axis. We also show that higher expression of CD300A was associated with decreased tumor-infiltrating Treg cells and longer survival time in patients with melanoma. Our findings reveal the role of TEV and CD300a on DCs in Treg cell activation in the tumor microenvironment.

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many different disease contexts. A rapidly growing body of design work is examining every modular component of traditional single-chain CARs as well as expanding out into many new and innovative engineered immunoreceptor designs that depart from this template. New approaches to immune cell and receptor engineering are being reported with rapidly increasing frequency, and many recent high-quality reviews (including one in this special issue) provide comprehensive coverage of the history and current state of the art in CAR-T and related cellular immunotherapies. In this review, we step back to examine our current understanding of the structure-function relationships in natural and engineered lymphocyte-activating receptors, with an eye towards evaluating how well the current-generation CAR designs recapitulate the most desirable features of their natural counterparts. We identify key areas that we believe are under-studied and therefore represent opportunities to further improve our grasp of form and function in natural and engineered receptors and to rationally design better therapeutics.

Aggregation of cell surface receptor proteins by multivalent antigens is an essential early step for immune cell signalling. A number of experimental and modelling studies in the past have investigated multivalent ligand-mediated aggregation of IgE receptors (FcɛRI) in the plasma membrane of mast cells. However, understanding of the mechanisms of FcɛRI aggregation remains incomplete. Experimental reports indicate that FcɛRI forms relatively small and finite-sized clusters when stimulated by a multivalent ligand. By contrast, modelling studies have shown that receptor cross-linking by a trivalent ligand may lead to the formation of large receptor superaggregates that may potentially give rise to hyperactive cellular responses. In this work, we have developed a Brownian dynamics-based spatio-temporal model to analyse FcɛRI aggregation by a trivalent antigen. Unlike the existing models, which implemented non-spatial simulation approaches, our model explicitly accounts for the coarse-grained site-specific features of the multivalent species (molecules and complexes). The model incorporates membrane diffusion, steric collisions and sub-nanometre-scale site-specific interaction of the time-evolving species of arbitrary structures. Using the model, we investigated temporal evolution of the species and their diffusivities. Consistent with a recent experimental report, our model predicted sharp decay in species mobility in the plasma membrane in response receptor cross-linking by a multivalent antigen. We show that, due to such decay in the species mobility, post-stimulation receptor aggregation may become self-limiting. Our analysis reveals a potential regulatory mechanism suppressing hyperactivation of immune cells in response to multivalent antigens.

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds such as PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb crosslinking induced NET formation similarly to PMA stimulation. Direct crosslinking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of ERK and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb crosslinkng induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β, induced TAK1 phosphorylation. A MEK (ERK kinase) specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb crosslinking activates TAK1 and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

The signaling lymphocytic activation molecule (SLAM), present on the surface of hematopoietic cells, can regulate some events of the immune responses. This modulatory action is associated with the capacity of SLAM to interact with an intracytoplasmic adapter, such as SLAM-associated protein (SAP). SLAM is constitutively expressed in most of these cells, is rapidly induced after antigenic or inflammatory stimuli, and participates in the immunological synapse. Defects in the function of the SLAM-SAP pathway contribute to immunological abnormalities, resulting in autoimmune diseases, tumors of the lymphoid tissues and inadequate responses to infectious agents. Initially, the role of SLAM was investigated using an anti-SLAM monoclonal antibody (α-SLAM mAb) identified as an agonist of the SLAM-SAP pathway, which could induce the production of interferon-γ and could redirect the immune response to a T helper 1 (Th1) cell profile. However, in this review we postulate that the SLAM-SAP pathway primarily induces a Th2 response and secondarily suppresses the Th1 response.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen--antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis. [ABSTRACT FROM AUTHOR]

Phagocytosis is a remarkably complex and versatile process: it contributes to innate immunity through the ingestion and elimination of pathogens, while also being central to tissue homeostasis and remodeling by clearing effete cells. The ability of phagocytes to perform such diverse functions rests, in large part, on their vast repertoire of receptors. In this review, we address the various receptor types, their mobility in the plane of the membrane, and two modes of receptor crosstalk: priming and synergy. A major section is devoted to the actin cytoskeleton, which not only governs receptor mobility and clustering but also is instrumental in particle engulfment. Four stages of the actin remodeling process are identified and discussed: (i) the 'resting' stage that precedes receptor engagement, (ii) the disruption of the cortical actin prior to formation of the phagocytic cup, (iii) the actin polymerization that propels pseudopod extension, and (iv) the termination of polymerization and removal of preassembled actin that are required for focal delivery of endomembranes and phagosomal sealing. These topics are viewed in the larger context of the differentiation and polarization of the phagocytic cells. [ABSTRACT FROM AUTHOR]

Abstract: Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially impact on the quality of the T cell response. In the present study, we prepared antibodies specific for each of the six different phosphotyrosines of the mouse CD3ζ chain. The antibodies were characterized with respect to their cross-reactivity, ability to recognize the phosphorylated versus non-phosphorylated forms of tyrosine-containing motifs, and cross-reactivity with the homologous phospho-motifs on the human CD3ζ protein. The antibodies were found to be specific and selective for phospho-CD3ζ. They can serve as useful tools for distinguishing between the six potential tyrosine phosphorylation sites on the CD3ζ chain, and for correlating the phosphorylation of specific CD3ζ tyrosine residues with activation of signaling pathways that dictate T cell differentiation into responding, anergic, or apoptotic cells. [Copyright &y& Elsevier]

Spleen tyrosine kinase (SYK) is crucial to cellular functions mediated by immunoreceptors and integrins. We have developed and characterized a new genetically-encoded Förster resonance energy transfer (FRET)-based biosensor for studying the dynamics of SYK activities in living cells at a subcellular level. It contains an N-terminal ECFP, SH2 domain, a peptide derived from a SYK substrate VAV2, and a C-terminal YPet. Upon the specific phosphorylation by SYK in vitro, the biosensor substrate peptide bound to the intramolecular SH2 domain to reduce the FRET efficiency. Transfection of the biosensor did not affect activation of the endogenous SYK in host cells. Phosphorylation of the biosensor followed the same kinetics as the endogenous VAV2. Using FRET imaging and ratiometric analysis with this SYK biosensor, we visualized and quantified the real-time activation of SYK in K562 cells upon IgG Fc engagement of Fcγ receptor IIA and in mouse embryonic fibroblasts upon stimulation by the platelet derived growth factor. These results demonstrate our biosensor as a powerful tool for studying cellular signaling that involves SYK. [ABSTRACT FROM AUTHOR]

Abstract: An increasing number of data involve immunoreceptors in brain development, synaptic plasticity and behavior. However it has yet to be determined whether these proteins in fact transmit an immunoreceptor-like signal in non-hematopoietic neuronal cells. The recruitment and activation of the Syk family tyrosine kinases, Syk and ZAP-70, being a critical step in this process, we conducted a thorough analysis of Syk/ZAP-70 expression pattern in nervous tissues. Syk/ZAP-70 is present in neurons of different structures including the cerebellum, the hippocampus, the visual system and the olfactory system. During the olfactory system ontogeny the protein is detected from the 16th embryonic day and persists in adulthood. Importantly, Syk was phosphorylated on tyrosine residues representative of an active form of the kinase in specialized neuronal subpopulations comprising rostral migratory stream neuronal progenitor cells, hippocampal pyramidal cells, retinal ganglion cells and cerebellar granular cells. Phospho-Syk staining was also observed in synapse-rich regions such as the olfactory bulb glomeruli and the retina inner plexiform layer. Furthermore, our work on cultured primary hippoccampal neurons indicates that as for hematopoietic cells, Syk phosphorylation is readily induced upon pervanadate treatment. Therefore, Syk appears to be a serious candidate in connecting immunoreceptors to downstream adaptor/effector molecules in neurons. [Copyright &y& Elsevier]

Abstract: An interaction of multivalent antigen with its IgE bound to the high-affinity IgE receptor (FcεRI) on the surface of mast cells or basophils initiates a series of signaling events leading to degranulation and release of inflammatory mediators. Earlier studies showed that the first biochemically defined step in this signaling cascade is tyrosine phosphorylation of the FcεRI β subunit by Src family kinase Lyn. However, the processes affecting this step remained elusive. In this review we critically evaluate three current models (transphosphorylation, lipid raft, and our preferential protein tyrosine kinase–protein tyrosine phosphatase interplay model) substantiating three different mechanisms of FcεRI phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract: Immunoreceptor engagement leads to the activation of multiple second messenger cascades, and integration of these pathways requires proper function of a number of adapter proteins. Although adapters possess no intrinsic enzymatic function, they nucleate the formation of multi-molecular protein complexes to support downstream signaling. Since adapters contain functionally distinct domains, intense investigation has been devoted to understanding how these regions act to integrate signals. This review describes the evolution of studies investigating one of these adapters, the SH2 domain-containing leukocyte protein of 76kDa. Through utilizing biochemical, genetic and imaging techniques, a model has emerged describing how this adapter regulates signals resulting in complex immune responses. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptors (CARs, immunoreceptors) are frequently used to redirect T cells with pre-defined specificity, in particular towards tumour cells for use in adoptive immunotherapy of malignant diseases. Specific targeting is mediated by an extracellularly located antibody-derived binding domain, which is joined to the transmembrane and intracellular CD3ζ moiety for T-cell activation. Stable CAR expression in T cells, however, requires a spacer domain interposed between the binding and the transmembrane domain and which is commonly the constant IgG1 Fc domain. We here revealed that CARs with Fc spacer domain bind to IgG Fc gamma receptors (FcγRs), thereby unintentionally activating innate immune cells, including monocytes and natural killer (NK) cells, which consequently secrete high amounts of pro-inflammatory cytokines. Engineered T cells, on the other hand, are likewise activated by FcγR binding resulting in cytokine secretion and lysis of monocytes and NK cells independently of the redirected specificity. To reduce FcγR binding, we modified the spacer domain without affecting CAR expression and antigen binding. Engineered with the modified CAR, T cells are not activated in presence of FcγR+ cells, thereby minimizing the risk of off-target activation while preserving their redirected targeting specificity. [ABSTRACT FROM AUTHOR]

The protein tyrosine kinase ZAP70 became the subject of intense scrutiny in the early nineties, when ZAP70 mutations were characterized in several young patients presenting with severe T cell immunodeficiencies. The association of a lack of expression of ZAP70 with an immunodeficiency consisting in a markedly reduced T lymphocyte-mediated immunity highlighted the crucial role of this tyrosine kinase in T cell development and function. This discovery was soon accompanied by the characterization of the substrates of ZAP70 and the signalling cascades that depend on ZAP70 activity. These studies demonstrated that ZAP70 was indeed at the crossroad of several signalling pathways that control T lymphocyte development and function. Recently, a revival of interest for this protein came again from studies associating abnormal ZAP70 expression with pathological conditions. Some chronic lymphocytic leukemia B cells were shown to express ZAP70, and this expression was correlated with bad prognosis. Mouse models also revealed that partial defects in ZAP70 activity can be associated with autoimmunity. These last results suggested that ZAP70 is involved in the fine balance between immunity and tolerance. In this review, we will discuss the role of ZAP70 in T cell activation and focus on what we learnt from pathological conditions associated with defective expression or activity of the ZAP70 kinase. [ABSTRACT FROM AUTHOR]

Osteoclasts mediate bone resorption, which is critical for bone development, maintenance, and repair. Proper control of osteoclast development and function is important and deregulation of these processes may lead to bone disease, such as osteoporosis. Previous studies have shown that the cytosolic protein tyrosine phosphatase SHP-1 acts as a suppressor of osteoclast differentiation and function, but putative inhibitory receptors that mediate recruitment and activation of SHP-1 in osteoclasts have remained unknown. In the present study, we identify the SHP-l-recruiting inhibitory immunoreceptor signal regulatory protein (SIRP) α as a negative regulator of osteoclast activity. SIRPα is expressed by osteoclasts, and osteoclasts from mice lacking the SIRPα cytoplasmic tail and signaling capacity display enhanced bone resorption in vitro. Consequently, SIRPα-mutant mice have a significantly reduced cortical bone mass. Furthermore, osteoclasts from SIRPα-mutant mice show an enhanced formation of actin rings, known to be instrumental in bone resorption. SIRPα mutation did not significantly affect osteoclast formation, implying that the role of SIRPα was limited to the regulation of mature osteoclast function. This identifies SIRPα as a bona fide inhibitory receptor that regulates the bone-resorption activity and supports a concept in which osteoclast function is balanced by the signaling activities of activating and inhibitory immunoreceptors. [ABSTRACT FROM AUTHOR]

Human T lymphocytes can be redirected with a new defined specificity by expression of a chimeric T-cell receptor (immunoreceptor) for the use in adoptive immunotherapy of cancer. Whereas standard procedures use retroviral gene transduction to constitutively express immunoreceptors in T cells, we here explored for the first time mRNA electroporation to achieve transient immunoreceptor expression, and thereby minimizing the risk of persistence of potential autoaggression. CD4+ and CD8+ T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Immunoreceptor-transfected T cells were specifically activated upon coincubation with ErbB2+ and CEA+ tumor cells, respectively, resulting in secretion of interferon-γ (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNFα). Furthermore, immunoreceptor-transfected CD8+ T cells specifically lysed ErbB2+ and CEA+ tumor cells, respectively. The RNA-transfected T cells retained their cytotoxic function after 2 days of activation and exhibited cytolytic activities like retrovirally transduced T cells. RNA electroporation of T cells thereby provides a versatile tool for transient immunoreceptor expression, which may be of advantage in avoiding the persistence of unintended autoaggression.Gene Therapy (2009) 16, 596–604; doi:10.1038/gt.2008.189; published online 22 January 2009 [ABSTRACT FROM AUTHOR]

This year marks the 25th anniversary of the first Annual Review of Immunology article to describe features of the T cell antigen receptor (TCR). In celebration of this anniversary, we begin with a brief introduction outlining the chronology of the earliest studies that established the basic paradigm for how the engaged TCR transduces its signals. This review continues with a description of the current state of our understanding of TCR signaling, as well as a summary of recent findings examining other key aspects of T cell activation, including cross talk between the TCR and integrins, the role of costimulatory molecules, and how signals may negatively regulate T cell function. [ABSTRACT FROM AUTHOR]

Abstract: In specific cell types infected with the alphaherpesviruses herpes simplex virus and pseudorabies virus (PRV), addition of virus-specific antibodies results in redistribution of cell-surface-anchored viral proteins. This redistribution is triggered by the viral protein gE and consists of the directional movement of the antibody–antigen complexes to one pole of the cell. This viral capping process has been associated with increased antibody-resistant virus spread and strongly resembles immunoreceptor capping, a process that is crucial in activation of different immune cells (e.g. capping of Fcγ-receptors, B and T cell receptors). Here, we report that the PRV gE-mediated viral capping process results in increased Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of gE and that a fraction of gE associates with lipid rafts, all very reminiscent of immunoreceptor capping. These results provide evidence that gE-mediated capping is a viral mimicry of immunoreceptor capping. [Copyright &y& Elsevier]

Redirecting T cell effector functions towards pre-defined target cells represents an attractive concept in the adoptive immunotherapy of malignant diseases. Our understanding of the mechanisms of T cell activation and costimulation as well as the design of recombinant T cell receptors have made major progress in the last years. Translating recent concepts of T cell stimulation into recombinant protein design provides the basis to engineer T cells with both pre-defined specificity and costimulatory capacity in order to enhance anti-tumor immunity and to break tolerance. Dual signaling immunoreceptors providing the CD3ζ signal simultaneously with an appropriate costimulatory signal moreover allows to modulate the quality of the anti-tumor T cell response in a predicted fashion. [ABSTRACT FROM AUTHOR]

Abstract: Phosphoinositide 3-kinases (PI3Ks) generate several distinct lipid second messengers including phosphatidylinositol (3,4,5) trisphosphate (PIP3) and phosphatidylinositol (3,4) bisphosphate PI(3,4)P2. PI(3,4)P2 is produced with distinct kinetics and binds to distinct PH domain effector proteins; however, the regulation of this signaling pathway is poorly understood. Superoxides such as hydrogen peroxide are transiently produced after activation through various cell surface receptors and play important roles in immune and inflammatory responses. Here we use quantitative microscopy to examine the effect of peroxide on PI(3,4)P2-mediated mobilization of signaling proteins in B lymphocytes. Peroxide was found to induce dose-dependant membrane recruitment of the PI(3,4)P2-binding PH domain proteins Bam32, TAPP2 and Akt/PKB but not the PIP3-binding PH domain of Btk. Peroxide-induced membrane recruitment was found to be dependant on PI3K activity, with the p110δ isoform contributing much of the activity in the BJAB human B lymphoma model. Strikingly, peroxide co-stimulation enhanced antigen receptor-induced membrane recruitment of Bam32 and TAPP2, with combined stimulation exceeding the maximum achievable with either stimulus alone. Expression of the lipid phosphatase PTEN led to reduction of antigen receptor-induced membrane recruitment of TAPP2; however, peroxide costimulation could overcome the inhibitory effect of PTEN. Inhibition of the NADPH oxidase led to reduction of antigen receptor-induced membrane recruitment of TAPP2. Our results indicate that exogenous and endogenous superoxides can modulate the quality of the PI3K signal in lymphocytes by selectively increasing PI(3,4)P2-dependant signaling. [Copyright &y& Elsevier]

The signaling lymphocyte-activating molecule (SLAM) family immunoreceptors are expressed in a wide array of immune cells, including both T and B lymphocytes. By virtue of their ability to transduce tyrosine phosphorylation signals through the so-called ITSM (immunoreceptor tyrosine-based switch motif) sequences, they play an important part in regulating both innate and adaptive immune responses. The critical role of the SLAM immunoreceptors in mediating normal immune reactions was highlighted in recent findings that SAP, a SLAM-associated protein, modulates the activities of various immune cells through interactions with different members of the SLAM family expressed in these cells. Importantly, mutations or deletions of the sap gene in humans result in the X-linked lymphoproliferative syndrome. In this review, we summarize current knowledge and survey the latest developments in signal transduction events triggered by the activation of SLAM family receptors in different cell types. [ABSTRACT FROM AUTHOR]

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called‘natural’ killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb. [ABSTRACT FROM AUTHOR]

Protein tyrosine and lipid phosphorylations are early and critical events in type 1 Fcε receptor (FcεRI)-mediated activation of mast cells and basophils. Tyrosine phosphorylation of FcεRI subunits as well as other signal transduction molecules reflects the balance between the action of protein tyrosine kinases and phosphatases. Similarly, the phosphate content of inositol phospholipids, involved in the recruitment of signalling molecules to the plasma membrane and the generation of secondary messengers, is the net result of the opposing effects of phosphoinositide kinases and lipid phosphatases. This review summarizes the current understanding of the structural and functional aspects of nonreceptor protein tyrosine phosphatases (SHP-1, SHP-2, HePTP, PTP20, PRL1, PRL2, PTP-MEG1 and PTP-MEG2) and lipid phosphatases (SHIP and SHIP2) in the activation of mast cells and basophils after FcεRI aggregation. New approaches towards a deeper understanding of the role of phosphatases in mast cell physiology are also discussed.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Although tumor-infiltrating regulatory T (Treg) cells play a pivotal role in tumor immunity, how Treg cell activation are regulated in tumor microenvironments remains unclear. Here, we found that mice deficient in the inhibitory immunoreceptor CD300a on their dendritic cells (DCs) have increased numbers of Treg cells in tumors and greater tumor growth compared with wild-type mice after transplantation of B16 melanoma. Pharmacological impairment of extracellular vesicle (EV) release decreased Treg cell numbers in CD300a-deficient mice. Coculture of DCs with tumor-derived EV (TEV) induced the internalization of CD300a and the incorporation of EVs into endosomes, in which CD300a inhibited TEV-mediated TLR3-TRIF signaling for activation of the IFN-β-Treg cells axis. We also show that higher expression of CD300A was associated with decreased tumor-infiltrating Treg cells and longer survival time in patients with melanoma. Our findings reveal the role of TEV and CD300a on DCs in Treg cell activation in the tumor microenvironment., Competing Interests: YN, NN, HK, KK, CN, AS No competing interests declared, (© 2021, Nakazawa et al.)

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many different disease contexts. A rapidly growing body of design work is examining every modular component of traditional single-chain CARs as well as expanding out into many new and innovative engineered immunoreceptor designs that depart from this template. New approaches to immune cell and receptor engineering are being reported with rapidly increasing frequency, and many recent high-quality reviews (including one in this special issue) provide comprehensive coverage of the history and current state of the art in CAR-T and related cellular immunotherapies. In this review, we step back to examine our current understanding of the structure-function relationships in natural and engineered lymphocyte-activating receptors, with an eye towards evaluating how well the current-generation CAR designs recapitulate the most desirable features of their natural counterparts. We identify key areas that we believe are under-studied and therefore represent opportunities to further improve our grasp of form and function in natural and engineered receptors and to rationally design better therapeutics. [ABSTRACT FROM AUTHOR]

Aggregation of cell surface receptor proteins by multivalent antigens is an essential early step for immune cell signalling. A number of experimental and modelling studies in the past have investigated multivalent ligand-mediated aggregation of IgE receptors (Fc ɛ RI) in the plasma membrane of mast cells. However, understanding of the mechanisms of Fc ɛ RI aggregation remains incomplete. Experimental reports indicate that Fc ɛ RI forms relatively small and finite-sized clusters when stimulated by a multivalent ligand. By contrast, modelling studies have shown that receptor cross-linking by a trivalent ligand may lead to the formation of large receptor superaggregates that may potentially give rise to hyperactive cellular responses. In this work, we have developed a Brownian dynamics-based spatio-temporal model to analyse Fc ɛ RI aggregation by a trivalent antigen. Unlike the existing models, which implemented non-spatial simulation approaches, our model explicitly accounts for the coarse-grained site-specific features of the multivalent species (molecules and complexes). The model incorporates membrane diffusion, steric collisions and sub-nanometre-scale site-specific interaction of the time-evolving species of arbitrary structures. Using the model, we investigated temporal evolution of the species and their diffusivities. Consistent with a recent experimental report, our model predicted sharp decay in species mobility in the plasma membrane in response receptor cross-linking by a multivalent antigen. We show that, due to such decay in the species mobility, post-stimulation receptor aggregation may become self-limiting. Our analysis reveals a potential regulatory mechanism suppressing hyperactivation of immune cells in response to multivalent antigens., Competing Interests: We declare we have no competing interests.