Search

Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) can be modulated by annular lipids sheathing their hydrophobic moiety. Here, we present evidence for this prediction in a natural membrane by investigating the mechanism that maintains steady amount of active isoform of Lck kinase (LckA) by Lck trans-autophosphorylation offset by the phosphatase CD45. We gauged experimental suitability by quantitation of CD45 and LckAsubcellular localisation, LckAgeneration as a function of Lck and pharmacological perturbation. Steady LckAwas challenged by swapping Lck membrane anchor with structurally divergent ones expected to substantially modify Lck annular lipids, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45, respectively. The data showed only small alteration of LckA, except for CD45 hydrophobic anchor that thwarted LckA, due to excessive lateral proximity to CD45. The data are best explained by annular lipids facilitating or penalising IMPs’ lateral proximity, hence modulating IMPs protein-protein functional interactions. Our findings can contribute to improve the understanding of biomembranes’ organisation.

Bottom-up proteomics analyses have been proved over the last years to be a powerful tool in the characterization of the proteome and are crucial for understanding cellular and organism behaviour. Through differential proteomic analysis researchers can shed light on groups of proteins or individual proteins that play key roles in certain, normal or pathological conditions. However, several tools for the analysis of such complex datasets are powerful, but hard-to-use with steep learning curves. In addition, some other tools are easy to use, but are weak in terms of analytical power. Previously, we have introduced ProteoSign, a powerful, yet user-friendly open-source online platform for protein differential expression/abundance analysis designed with the end-proteomics user in mind. Part of Proteosign's power stems from the utilization of the well-established Linear Models For Microarray Data (LIMMA) methodology. Here, we present a substantial upgrade of this computational resource, called ProteoSign v2, where we introduce major improvements, also based on user feedback. The new version offers more plot options, supports additional experimental designs, analyzes updated input datasets and performs a gene enrichment analysis of the differentially expressed proteins. We also introduce the deployment of the Docker technology and significantly increase the speed of a full analysis. ProteoSign v2 is available at http://bioinformatics.med.uoc.gr/ProteoSign., Graphical Abstract Graphical AbstractProteoSign v2: a faster and evolved user-friendly online tool for statistical analyses of differential proteomics.

International audience; Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction whilst investigating the mechanism for maintaining a steady amount of the active form of IMP Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behaviour fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.

Summary The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation., Graphical abstract, Highlights • Mutations in TCRβ and CD3ζ TMRs that reduce their interaction augment signaling • pMHC and anti-CD3 binding to TCR-CD3 induce similar quaternary structure relaxation • Soluble monovalent pMHC alone signals and reduces TCRαβ cohesion with CD3ζ • Allosteric changes in TCR-CD3 dynamics instigate T cell activation, T cell antigen receptor is central in adaptive immunity; however, the mechanism that couples ligand binding and intracellular signaling is still controversial. Here, Lanz et al. have taken an interdisciplinary approach demonstrating that binding of soluble monovalent pMHC to TCR-CD3 allosterically reduces TCRαβ cohesion with CD3ζ and initiates signal transduction.

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn−/− cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations – one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism., Egress of flaviviruses and involved host pathways are not well understood. Here, the authors show that Lyn is a critical host kinase for Dengue and Zika virus egress resulting in infectious virus progenies within autophagosome-derived vesicles, which might help the virus to evade antibody responses.

The machinery involved in cytotoxic T-cell activation requires three main characters such as: the major histocompatibility complex class I (MHC I) bound to the peptide (p), the T-cell receptor (TCR), and the CD3-complex which is a multidimer interfaced with the intracellular side. The pMHC:TCR interaction has been largely studied both in experimental and computational models, giving a contribution in understanding the complexity of the TCR triggering process. Nevertheless, a detailed study of the structural and dynamical characterization of the full complex (pMHC:TCR:CD3-complex) is still missing, due to insufficient data available on the CD3-chains arrangement around the TCR. The recent determination of the TCR:CD3-complex structure by means of Cryo-EM technique has given a chance to build the entire proteins system essential in the activation of T-cell, and thus in the adaptive immune response. Here, we present the first full model of the pMHC interacting with the TCR:CD3-complex, built in a lipid environment. To describe the conformational behaviour associated with the unbound and the bound states, all atoms Molecular Dynamics simulations were performed for the TCR:CD3-complex and for two pMHC:TCR:CD3-complex systems, bound to two different peptides. Our data point out that a conformational change affecting the TCR Constant β (Cβ) region occurs after the binding to the pMHC, revealing a key role of such a region in the propagation of the signal. Moreover, we found that the TCR reduces the flexibility of the MHC I binding groove, confirming our previous results.

Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.

The major defense system against microbial pathogens in vertebrates is the adaptive immune response and represents an effective mechanism in cancer surveillance. T cells represent an essential component of this complex system. They can recognize myriads of antigens as short peptides (p) originated from the intracellular degradation of foreign proteins presented by major histocompatibility complex (MHC) proteins. The clonotypic T-cell antigen receptor (TCR) is specialized in recognizing pMHC and triggering T cells immune response. It is still unclear how TCR engagement to pMHC is translated into the intracellular signal that initiates T-cell immune response. Some work has suggested the possibility that pMHC binding induces in the TCR conformational changes transmitted to its companion CD3 subunits that govern signaling. The conformational changes would promote phosphorylation of the CD3 complex &zeta, chain that initiates signal propagation intracellularly. Here, we used all-atom molecular dynamics simulations (MDs) of 500 ns to analyze the conformational behavior of three TCRs (1G4, ILA1 and ILA1&alpha, 1&beta, 1) interacting with the same MHC class I (HLA-A*02:01) bound to different peptides, and modelled in the presence of a lipid bilayer. Our data suggest a correlation between the conformations explored by the &beta, chain constant regions and the T-cell response experimentally determined. In particular, independently by the TCR type involved in the interaction, the TCR activation seems to be linked to a specific zone of the conformational space explored by the &beta, chain constant region. Moreover, TCR ligation restricts the conformational space the MHC class I groove.

SummaryAmong the various host cellular processes that are hijacked by flaviviruses, very few mechanisms have been described with regard to viral secretion. Here we investigated how flaviviruses exploit the Src family kinases (SFKs) for exit from infected cells. We isolated three members of the SFK family – Src, Fyn and Lyn – that were specifically activated during secretion of Dengue and Zika or their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs blocked virus secretion, most significantly upon Lyn-deficiency. Lyn-/-cells were severely impaired in virus release, and were rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We further established that Lyn, via its palmitoylation-dependent membrane association, triggered post-Golgi virus transport in specialised Rab11 and Transferrin receptor positive organelles resembling secretory autophagosomes, and distinct from conventional exocytic vesicles. In the absence of Lyn activity or its aberrant membrane association, virions were sorted into the lysosomal pathway for degradation. This mode of export was specifically triggered by processed, and mature, but not by furin-resistant virus particles, and occurred with significantly faster kinetics than the conventional secretory pathway. Our study therefore charts a previously undiscovered Lyn-dependent exit strategy, triggered by flaviviruses in secretory autophagosomes that might enable them to evade circulating antibodies and dictate tissue tropism.

SummaryThe in-depth analysis of costimulatory signaling enhancing the activity of cytotoxic T cells (CTLs) represents a major approach towards immunotherapy development. Here we report that CD2 costimulation plays a critical role in killing by freshly isolated human CTLs, which represent a challenging but valuable study model to gain insight into CTL biology. We show that CD2 triggering critically aids signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules towards the microtubule-organizing center (MTOC). To gain insight into the underlying elusive mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeleton organization, autophagy and metabolism. Strikingly, signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) represents a functionally critical node of the CD2 network which regulates granule polarization towards the MTOC in CTLs. Granule trafficking is driven by active AMPK enriched on adjacent lysosomes, illustrating a novel signaling cross-talk between vesicular compartments in CTLs. Our results thus establish CD2 signaling as key for regulating cytotoxic killing and granule polarization in freshly isolated CTLs and strengthens the rationale to choose CD2 and AMPK as therapeutic targets to boost CTL activity.

Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR α and β chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and β chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols.

Multiple autoimmune pathologies are associated with single nucleotide polymorphisms of the human gene TAGAP, which encodes TAGAP, a guanosine triphosphatase (GTPase)-activating protein (GAP). Here, we showed in mice that Tagap-mediated signaling by the sema3E–plexin-D1 ligand-receptor complex attenuates thymocyte adhesion to the cortex through their β(1)-containing integrins. By promoting thymocyte detachment within the cortex of the thymus, Tagap-mediated signaling enabled their translocation to the medulla, which is required for continued thymic selection. Tagap physically interacted with the cytoplasmic domain of plexin-D1 and directly stimulated the activity and signaling of the GTPase RhoA. In addition, Tagap indirectly mediated the activation of Cdc42 in response to the binding of sema3E to plexin-D1. Both RhoA and Cdc42 are key mediators of cytoskeletal and integrin dynamics in thymocytes. Knockdown of Tagap in mice suppressed the sema3E- and plexin-D1–mediated release of thymocytes that adhered within the cortex through β(1)-containing integrins. This suppression led to the impaired translocation of thymocytes from the cortex to the medulla and resulted in the formation of ectopic medullary structures within the thymic cortex. Our results suggest that TAGAP variation modulates the risk of autoimmunity by altering thymocyte migration during thymic selection.

Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono-methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA-binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg-Gly-Gly motifs or Asn-rich contexts.

The organization of the T-cell's plasma membrane continues to nourish the curiosity of immunologists, cell biologists and biophysicists. The main reason is the biological and biomedical interest to understand the workings of the cell–cell communication network activated by T-cells during an immune response. The molecular armamentarium of the T-cell plasma membrane helps to identify with high sensitivity, specificity and rapidity antigens from invading microbial pathogens and prepare adequate countermeasures to fend them off, while protecting from attacks against our normal tissues. Many T-cell membrane proteins act as receptors to carry out and finely tune these complex tasks. However, the TCR (T-cell receptor) holds a decisive hegemony for its crucial contribution in steering T-cell function and fate. An emerging notion is that TCR proximal signalling occurs at submicrometre-scale membrane domains. In the present chapter, we discuss the current knowledge on the TCR structure and the associated signal transduction machinery and how the notion of membrane nanodomains has decisively contributed to further understand the molecular basis of T-cell activation.

Sustained signalling at the immune synapse (IS) requires the synaptic delivery of recycling endosome-associated T cell antigen receptors (TCRs). IFT20, a component of the intraflagellar transport system, controls TCR recycling to the IS as a complex with IFT57 and IFT88. Here, we used quantitative mass spectrometry to identify additional interaction partners of IFT20 in Jurkat T cells. In addition to IFT57 and IFT88, the analysis revealed new binding partners, including IFT54 (also known as TRAF3IP1), GMAP-210 (also known as TRIP11), Arp2/3 complex subunit-3 (ARPC3), COP9 signalosome subunit-1 (CSN1, also known as GPS1) and ERGIC-53 (also known as LMAN1). A direct interaction between IFT20 and both IFT54 and GMAP-210 was confirmed in pulldown assays. Confocal imaging of antigen-specific conjugates using T cells depleted of these proteins by RNA interference showed that TCR accumulation and phosphotyrosine signalling at the IS were impaired in the absence of IFT54, ARPC3 or ERGIC-53. Similar to in IFT20-deficient T cells, this defect resulted from a reduced ability of endosomal TCRs to polarize to the IS despite a correct translocation of the centrosome towards the antigen-presenting cell contact. Our data underscore the traffic-related role of an IFT20 complex that includes components of the intracellular trafficking machinery in IS assembly.

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αβ-expressing 'single-positive' thymocytes from CD4(+)CD8αβ(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli. © 2013 Nature America, Inc. All rights reserved.

T-cell activation requires the translation of antigen binding to the T-cell receptor (TCR) into intracellular signaling. However, how antigen recognition and signal transduction are mechanistically linked is poorly understood. Here, we used single-molecule localization microscopy to link TCR clustering to signaling. We found that the likelihood of a single receptor to initiate signaling upon ligand binding depended on receptor-to-receptor spacing, with TCRs in dense clusters having the highest signaling efficiency. This means that antigen recognition must first be translated into a spatial reorganization of receptors into dense, signaling-competent clusters before signaling can begin. Thus, the quality of an antigen in terms of signaling is given by its ability to densely cluster receptors.

Thymocyte selection involves the positive and negative selection of the repertoire of T cell receptors (TCRs) such that the organism does not suffer autoimmunity, yet has the benefit of the ability to recognize any invading pathogen. The signal transduced through the TCR is translated into a number of different signaling cascades that result in transcription factor activity in the nucleus and changes to the cytoskeleton and motility. Negative selection involves inducing apoptosis in thymocytes that express strongly self-reactive TCRs, whereas positive selection must induce survival and differentiation programs in cells that are more weakly self-reactive. The TCR recognition event is analog by nature, but the outcome of signaling is not. A large number of molecules regulate the strength of the TCR-derived signal at various points in the cascades. This review discusses the various factors that can regulate the strength of the TCR signal during thymocyte development.

Thymocyte development and maintenance of peripheral T-cell numbers and functions are critically dependent on T-cell receptor (TCR) signal strength. SHP1 (Src homology region 2 domain-containing phosphatase-1), a tyrosine phosphatase, acts as a negative regulator of TCR signal strength. Moreover, germline SHP1 knockout mice have shown impaired thymic development. However, this has been recently questioned by an analysis of SHP1 conditional knockout mice, which reported normal thymic development of SHP1 deficient thymocytes. Using this SHP1 conditional knockout mice, in this issue of the European Journal of Immunology, Martinez et al. [Eur. J. Immunol. 2016. 46: 2103-2110] show that SHP1 indeed does have a role in the negative regulation of TCR signal strength in positively selected thymocytes, and in the final maturation of single positive thymocytes. They report that thymocyte development in such mice shows loss of mature, post-selection cells. This is due to increased TCR signal transduction in thymocytes immediately post positive-selection, and increased cell death in response to weak TCR ligands. Thus, SHP1-deficiency shows strong similarities to deficiency in the T-cell specific SHP1-associated protein Themis.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Ligation of the T cell antigen receptor (TCR) stimulates protein tyrosine kinases (PTKs), which regulate intracellular calcium and control the activity of protein kinase C (PKC) isozymes. PTKs activated by antigen receptors and costimulatory molecules also couple to phosphatidylinositol-3 kinase (PI3K) and control the activity of Ras- and Rho-family GTPases. T cell signal transduction is triggered physiologically by antigen in the context of antigen presenting cells (APC). The formation of stable and prolonged contacts between T cells and APCs is not neccessary to initiate T cell signaling but is required for effective T cell proliferation and differentiation. The stabilization of the T cell/ APC conjugate is regulated by intracellular signals induced by antigen receptors and costimulators. These coordinate the regulation of the actin and microtubule cytoskeleton and organize a specialized signaling zone that allows sustained TCR signaling.

Recent advances in peptide-based (bottom-up) quantitative proteomics and bioinformatics have opened unprecedented opportunities for extensive investigation of cellular proteomes and their dynamics. Here we discuss two approaches currently used to investigate the global dynamics of phosphorylation based on the isolation of phosphorylated proteins or peptides. We evaluate the accuracy of these methodologies to grasp the global dynamics of phosphorylation, and we raise awareness on ambiguities inherent to these analyses. We conclude that further development of targeted approaches should prevent inaccurate conclusions about the nature of biological regulations and in particular kinase-substrate networks.

Normalized spectral index quantification was recently presented as an accurate method of label-free quantitation, which improved spectral counting by incorporating the intensities of peptide MS/MS fragment ions into the calculation of protein abundance. We present SINQ, a tool implementing this method within the framework of existing analysis software, our freely available central proteomics facilities pipeline (CPFP). We demonstrate, using data sets of protein standards acquired on a variety of mass spectrometers, that SINQ can rapidly provide useful estimates of the absolute quantity of proteins present in a medium-complexity sample. In addition, relative quantitation of standard proteins spiked into a complex lysate background and run without pre-fractionation produces accurate results at amounts above 1 fmol on column. We compare quantitation performance to various precursor intensity- and identification-based methods, including the normalized spectral abundance factor (NSAF), exponentially modified protein abundance index (emPAI), MaxQuant, and Progenesis LC-MS. We anticipate that the SINQ tool will be a useful asset for core facilities and individual laboratories that wish to produce quantitative MS data, but lack the necessary manpower to routinely support more complicated software workflows. SINQ is freely available to obtain and use as part of the central proteomics facilities pipeline, which is released under an open-source license.

A 1000-fold purification of human T-cell growth factor (TCGF) was achieved starting from supernatants of human spleen cells stimulated with phytohaemagglutinin (PHA) in culture medium containing 0.5% serum. The purification scheme involved precipitation with ammonium sulphate, gel filtration and blue-Sepharose chromatography. The use of polyethylene glycol 6000 (PEG 6000) was critical during the chromatographic steps in order to obtain high final recoveries or activity (40-50%). Purified preparations of TCGF labelled with 125I by the chloramine T method revealed that the activity co-migrated with 2 molecular species of 14,000-17,000 daltons in SDS-PAGE under non-reducing conditions.

Renal cell carcinoma (RCC) infiltrating lymphocytes (TILs) express killer cell immunoglobulinlike receptors (KIRs) that inhibit the antitumor CD8+ T-cell lysis. In the present study, to better examine the functional consequences of KIR engagement on cytotoxic T lymphocyte (CTL)/tumor interaction, we have investigated the influence of KIR CD158a on early steps of T-cell activation. We show that coengagement of T-cell receptor (TCR) and CD158a by tumor cells inhibited tyrosine phosphorylation of early signaling proteins ZAP-70 and LAT, lipid raft coalescence, and TCR/CD3 accumulation at the CTL/tumor cell interface. In addition, the guanine exchange factor Vav was not phosphorylated, and no actin cytoskeleton rearrangement was observed. Our data indicate a role of KIR CD158a in the dynamic events induced by TCR triggering, preventing CTL membrane reorganization, and subsequent completion of CTL activation program. Accordingly, the expression of CD158 by TILs may favor tumor cell escape to the immune response.

The T-cell antigen receptor (TcR) complex present on most T-cells is formed by a clone-specific disulfide-linked alpha/beta heterodimer noncovalently associated to the CD3 complex, the latter composed of five invariant polypeptides: gamma, delta, epsilon, zeta/zeta, or zeta/eta. The presence of conserved, oppositely charged, amino acids in the predicted transmembrane domains of all the subunits of the TcR.CD3 complex suggests that these residues may have a critical function in the assembly and/or stabilization of the complex. In order to analyze the role of the transmembrane-charged amino acids in the association and cell surface expression of the TcR.CD3 complex, we have carried out site-directed mutagenesis of Lys271 in the transmembrane domain of the TcR beta chain and analyzed the capacity of the altered chain to assemble in a TcR beta-negative T-cell line. Here we show that substitution of this positively charged residue by alanine or glutamine does not prevent cytoplasmic association of alpha and beta chains to form disulfide-linked heterodimers, but does abolish formation of an alpha/beta.CD3 complex and, consequently, its expression on the cell surface.

Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes. mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission. To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi). Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression. In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified. In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited. This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.

CD4 and CD8 are thought to function as coreceptors by binding to the cognate major histocompatibility complex (MHC) molecules recognized by the T cell antigen receptor (TCR) and initiating the signal transduction cascade. We report that during T cell–antigen-presenting cell interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics. This coordinated disappearance takes place whenever the TCR is triggered, even when the coreceptor does not engage the cognate MHC molecule and is the consequence of binding of the coreceptor-associated Lck to ZAP-70. The interaction of coreceptor and cognate MHC molecules is dispensable when T cells are stimulated by optimal ligands, but becomes crucial when suboptimal ligands are used. In the latter case the coreceptor increases the efficiency of TCR triggering without changing the activation threshold or the quality of the T cell response.

The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain through its two src-homology 2 (SH2) domains results in its activation coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substrate phosphorylation assay. Under these conditions, we observed a lag phase of several minutes before full ZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggesting that it might be due to an intramolecular interaction involving the interdomain A and SH2 region. Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. The same results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findings are consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s) other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR, can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.

Approaches that allow global analysis of cell transcriptomes are greatly accelerating the dissection of lymphocyte signaling. Using serial analysis of gene expression, a recent study has provided evidence that most plasma membrane proteins of T cells have now been identified. Is this also true for the proteins governing intracellular signaling circuitries? This article comments on the recent study and describes other high-throughput screenings aimed at identifying and functionally characterizing signaling proteins.

Several surface molecules appear to be involved in antigen recognition by human T lymphocytes including the monomorphic 20/25K T3 structure present on all mature T lymphocytes and the subset-specific associative recognition elements, T4 and T8 (refs 1-8). More recently, Ti1, a clonally unique antigen recognition structure comprised of a 49,000 molecular weight (49K) alpha-chain and a 43K beta-chain, linked to T3 was identified on a major histocompatibility complex (MHC) class I specific T8+ T-cell clone, CT8III (ref. 9). To determine whether analogous receptor molecules could be found on other T-cell clones of differing specificity, we produced monoclonal antibodies against a clonal structure (Ti2) on an MHC class II specific T4+ lymphocyte, CT4II, derived from the same donor as CT8III. The Ti2 structure on CT4II is shown here to be a disulphide-linked heterodimer like Ti1 on CT8III and is composed of subunits of similar molecular weight. Monoclonal antibodies against Ti2 or Ti1 block antigen specific functions of the respective clone without showing any cross-reactivity. These findings suggest that each T lymphocyte, regardless of subset derivation or specificity, uses an analogous Ti heterodimer for antigen specific function. The latter is linked to T3 and expressed on the cell surface at an identical density (30,000-40,000 sites per cell).

T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylatedImmunoreceptor Tyrosine-basedActivation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-ζ chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the ζ chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other.