Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells.

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.

TCR dynamics on the surface of living T cells.

T lymphocyte activation by specific antigen requires prolonged TCR occupancy and sustained signaling. This is accomplished by the formation of a specialized signaling domain, the immunological synapse, at the T cell-antigen-presenting cell contact site. Surface receptors and signaling components are progressively recruited into this domain where they are organized in defined three-dimensional structures. To better understand how TCR are supplied to the signaling domain during the activation process, we measured (using confocal microscopy and photo-bleaching recovery techniques) lateral mobility of GFP-tagged TCR on living Jurkat cell surface. We show that: (i) surface-expressed TCR exhibit an intrinsic, actin cytoskeleton-independent, lateral mobility which allows them to passively diffuse over the entire T cell surface within approximately 60 min and (ii) non-stimulated TCR rapidly enter the signaling domain. Our results indicate that TCR lateral mobility per se is sufficient to ensure TCR supply to the immunological synapse in the course of sustained T cell activation.

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes.

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.

Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes.

T lymphocytes are activated by the engagement of their antigen receptors (TCRs) with complexes of peptide and major histocompatibility complex (MHC) molecules displayed on the cell surface of antigen-presenting cells (APCs) [1]. An unresolved question of antigen recognition by T cells is how TCR triggering actually occurs at the cell-cell contact area. We visualized T-cell-APC contact sites using confocal microscopy and three-dimensional reconstruction of z-sections. We show the rapid formation of a specialized signaling domain at the T-cell-APC contact site that is characterized by a broad and sustained area of tyrosine phosphorylation. The T-lymphocyte cell-surface molecule CD2 is rapidly recruited into this signaling domain, whereas TCRs progressively percolate from the entire T-cell surface into the phosphorylation area. Remarkably, the highly expressed phosphatase CD45 is excluded from the signaling domain. Our results indicate that physiological TCR triggering at the T-cell-APC contact site is the result of a localized alteration in the balance between cellular kinases and phosphatases. We therefore provide experimental evidence to support current models of T-cell activation based on CD45 exclusion from the TCR signaling area [2] [3] [4].

Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Optimal activation of tumor-reactive T cells by selected antigenic peptide analogues.

Many mechanisms have been proposed to explain why immune responses against human tumor antigens are generally ineffective. For example, tumor cells have been shown to develop active immune evasion mechanisms. Another possibility is that tumor antigens are unable to optimally stimulate tumor-specific T cells. In this study we have used HLA-A2/Melan-A peptide tetramers to directly isolate antigen-specific CD8(+) T cells from tumor-infiltrated lymph nodes. This allowed us to quantify the activation requirements of a representative polyclonal yet monospecific tumor-reactive T cell population. The results obtained from quantitative assays of intracellular Ca(2+) mobilization, TCR down-regulation, cytokine production and induction of effector cell differentiation indicate that the naturally produced Melan-A peptides are weak agonists and are clearly suboptimal for T cell activation. In contrast, optimal T cell activation was obtained by stimulation with recently defined peptide analogues. These findings provide a molecular basis for the low immunogenicity of tumor cells and suggest that patient immunization with full agonist peptide analogues may be essential for stimulation and maintenance of anti-tumor T cell responses in vivo.

Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation.

Using human CD4+ T-cell clones and peptide-pulsed antigen-presenting cells (APC) we measured, at the single cell level, different steps in the T-cell activation cascade. Simultaneous analysis of T-cell antigen receptor (TCR) down-regulation and interferon-gamma (IFN-gamma) production shows that both the level of TCR occupancy and the amount of IFN-gamma produced by single T cells increase in an antigen dose-dependent fashion. Conversely, commitment of T cells to IFN-gamma production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustained signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose-dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen-stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy. PTK activation is sustained for at least 2 hr after conjugation, and is required to maintain a sustained increase in intracellular Ca2+ concentration. Our results show, for the first time, a direct correlation between the level of TCR occupancy and the activation of PTK in individual T cells and offer an explanation for how the number of triggered TCR can be 'counted' and integrated in a corresponding biological response.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

HLA-restricted, processing- and metabolism-independent pathway of drug recognition by human alpha beta T lymphocytes.

T cell recognition of drugs is explained by the hapten-carrier model, implying covalent binding of chemically reactive drugs to carrier proteins. However, most drugs are nonreactive and their recognition by T cells is unclear. We generated T cell clones from allergic individuals specific to sulfamethoxazole, lidocaine (nonreactive drugs), and cef-triaxone (per se reactive beta-lactam antibiotic) and compared the increase of intracellular free calcium concentration ([Ca2+]i) and the kinetics of T cell receptor (TCR) downregulation of these clones by drug-specific stimulations. All drugs tested induced an MHC-restricted, dose- and antigen-presenting cell (APC)-dependent TCR downregulation on specific CD4(+) and CD8(+) T cell clones. Chemically nonreactive drugs elicited an immediate and sustained [Ca2+]i increase and a rapid TCR downregulation, but only when these drugs were added in solution to APC and clone. In contrast, the chemically reactive hapten ceftriaxone added in solution needed > 6 h to induce TCR downregulation. When APC were preincubated with ceftriaxone, a rapid downregulation of the TCR and cytokine secretion was observed, suggesting a stable presentation of a covalently modified peptide. Our data demonstrate two distinct pathways of drug presentation to activated specific T cells. The per se reactive ceftriaxone is presented after covalent binding to carrier peptides. Nonreactive drugs can be recognized by specific alphabeta+ T cells via a nonconventional presentation pathway based on a labile binding of the drug to MHC-peptide complexes.

The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement.

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.

Agonist-induced T cell receptor down-regulation: molecular requirements and dissociation from T cell activation.

T cell receptor (TCR) down-regulation is a consequence of specific receptor engagement and plays an important role in modulating the T cell response. We have investigated the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the induction of TCR down-regulation. We report that the mutation of S126 in the CD3-gamma chain that is known to inhibit phorbol-12-myristate 13-acetate-induced TCR down-regulation does not affect down-regulation induced by a specific agonist. In addition, agonist-induced TCR down-regulation is not affected by blockade or depletion of PKC, neither by blockade or lack of PTK, while the same treatments efficiently interfere with T cell activation. These results demonstrate that TCR down-regulation is induced by early events which follow specific engagement by an agonist and can be dissociated from those required for full T cell activation.

Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation.

T cell activation by specific antigen results in a rapid and long-lasting downregulation of triggered T cell receptors (TCRs). In this work, we investigated the fate of downregulated TCR- CD3-zeta complexes. T cells stimulated by peptide-pulsed antigen-presenting cells (APCs) undergo an antigen dose-dependent decrease of the total cellular content of TCR-beta, CD3-epsilon, and zeta chains, as detected by FACS(R) analysis on fixed and permeabilized T-APC conjugates and by Western blot analysis on cell lysates. The time course of CD3-zeta chain consumption overlaps with that of TCR downregulation, indicating that internalized TCR-CD3 complexes are promptly degraded. Inhibitors of lysosomal function (bafilomycin A1, folimycin) markedly reduced zeta chain degradation, leading to the accumulation of zeta chain in large Lamp1(+) vesicles. These results indicate that in T cell-APC conjugates, triggered TCRs are rapidly removed from the cell surface and are degraded in the lysosomal compartment.

Signal extinction and T cell repolarization in T helper cell-antigen-presenting cell conjugates.

We have previously demonstrated that in T cell-antigen-presenting cell (APC) conjugates many T cell receptors (TCR) are serially triggered by a few peptide-MHC complexes, resulting in sustained signaling. Here, we investigate the mechanisms that determine the duration and extent of signaling. We show that in the course of the T helper cell-APC interaction, down-regulation of triggered TCR leads to extinction of signaling. However, T cells that have been activated by a previous encounter with peptide-pulsed APC and have extinguished signaling can swiftly repolarize towards APC displaying higher antigen concentrations and dedicate their help to these cells. These results demonstrate that TCR down-regulation allows T cells to calibrate their response and dedicate their help to APC offering the highest stimulus.

Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy.

We have investigated the level of TCR occupancy required to elicit different biological responses in human CTL clones specific for an influenza matrix peptide. Specific cytotoxicity could be detected at extremely low peptide concentrations (10(-12) to 10(-15) M). However, IFN-gamma production, responsiveness to IL-2 and Ca++ fluxes were observed only at peptide concentrations > 10(-9) M, while autonomous proliferation required even higher peptide concentrations. In parallel experiments we measured TCR downregulation to estimate the number of TCRs triggered. We observed that at low peptide concentrations, where only cytotoxicity is triggered, TCR downregulation was hardly detectable. Conversely, induction of IFN-gamma production and proliferation required triggering of at least 20-50% of TCRs. Taken together these results indicate that a single CTL can graduate different biological responses as a function of antigen concentration and that killing of the specific target does not necessarily result in full activation.

Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines.

Trafficking to tissues and then to lymph nodes is a crucial aspect of the immunobiology of dendritic cells. The present study was designed to identify molecules able to direct the migration of human blood-derived dendritic cells. fMLP (representative of formyl peptides of bacterial origin), C5a, and the C-C chemokines monocyte chemotactic protein (MCP)-3, MIP-1 alpha/LD78, and RANTES elicited chemotactic migration and a rise of intracellular free calcium in dendritic cells. In contrast, the C-X-C chemokines IL-8 and IP-10 and the C-C chemokines MCP-1 and MCP-2 were inactive as chemoattractants. Thus, dendritic cells respond to classical chemotactic signals and to a set of chemokines distinct from that active on monocytes and neutrophils. Chemoattractants are likely to contribute to localization and trafficking of dendritic cells and provide tools to recruit these cells in the design of immunization strategies.

Distinct signaling properties identify functionally different CD4 epitopes.

The CD4 coreceptor interacts with non-polymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF-AT transcription factor which is required for interleukin-2 gene expression. Whereas different anti-CD4 mAb or HIV-1 gp120 could all trigger activation of the protein tyrosine kinases p56lck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF-AT. Lack of full activation of NF-AT could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and calcium pathways.

Serial triggering of many T-cell receptors by a few peptide-MHC complexes.

T lymphocytes can recognize and be activated by a very small number of complexes of peptide with major histocompatibility complex (MHC) molecules displayed on the surface of antigen-presenting cells (APCs). The interaction between the T-cell receptor (TCR) and its ligand has low affinity and high off-rate. Both findings suggest that an extremely small number of TCRs must be engaged in interaction with APCs and raise the question of how so few receptors can transduce an activation signal. Here we show that a small number of peptide-MHC complexes can achieve a high TCR occupancy, because a single complex can serially engage and trigger up to approximately 200 TCRs. Furthermore, TCR occupancy is proportional to the T cell's biological response. Our findings suggest that the low affinity of the TCR can be instrumental in enabling a small number of antigenic complexes to be detected.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion.

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein.