Salvatore Valitutti

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 κB 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.

Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition

Abstract Sensitivity of antigen detection by B cells correlates with high-affinity binding. This paradigm does not appear to hold for the T-cell receptor (TCR), which is able to bind its ligand — peptide in the context of major histocompatibility complex (MHC) — with low affinity. Here, Salvatore Valitutti and Antonio Lanzavecchia propose that the efficiency of T-cell antigen recognition is dependent upon optimal kinetics of the TCR—peptide—MHC interaction, allowing serial engagements and triggering of many TCRs by a few peptide—MHC complexes.

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].

Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence

Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes, such as tryptase and chymase, at the cell-cell contact site. This previously unidentified mast cell effector mechanism, which we name the antibody-dependent degranulatory synapse (ADDS), is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably, IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence.

Uncoupling between Immunological Synapse Formation and Functional Outcome in Human γδ T Lymphocytes

Human T lymphocytes expressing the Vγ9Vδ2 TCR recognize non-peptidic Ags, referred to as phosphoantigens, produced by microbial pathogens and by human tumor cells. Here we show that γδ T cells establish a mature immunological synapse (IS) with the myelomonocytic THP-1 tumoral cell line. This synapse is characterized by an enrichment for phosphotyrosine, CD2, and γδ TCR together with the exclusion of CD45. The CD94 and NKG2D receptors are also recruited to the signaling area, while the C-lectin-like activation marker CD69 segregates out of the synapse. γδ T cell conjugation to THP-1 increases upon stimulation by soluble phosphoantigen, is paralleled by the metabolic activation of γδ T cells and leads to cytokine production. Molecular segregation of the above molecules also occurs at the γδ T cell/THP-1 interface in the absence of exogenously added phosphoantigen, although it does not result in intracellular signaling and cytokine production under these conditions. Hence the molecular interactions at the γδ T cell-THP-1 target cell interface are sufficient to induce the formation of an IS, but cytokine production requires the full engagement of γδ TCR by a strong agonist. Thus in γδ T cells, formation of the IS is uncoupled from its functional outcome.

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‐γ (IFN‐γ) production shows that both the level of TCR occupancy and the amount of IFN‐γ produced by single Tu2003cells increase in an antigen dose‐dependent fashion. Conversely, commitment of Tu2003cells to IFN‐γ 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 Tu2003cells undergo an antigen dose‐dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen‐stimulated Tu2003cells 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 2u2003hr 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 Tu2003cells and offer an explanation for how the number of triggered TCR can be ‘counted’ and integrated in a corresponding biological response.

Human regulatory T cells inhibit polarization of T helper cells toward antigen-presenting cells via a TGF-β-dependent mechanism

The molecular mechanisms used by regulatory T cells (Treg) to inhibit the effector phase of adaptive immune responses are still elusive. In the present work, we investigated the possibility that Treg may interfere with a basic biological function of T helper cells (TH): polarization of secretory machinery for dedicated help delivery. To address this question, we visualized by confocal microscopy different parameters of activation in TH and Treg cells interacting simultaneously with individual antigen-presenting cells (APC). Our results show that, although productive TCR engagement in TH/APC conjugates was unaffected by the presence of adjacent Treg, the reorientation of TH secretory machinery toward APC was strongly inhibited. Blocking TGF-β completely reverted Treg induced inhibition of TH polarization. Our results identify a previously undescribed mechanism by which Treg inhibit effector T cells. TGF-β produced by adjacent Treg interferes with polarization of TH secretory machinery toward APC, thus affecting a crucial step of TH-mediated amplification of the immune response.

Reciprocal Polarization of T and B Cells at the Immunological Synapse

Cognate interactions between T and B lymphocytes lead to the formation of the immunological synapse (IS) where bidirectional activation signals are exchanged. Although the molecular architecture and the function of the IS have been studied extensively on the T cell side, little is known about events occurring during synapse formation in Ag-presenting B cells. We investigated the impact of BCR and TLR signaling on human B cell activation and on the T and B cell side of the IS. On the T cell side, we observed that T cells polarized toward both naive and previously activated B cells. Nevertheless, when T cells interacted with different B cells simultaneously, T cells selectively polarized their secretory machinery toward preactivated B cells. Furthermore, both naive and preactivated B cells reoriented their microtubule-organizing center toward the synaptic T cell during cognate interactions. This phenomenon was rapid and not dependent on T cell secretory activity. Interestingly, not only the microtubule-organizing center but also the Golgi apparatus and Lamp-3+ and MHC class II+ vesicles all repositioned beneath the IS, suggesting that the entire endocytic/exocytic B cell compartment was reoriented toward the T cell. Taken together, our results show that the B cell activation status fine-tunes T cell polarization responses and reveal the capacity of naive and activated B cells to polarize toward T cells during cognate interactions.

Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse

Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients.

New insights to the functional role of the T cell-Antigen Presenting Cell immunological synapse

Three innovative and complementary morphological approaches were employed to study the T cell/antigen presenting cell (APC) interaction: (i) high resolution three-dimensional confocal microscopy of the T cell-APC contact site; (ii) time lapse video recording in living T cells of [Ca2+]I and changes in distribution of various GFP fusion proteins with TCR/CD3-zeta complex associated- and other signaling components; (iii) measurement of lateral TCR mobility and that of recruited signaling components using techniques based on fluorescence recovery after photo-bleaching. These approaches were combined with biochemical and functional experiments to investigate two principal issues: (A) Recruitment and the three-dimensional arrangement of receptors and signaling components at the contact site between human CD4+ T lymphocytes and APCs, (B) Structure of the immunological synapse formed at the contact site between cytotoxic T lymphocytes (CTLs) and target cells. We discuss evidence indicating that TCR engagement and triggering can occur in the absence of large-scale molecular segregation into the T cell-APC contact site. Taken together our results indicate that although not required for TCR engagement and triggering, formation of the IS is important to reinforce TCR-mediated signal transduction and achieve full T cell activation.