Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigens bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their complex functional responses.National Institute of Allergy and Infectious Diseases (U.S.) (5P01AI045757)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051

La costimolazione dei linfociti T alla sinapsi immunologica: CD28, rafts di membrana e recettori chemochinici

T lymphocytes are activated when their T-cell receptors (TCRs) recognize and interact with the specific antigenic complexes formed by antigen-derived peptides bound to proteins of the major histocompatibility complex (MHC) and exposed on the surface of an antigen-presenting cell (APC). The contact site between these two cells is referred as immunological synapse (IS) and represents a highly specialized cellular junction, where T lymphocyte receives and integrates several signals provided by cellular partner, in order to choose between tolerance and immunity. These signals, additional to TCR, are substantially provided by costimulatory molecules that are tuned by inflammatory environment, thus coding the context of antigenic presentation. The immune system has developed different strategies for such a complex function and recently new costimulatory mechanisms, different from the classical ones based on membrane receptors, have been identified, such as signal amplification by membrane rafts and costimulation through chemokines. In this thesis I have been interested in defining the molecular mechanisms underlying these new costimulatory strategies at the IS. The cellular plasma membrane contains small, heterogeneous and highly dynamic, microdomains, enriched in sterol and sphingolipid and in selective proteins, defined as membrane rafts. In T-cell plasma membrane these microdomains are recruited into the IS, forming a platform where TCR signal is protected and amplified, and thus contribute to costimulation. In our group it has been proposed that CD28, the main costimulatory molecule for naive T lymphocytes, amplifies TCR signal by inducing membrane rafts rearrangement and recruitment into the IS. In order to identify the molecular mechanism allowing CD28-mediated rafts recruitment into the IS, and considering the essential role played by actin cytoskeleton in molecule mobilization toward IS, we focused on interaction among CD28, cytoskeleton and rafts. In this thesis it is demonstrated that CD28 binds to filamin-A (FLNa), an actin-binding protein able to induce actin crosslinking and to stabilize the cortical cytoskeleton, and recruits FLNa into the IS. The interaction between CD28 and FLNa, as well as the recruitment of FLNa into the IS, require the same CD28 prolin-rich motif needed for membrane rafts mobilization into the IS. Moreover FLNa silencing by small interference (si)RNA inhibits CD28-induced rafts recruitment into the IS, Cdc42 activation (that regulates cytoskeletal rearrangements) and CD28 costimulation. These results indicate that CD28 uses FLNa to integrate signalling pathways, resulting in actin crosslinking and lipid raft recruitment into the IS, thus sustaining TCR signaling and lowering the T-cell activation threshold. The costimulatory properties of chemokines have been recently demonstrated in human T lymphocytes. In these cells, the chemokine receptor CXCR4 is constitutively expressed and regulates lymphocyte migration towards gradients of CXCL12; in contrast, CCR5 is expressed only in activated T cells and leads their migration towards gradients of CCL3, CCL4 and CCL5. These two receptors are involved in several pathological conditions, such as autoimmunity, cancer and HIV (human immunodeficiency virus). Our group has demonstrated that during T-cell stimulation both CXCR4 and CCR5 are recruited and trapped into the IS, through a mechanism that requires chemokine secretion by APC. The CXCR4 and CCR5 recruitment into the IS results in stronger interactions between T cell and APC, in reduced responsiveness to chemotactic gradients and in higher levels of T-cell proliferation and IFN-? (interferon-?) production. Interestingly, we found that during T cell activation chemokine receptors are coupled with Gq instead of Gi, the classical G protein coupled to these receptors during cell migration. The aim of my thesis was to study the mechanism for CXCR4 and CCR5 versatility in function and signaling, and to identify the requirements for chemokine-induced T-cell costimulation. Since chemokine receptors can form receptor complexes with specific pharmacological and signaling properties through homo- and hetero-dimerization, we hypothesized that molecular complexes between CXCR4 and CCR5 in T lymphocytes are required for their costimulation at the IS. In this thesis it is demonstrated that, in contrast with CXCR4 and CCR5 chemotactic functions, which depends on receptor homodimers, the costimulatory function of these receptors requires their functional collaboration: CXCR4 and CCR5 must be co-recruited into the IS and must be co-expressed by T cell to costimulate cytokine production. Moreover it has been demonstrated that co-expressed CXCR4 and CCR5 form constitutive complexes (hetero-dimers or hetero-oligomers), suggesting that cooperation between receptors represents one key strategy for the functional plasticity of chemokines. In conclusion, my study on novel T-cell costimulation mechanisms highlight the complexity of the process leading to transmission of signals at the IS. This integrated and dynamic process involves soluble mediators, membrane receptors and the cell cytoskeleton, and generates micro-environments specific for signal amplification by locally modifying the cell membrane composition and its signaling complexes

T cells and their partners: the chemokine dating agency

Chemokines and their receptors have long been recognized as key molecules directing leukocyte migration between blood, lymph and tissues. Evidence accumulated in recent years indicates that, in addition to their chemotactic functions, chemokine receptors are highly versatile players fine-tuning immune responses. Chemokine receptors and ligands have been implicated in dendritic-cell maturation, signal transmission at the immunological synapse between T lymphocytes and their cellular partners, and in the polarization of immune responses. These findings identify new roles for chemokines in T-cell triggering and activation of effector functions, and suggest that these small cytokines represent key conductors of adaptive immunit

Chemokines: coded messages for T-cell missions

Chemokines and their receptors control leukocyte migration and homing throughout the body in both physiological and pathological conditions. In the context of the adaptive immune system, which requires high efficiency and control, chemokines and chemokine receptors represent a versatile code that orchestrates the "who, where and when" of the immune response by providing the spatio-temporal guidance for T-cell development, priming and effector functions. In addition to their chemotactic properties, chemokines can directly modulate T-cell responses by amplifying signals at the immune synapse and tuning Th1/Th2 polarization. In this review we will discuss the role of chemokines in T-cell biology, following an ideal pilgrimage that spans the key steps of the T-cell life

Adhesion shapes T cells for prompt and sustained T-cell receptor signalling

Chemokine receptors and the integrin LFA-1 establish cell polarity in T lymphocytes independently of T-cell receptor (TCR) stimulation. The integrin-mediated adhesion and cell polarity is needed for optimum TCR signalling