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.