Main results

Throughout its life, a naïve T lymphocyte continually circulates between blood and lymph nodes, searching for the right partner, an antigen-presenting cell (APC) carrying peptide-MHC complexes specific for its antigen-recognition receptors. After this encounter occurring in a specialized area of the lymph nodes, T cells migrate into the inflamed tissue and exert their effector functions. We can therefore identify three distinct phases in T lymphocyte physiology: 1) migration; 2) priming; 3) effector functions. My group studies signals modulating these processes in physiopathological conditions.

 

1. CD28 interaction with filamin-A controls lipid raft accumulation at the T-cell immunological synapse.

During physiological T cell stimulation by APCs, a major T cell membrane rearrangement is known to occur leading to the organization of ‘supramolecular activation clusters’ at the immunological synapse (IS). A possible role for the synapse is the generation of membrane compartments where signaling might be organized and propagated. Thus, at the IS, engagement of the costimulatory molecule CD28 promotes the organization of a signaling compartment by inducing cytoskeletal changes and lipid raft accumulation. We identified the actin-binding protein Filamin-A (FLNa) as a novel molecular partner of CD28. We found that, after physiological stimulation, CD28 associated with and recruited FLNa into the IS, where FLNa organized CD28 signaling. Notably, FLNa knockdown by short interfering (si)RNA inhibited CD28-mediated raft accumulation at the IS and T cell costimulation. Together, our data indicate that CD28 binding to FLNa is required to induce the T cell cytoskeletal rearrangements leading to recruitment of lipid microdomains and signaling mediators into the IS.

 

2.   T cell costimulation by chemokine receptors.

Recently, we have proposed a new role for chemokines during the early phases of T cell activation by APCs. During T-cell activation, CXCR4 and CCR5 chemokine receptors are recruited into the IS by APC-released chemokines. Recruitment of chemokine receptors into the IS has two important consequences for T cells: it results in stronger interaction with the APC and in enhanced activation, in terms of proliferation and cytokine production. Accumulation of chemokine receptors at the IS requires chemokine secretion by the APC, indicating that costimulatory signals can be not only delivered through membrane-bound proteins, but also through soluble immunotransmitters, Interestingly, TCR triggering seems to modify chemokine receptor signaling properties. Chemokine receptors are seven-transmembrane molecules coupled to heterotrimeric G proteins. Chemokine binding to chemokine receptors dissociates G _i, the Gmost commonly associated with those receptors, and G subunits of the heterotrimeric G proteins, leading to calcium flux and activation of several signaling pathways. Consistent with G _i association, the vast majority of chemokine responses are inhibited by treatment with pertussis toxin (PTx). However, chemokine receptor accumulation at the IS is insensitive to PTx treatment, indicating that the process does not involve G _i-mediated signaling. Compatible with this, we found that TCR signaling induces coupling of CCR5 with G _q/11, thus modifying the chemokine receptor signaling pathway transduced at the IS.

We have also demonstrated that the costimulatory properties of CCR5 and CXCR4 chemokine receptors depend on their ability to form heterodimers. CXCR4 and CCR5 homodimers are not recruited into the IS and do not costimulate T cell activation, although they are perfectly functional in inducing chemotactic responses. These data indicate that chemokine receptor homo- and heterodimers have distinctive signaling and biological properties and that the functional outcome of stimulation of one particular chemokine receptor will also depend on the expression of its potential partners.

 

3. S YBILLA (Systems Biology of T-cell activation in health and disease): The Lck kinase.

SYBILLA is a consortium of 17 scientific and industrial partners. Through a multidisciplinary effort, it aims to understand at systems level how T-cells discriminate foreign- from self-peptides by activating quantitatively distinct signaling pathways. Proteomics, transcriptomics, imaging and multiplexed biochemical techniques will be applied to obtain holistic maps of the T-cell signaling network and to achieve a quantitative and dynamic understanding of network composition and regulation in response to different signal inputs.

The Src-family tyrosine kinase Lck is responsible
for initiating the signaling cascade upon TCR engagement by peptide/MHC. The catalytically active form of Lck (phophoryated on tyrosine 394, pY394)
 phosphorylates ITAM sequences of the 
intracellular tails of the CD3-zeta subunits of
the TCR, the first event in T cell activation. The conventional textbook view recites that Lck
 is activated when the TCR becomes engaged. 
However, this is not been thoroughly investigated and it is still unknown how Lck is
regulated during TCR engagement.
 We have analyzed by biochemical and imaging
 approaches the functional status of Lck  in
 unstimulated T cell lines, primary T cells and 
thymocytes and found that active Lck is readily
 detectable in these cells as well as in situ in
 lymphoid tissues. Our data provide evidence that T cells 
express a congruous amount of activated Lck that is generated without the need for TCR engagement. These
data force a revision of the conventional view 
of TCR-induced activation.

 

4.   Orchestration of leukocyte chemotaxis by mitochondrial dynamics.

Leukocyte traffic is required to maintain homeostasis and perform appropriate immunological reactions. To migrate into inflamed tissues, leukocytes must acquire spatial and functional asymmetries. Mitochondria are highly dynamic organelles that distribute in the cytoplasm to meet specific cellular needs, albeit whether this is essential to leukocyte functions is unknown. We show that during leukocyte migration mitochondria specifically concentrate at the uropod by a process involving rearrangements of their shape. Mitochondrial fission facilitates relocation of the organelles and promotes lymphocyte chemotaxis, whereas mitochondrial fusion inhibits both the processes. Our data substantiate a new role for mitochondrial dynamics and suggest that mitochondria redistribution is required to regulate the motor of migrating cells.

 

5.   Boosting anti-tumor responses of T lymphocytes infiltrating human cancers.

An important emerging concept is that the altered metabolism in the tumor microenvironment has a profound impact on antitumor immunity. We have investigated the role of tumor microenvironment in human prostate cancer and found a high local production of reactive nitrogen species (RNS). By inhibiting the activity of arginase and nitric oxide synthase, key enzymes of L-arginine metabolism that are highly expressed in malignant but not in normal prostates, reduced tyrosine nitration and restoration of T cell responsiveness to tumor were achieved. These results identify a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy.

We speculated that RNS produced in the tumor microenviroment modify chemokine structure, inhibiting their chemoattractant functions. We found that the chemoattractants CXCL12, CCL21 and CCL2 loose their ability to recruit T lymphocytes if exposed to peroxynitrite. However, the modified chemokine CCL2 retains its capacity of recruiting myeloid-derived suppressor cells. These data suggest that the modified chemokines may modify the tumor microenvironment and favor immunosuppression.

Main objectives and research lines

 

·    The role and mechanism of function of CXCR4 in the adaptive response-related pathology of WHIM syndrome patients.

The WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) syndrome is a rare human disease characterised by symptoms including neutropenia, recurring infections and warts. It is caused by dominant mutations in the chemokine receptor CXCR4, which result in C-terminal truncation of the receptor. This has been shown to impair the intracellular trafficking of the receptor in neutrophils, leading to increased responsiveness to chemokine. Whist this leads to retention of neutrophils in the bone marrow and consequent neutropenia, the recurrent infections and warts, the reduced immunoglobulin levels and impaired memory B/plasma cell function exhibited by most WHIM patients are indications of defects in the adaptive immune system, and thus cannot be explained by the above findings. We have previously demonstrated that the chemokine receptors CXCR4 and CCR5, in addition to their established function in leukocyte migration, have an important role in increasing the stability of immunological synapses between T cells and antigen presenting cells.
We hypothesised that the WHIM-associated mutations of CXCR4 may be affecting the role of the receptor in the stabilization of IS, leading to the observed adaptive response-related symptoms. We have found that the mutant CXCR4 on T cells is recruited to the immunological synapse in a normal manner, and that the ability of the receptor to enhance T cell co-stimulation is not impaired by the mutations. However, the stability of the T cell-antigen presenting cell conjugates formed in the presence of competing external chemokine signals is substantially impaired, as shown by in vitro studies, as well as ex vivo experiments using cells from WHIM patients. We are planning to use an experimental model for in vivo confirmation of our results.

 

·   Mitochondria localization at the immunological synapse

It has been reported that mitochondria accumulate under the IS in response to TCR stimulation. This process seems to be required to allow proper TCR-induced calcium influx in T cells in contact with APCs, because mitochondria can sequester calcium and thus keep CRAC channels open. Antigen-induced calcium signaling is however very fast, and clearly much faster than mitochondria translocation toward the IS. Thus, we speculated that other signals are involved in recruiting the organelles to the contact region between T cells and APCs. We found that the adhesion molecule LFA-1 induces localization of mitochondria at the IS. This process is antigen-independent and is enhanced by the presence of chemokines in the T cell environment. However, TCR triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. Our data suggest that, by recruiting mitochondria to the cell-cell contact region, LFA-1 prepares and facilitate TCR signaling. We are performing experiments to understand the signaling pathways involved in mitochondria translocation at the IS.

 

·   Control of T-cell chemotaxis.

We are interested in discovering new pathways controlling T cell migration. We are working on three different hypotheses.

1)  Role of serotonin: human T cells express functional serotonin receptors, including 5HT3 receptors, which are ligand-gated cation channels selectively inhibited by drugs such as granisetron and ondansetron. We have data indicating that peripheral blood T cells exert enhanced migration toward CXCL12 when 5HT3 are blocked. In accordance, serotonin or 5HT3 agonists reduce CXCL12-induced T cell migration. The effects on migration are paralleled by those on CXCL12-induced calcium influx, indicating that 5HT3 receptors control early events of chemokine receptor signaling. We are analyzing the physiological role of this novel signaling cross-talk.

2)   Role of calcium influx: whether calcium influx is required for T cell migration is still debated. Previous experiments performed in our lab using Jurkat and IP3R-KO DT40 cell lines suggested that calcium influx is irrelevant for lymphocyte migration. However, recent data obtained in our lab using peripheral blood T cells and the availability of reagents to selectively inhibit CRAC signaling prompted us to revisit this problem.

3)   Role of PIP5K: Type I phosphatidylinositol 4-phosphate 5-kinase has been involved in neutrophil migration. We are addressing the possibility that PIP5K regulates mitochondria localization at the uropod of migrating T cells.

 

·   Regulatory T cell-mediated modification of lymph node chemokine microenvironment.

We are currently addressing the effect of regulatory T cells on chemokines produced upon antigen-specific T cell activation in the lymph node, using a TCR transgenic model where regulatory T cells are generated in vivo. We have found that pro-inflammatory chemokines are secreted in the lymph node early (24h) after antigen-specific T cell activation, and that this secretion is significantly impaired in a manner directly proportional to the frequency of regulatory T cells specific for the same antigen. This in turn decreases significantly further recruitment of antigen presenting cells and other immune cells into the lymph node, suggesting that immune regulation involves not only a suppressive effect on a per cell basis but an effect on the overall magnitude of the early response.   

 

·   Boosting anti-tumor responses of T lymphocytes infiltrating human cancers.

Based on our findings, drugs controlling the in situ production of RNS might be useful to aid immunotherapeutic approaches for the treatment of cancer, by creating a favorable tumor environment for lymphocyte activation. Using in vitro screenings, we have developed novel small molecules aimed at interfering with multiple, interconnected metabolic pathways leading to RNS generation in the tumor microenvironment. We used one of this new compounds (AT38) to verify in vivo the hypothesis that peroxynitrites may keep T cell far from the tumor. Models inoculated subcutaneously with the colon carcinoma C26-GM were treated for 8 days with various doses of AT38. At the end of the treatment, models were sacrificed, the tumor explanted and the presence of peroxynitrites and lymphocytes was analyzed by immunohistochemistry. Tumors from models that did not receive AT38 treatment were characterized by intense nitrotyrosine staining and few TILs, whereas tumors from models that have been treated with AT38 for 8 days showed reduced numbers of nitrotyrosine-positive cells and were infiltrated by higher numbers of TILs. We would like to continue this translational project and demonstrate the feasibility, usefulness and action mechanisms of a combined therapy based on these novel immune modulators.

On the other hand, our data suggest that CCL2 modification by RNS can alter the balance between positive and negative effects caused by infiltration of inflammatory cells in cancers, because tumor-promoting cells, such as myeloid-derived suppressor cells and monocytes, will be preferentially recruited in the tumor microenvironment. In the next years, we will try to address three main questions: i) Why monocytes can still migrate toward RNS-modified CCL2 whereas T cells do not recognized the nitrated form of the chemokine? ii) do RNS and RNS-modified chemokines influence immunity against human cancers?

 

·   Requirements for Agrin in hematopoiesis.

Agrin is a heparan sulfate proteoglycan that is best known for its crucial involvement in the organization and maintenance of postsynaptic structures at the neuromuscular junction. Consistent with this role, agrin-deficient models die at birth due to respiratory failure. Here we examined the early postnatal hematopoiesis of agrin-deficient models in which perinatal death was prevented by transgenic Musk selective overexpression on muscle cells. Such transgenic, agrin-deficient models were born at Mendelian ratio but die shortly after birth.