Laura Patrussi

The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms

Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche.

Simvastatin inhibits T-cell activation by selectively impairing the function of Ras superfamily GTPases

Statins are widely used hypocholesterolemic drugs that inhibit 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase, a rate‐limiting enzyme of the mevalonate pathway whose biosynthetic end product is cholesterol. In addition to lowering circulating cholesterol, statins perturb the composition of cell membranes, resulting in disruption of lipid rafts, which function as signaling platforms in immunoreceptor signaling. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and hence activity of small GTPases, which control multiple pathways triggered by these receptors. T‐cell activation is crucially dependent on Ras, Rho and Rab GTPases. Furthermore TCR signaling is orchestrated at lipid rafts, identifying T‐cells as potential cellular targets of statins. Here we report that simvastatin suppresses T‐cell activation and proliferation as the result of its capacity to inhibit HMG‐CoA reductase. T‐cell treatment with simvastatin does not affect intracellular cholesterol levels or raft integrity nor, accordingly, the initial tyrosine phosphorylation‐dependent cascade. Conversely, inhibition of protein prenylation by simvastatin results in a dramatic impairment in the pathways regulated by small GTPases, including the Ras/MAP kinase pathway, the Rac/stress kinase pathway, and the Rab‐dependent pathway of receptor endocytosis. The results identify Ras superfamily GTPases as strategic molecular targets in T‐cell immunosuppression by statins.

p66SHC Promotes Apoptosis and Antagonizes Mitogenic Signaling in T Cells

ABSTRACT Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc−/− T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.

Simvastatin inhibits the MHC class II pathway of antigen presentation by impairing Ras superfamily GTPases

Statins are widely used hypocholesterolemic drugs that inhibit 3‐hydroxy‐3‐methyl‐glutaryl‐coenzymeu2004A reductase, a rate‐limiting enzyme of the mevalonate pathway whose biosynthetic endproduct is cholesterol. As a result of this activity, statins may perturb the composition of cell membranes, resulting in lipid raft disruption. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and activity of small GTPases. Antigen uptake, processing and presentation involve the interplay of Rab and Rho family GTPases. Furthermore, lipid rafts have been implicated both in antigen internalization by the BCR and in MHC classu2004II clustering at the immunological synapse. Here we have addressed the effects of simvastatin on antigen processing and presentation by human B cells and dendritic cells. The results show that simvastatin potently suppresses tetanus toxoid processing and presentation to CD4+ T cells by HLA‐DR by inhibiting protein antigen uptake through both receptor‐mediated endocytosis and macropinocytosis. This effect can be largely accounted for by defective prenylation of Rho and Rab GTPases in the absence of any measurable perturbation of lipid rafts. In addition, simvastatin was found to preferentially affect the invariant chain‐dependent MHC classu2004II pathway, thereby identifying this route of antigen processing and presentation as a selective target of statins.

Anthrax toxins inhibit immune cell chemotaxis by perturbing chemokine receptor signalling

Pathogenic strains of Bacillus anthracis produce two potent toxins, lethal toxin (LT), a metalloprotease that cleaves mitogen‐activated protein kinase kinases, and oedema toxin (ET), a calcium/calmodulin‐dependent adenylate cyclase. Emerging evidence indicates a role for both toxins in suppressing the initiation of both innate and adaptive immune responses, which are essential to keep the infection under control. Here we show that LT and ET inhibit chemotaxis of T‐cells and macrophages by subverting signalling by both CXC and CC chemokine receptors. The data highlight a novel strategy of immunosuppression by B.u2003anthracis based on inhibition of immune cell homing.

Specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system

ABSTRACT T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5+ endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis.

The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse.

ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. Highlighted Article: The ciliary regulator Rab8 is co-opted by T-cells during the assembly of the immune synapse to promote the VAMP-3-dependent step of polarized T-cell receptor recycling.

The CXCL12/CXCR4 axis as a therapeutic target in cancer and HIV-1 infection.

The seven-spanning transmembrane G-protein coupled receptor CXCR4, which specifically binds to the chemokine CXCL12, is expressed on many cell types, including various types of tumour cells. CXCR4 plays a crucial role in organ-specific metastasis, directing migration of malignant cells expressing this receptor toward microenvironments where the cognate ligand is secreted. CXCL12 has a direct growth and survival-promoting effect for various cancer cells and enhances moreover tumour angiogenesis by recruiting endothelial progenitor cells to tumours. Drugs which modulate the CXCL12/CXCR4 axis are therefore promising candidates in anti-cancer therapies. CXCR4 is also a coreceptor for human immunodeficiency virus type 1 (HIV-1) X4 virus and, as such, plays an important role in virus entry into target cells. Hence, antiviral agents that bind to CXCR4 are expected to inhibit HIV-1 entry. Here we review the structure, mechanism of action and biological activity of the main CXCR4 antagonists (peptide inhibitors, non-peptide antagonists, neutralizing antibodies, modified analogues of CXCL12) and agonists (CXCL12 peptide analogues) and discuss the CXCL12/CXCR4 axis as an important target in development of anti-tumoral and anti-HIV-1 therapies.

Cooperation and selectivity of the two Grb2 binding sites of p52Shc in T-cell antigen receptor signaling to Ras family GTPases and Myc-dependent survival.

Shc proteins participate in a variety of processes regulating cell proliferation, survival and apoptosis. The two ubiquitously expressed isoforms, p52Shc/p46Shc, couple tyrosine kinase receptors to Ras by recruiting Grb2/Sos complexes to a membrane-proximal localization. Tyrosine residues 239/240 and 317 become phosphorylated following receptor engagement and, as such, form two Grb2 binding sites, which have been proposed to be differentially coupled to Myc-dependent survival and to fos-dependent proliferation, respectively. Here, we have addressed the individual function of YY239/240 and Y317 in T-cell antigen receptor (TCR) signaling. We show that p52Shc is phosphorylated on both YY239/240 and Y317 following TCR engagement. Mutation of either YY239/240 or Y317 results in impaired interaction with Grb2 and inhibition of Ras/MAP kinase activation and CD69 induction, supporting a role for both Grb2 binding sites in this function. Substitution of either YY239/240 or Y317 also results in a defective activation of Rac and the coupled stress kinases JNK and p38. Furthermore, mutation of Y317 or, to a larger extent, of YY239/240, results in increased activation-induced cell death, which in cells expressing the FF239/240 mutant is accompanied by impaired TCR-dependent c-myc transcription. The data underline a pleiotropic and nonredundant role of Shc, mediated by both YY239/240 and Y317, in T-cell activation and survival.

S1P1 expression is controlled by the pro-oxidant activity of p66Shc and is impaired in B-CLL patients with unfavorable prognosis

Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.