Cristina Ulivieri

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.

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.

T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross‐linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck‐deficient Jurkat T cells. In wild‐type Jurkat cells, Lck was activated by CD81 cross‐linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl‐β‐cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3‐deficient Jurkat cells. CD81 cross‐linking increased and prolonged the anti‐CD3‐induced tyrosine phosphorylation of TCRζ and of other proteins, indicating that the CD81‐mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross‐linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.

Defective recruitment and activation of ZAP-70 in common variable immunodeficiency patients with T cell defects

We have previously identified a subset of common variable immunodeficiency (CVID) patients with defective T cell function associated with impaired activation of the TCR‐dependent tyrosine phosphorylation cascade. Here we have assessed the structural and functional integrity of the principal components involved in coupling the TCR/CD3 complex to intracellular tyrosine kinases in two of these patients. We show that ZAP‐70 fails to bind the signaling‐competent CD3ζ  tyrosine phosphorylation isoform and to become activated following TCR engagement, suggesting that defective recruitment of ZAP‐70 might underlie the TCR signaling dysfunction in these patients. Determination of the nucleotide sequences encoding the intracellular domains of the CD3/ζ  subunits and ZAP‐70 did not reveal any mutation. Furthermore, ZAP‐70 from these patients could interact in vitro with recombinant phospho‐ζ , ruling out genetic defects at the immunoreceptor tyrosine‐based activation motif/SH2 domain interface responsible for ZAP‐70 recruitment to the activated TCR. No defect was found in expression, activity or subcellular localization of Lck, which is thought to be primarily responsible for CD3ζ  phosphorylation. Hence, while the T cell defect in these CVID patients can be pinpointed to the interaction between ZAP‐70 and CD3ζ , the integrity in the components of the signaling machinery involved in this process suggests that additional components might be required for completion of this step.

Generation of a monoclonal antibody to a defined portion of the Helicobacter pylori vacuolating cytotoxin by DNA immunization

The capacity of mammalian cells to express proteins encoded by plasmid vectors injected as naked DNA has opened the possibility to induce an immune response against protein antigens by DNA vaccination. We have used DNA immunization to generate a strong antibody response to a defined portion of the Helicobacter pylori vacuolating cytotoxin. A high specific monoclonal antibody has been derived from the splenocytes of an immunized mouse. Our data show that DNA immunization offers the possibility to obtain monoclonal antibodies following a significant shortcut with respect to traditional immunization protocols.

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis.

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells’ Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.

Cytoskeleton rearrangement induced by tetraspanin engagement modulates the activation of T and NK cells

The hepatitis C virus (HCV) binds to human cells through the interaction of its envelope glycoprotein E2 with the tetraspanin CD81. We have previously reported that engagement of CD81 has opposite effects on T and NK cell function, as it enhances T cell receptor‐mediated T cell activation and inhibits CD16‐ or IL‐12‐mediated NK cell activation. We further investigated this dichotomy and found that another tetraspanin, CD82, induces the same opposing effects on human primary T and NK cells. Activation by other unrelated stimuli such as NKG2D‐ and beta‐1 integrin is also reduced by CD81 ligation on NK cells. CD81 engagement by monoclonal antibody or HCV‐E2 enhances zeta and Erk phosphorylation in T cells and reduces them in NK cells, reflecting the opposite functional outcomes. CD81 engagement induces dramatic morphological changes and local F‐actin accumulation in both NK and T cells, indicating rearrangement of the actin cytoskeleton. Pharmacological inhibition of actin polymerization reduces T cell activation, whereas it greatly enhances NK cell activation. Importantly, treatment with actin blockers abolishes the inhibitory effect of CD81 ligation on NK cells. We propose that tetraspanin engagement leads to comparable cytoskeleton reorganization in T and NK cells, which in turn results in opposite functional outcomes.

GluD1 is a common altered player in neuronal differentiation from both MECP2 -mutated and CDKL5 -mutated iPS cells

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

The proapoptotic and antimitogenic protein p66SHC acts as a negative regulator of lymphocyte activation and autoimmunity

The ShcA locus encodes 3 protein isoforms that differ in tissue specificity, subcellular localization, and function. Among these, p66Shc inhibits TCR coupling to the Ras/MAPK pathway and primes T cells to undergo apoptotic death. We have investigated the outcome of p66Shc deficiency on lymphocyte development and homeostasis. We show that p66Shc(-/-) mice develop an age-related lupus-like autoimmune disease characterized by spontaneous peripheral T- and B-cell activation and proliferation, autoantibody production, and immune complex deposition in kidney and skin, resulting in autoimmune glomerulonephritis and alopecia. p66Shc(-/-) lymphocytes display enhanced proliferation in response to antigen receptor engagement in vitro and more robust immune responses both to vaccination and to allergen sensitization in vivo. The data identify p66Shc as a negative regulator of lymphocyte activation and show that loss of this protein results in breaking of immunologic tolerance and development of systemic autoimmunity.

The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma

BackgroundBurkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression by its own encoded microRNAs.MethodsUsing microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNAs. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases.ResultsWe showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Rα and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression.ConclusionsOur preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.