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.

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 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.

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.

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 cellderived 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 IntroductionAlthough progressive accumulation of monoclonal CD5 ϩ B cells in the blood, peripheral lymphoid organs, and BM is the hallmark of chronic lymphocytic leukemia (CLL), the clinical course of this disorder is highly variable, ranging from a stable disease that may only require monitoring over time to a progressive, severe disease. 1,2 Several markers have been associated with poor prognosis. Coupled to the cytogenetic abnormalities, the mutational status of immunoglobulin heavy chain variable region (IGHV) genes is the most valuable marker presently available, with unmutated IGHV found in patients who develop aggressive disease. 3 Nevertheless, the onset of disease progression and response to treatment are to date largely unpredictable.At variance with other hematologic malignancies, CLL B cells are usually arrested at G 0 /G 1 and their accumulation is the result of an abnormally prolonged survival rather than uncontrolled proliferation. 1,2 Intrinsic defects in the apoptotic machinery underlie the prolonged lifespan of CLL B cells, a major target being the Bcl-2 family, in which overexpression of antiapoptotic members (Bcl-2 and Mcl-1) or impaired expression of proapoptotic members (Bax and Bak) tilts the balance toward cell survival. 4 Extrinsic factors consisting mainly of stromal cell-derived chemokines (CXCL12, CXCL13, CCL19, and CCL21) also to contribute to the extended lifespan of CLL B cells by providing survival cues during their transit through peripheral lymphoid tissues and BM. 5 Lymphocyte trafficking is tightly controlled by the chemokines present in the lymphoid microenvironment and the chemokine receptors expressed by the lymphocyte itself. 6 CLL B cells express increased levels of CXCR4, CCR7, and CXCR5, which has...

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Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eμ-TCL1/p66Shc−/− mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eμ-TCL1 mice. Eμ-TCL1/p66Shc−/− mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells’ level of p66Shc expression. p66Shc expression declined with disease progression in Eμ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.