No abstract

Most eukaryotic cells have a primary cilium which acts as a sensory organelle1. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors2. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly3 , 4, was found to colocalize with both the MTOC and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse (IS). IFT20 knockdown resulted in impaired TCR/CD3 clustering and signaling at the IS due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57, IFT88) and the TCR. The results identify IFT20 as a novel regulator of IS assembly in T-cells and provide the first evidence that IFT is implicated in membrane trafficking in cells lacking primary cilia, thereby opening a new perspective on IFT function beyond its role in ciliogenesis.When naive T-cells encounter antigen presenting cells (APC) bearing cognate MHC-bound peptide antigen, a dynamic rearrangement of membrane and cytosolic molecules occurs at the T-cell:APC contact area. This results in the formation of a highly organized interface known as immune synapse (IS), which acts as a platform for signal integration, fine-tuning and extinction5 , 6. A hallmark of the nascent IS is reorientation of the microtubule organizing center (MTOC) towards the APC7, which ensures targeted delivery of signaling molecules from intracellular pools to the IS8. This includes the TCR/CD3 complex itself, which is carried to the IS through polarized recycling9. Directional movement of structural and regulatory molecules, orchestrated by the MTOC, is a characteristic feature of primary cilia. These structures, present on most non-dividing cells, act as sensory organelles, relaying information from the external environment into the cell through intraflagellar transport (IFT), a process carried out by multimeric IFT particles and molecular motors2 , 10.At variance with most other eukaryotic cell types, hematopoietic cells lack primary cilia10. Surprisingly, IFT20, an IFT component essential for ciliary assembly3 ,4 , was found to be Correspondence should be addressed to CTB. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 1. Published in final edited form as:Nat Cell Biol. 2009 November ; 11(11): 1332-1339. doi:10.1038/ncb1977. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript expressed in human cells of both lymphoid (peripheral blood lymphocytes, Jurkat T-lymphoma cells) and myeloid (monocytes, monocyte-derived DC) lineages (Fig.1a). Similar results were obtained in the mouse, where IFT20 was detectable in central (thymus, bone marrow) and peripheral (lymph node, spleen) lymphoid organs (Fig.1a). Immunofluorescence analysis of Jurkat cells and human peripheral blood lymphocytes (PBL) showed that IFT20...

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.

IntroductionChronic lymphocytic leukemia (CLL) is the most common B-cell neoplasm in Europe and the United States, characterized by progressive accumulation of monoclonal CD5 ϩ B cells in peripheral blood, bone marrow, and peripheral lymphoid organs. The clinical course of CLL is highly variable, ranging from an indolent disease that may never require treatment to a rapidly progressive disease. 1 One of the principal prognostic features is the mutational status of the immunoglobulin heavy chain variable region genes (IGHV). CLL with poor prognosis has unmutated IGHV genes (U-CLL), whereas CLL with good prognosis carries somatic mutations in these genes (M-CLL). 2,3 Neoplastic CLL cells are typically arrested in the G 0 /G 1 phase of the cell cycle and accumulate in tissues because of prolonged survival. 1 Although extrinsic factors, such as escape from immune surveillance and chemotaxis to a favorable microenvironment, contribute to the extended survival of CLL B cells, 4 CLL is primarily a disease of defective apoptosis, and intrinsic defects in a number of components of the apoptotic circuitry have been identified, including overexpression of antiapoptotic proteins (eg, Bcl-2, Mcl-1, XIAP) and reduction in the expression of proapoptotic proteins (eg, Bax, DAPK-1). [5][6][7] Other apoptosis defects, including abnormalities in the p53 and ATM pathways in the most aggressive subsets, have also been implicated in the prolonged survival of malignant cells. [8][9][10] In this respect, apoptosis is emerging as a key therapeutic target in CLL, as witnessed by the ongoing clinical trials on Bcl-2 inhibitors. 11 p66Shc, a member of the Shc family of protein adapters, acts as antagonist of mitogenic signaling and positive regulator of oxidative stress-induced apoptosis in fibroblasts. 12,13 In T cells, where its expression is epigenetically controlled, 14,15 p66Shc uncouples the T-cell receptor from activation of the Ras/mitogen-activated protein kinase pathway by competitively inhibiting recruitment to the T-cell receptor of the mitogenic isoform, p52Shc. 16 Furthermore, p66Shc enhances T-cell susceptibility to apoptotic stimuli by increasing reactive oxygen species (ROS) production and impairing Ca 2ϩ homeostasis. 17 We have recently reported that p66Shc is expressed in murine B cells and that p66Shc deficiency results in enhanced proliferative responses of mouse B cells to B-cell receptor (BCR) engagement, 18 suggesting that p66Shc may display similar activities in T and B cells.Here, we have investigated the role of p66Shc in B-cell survival. We show that p66Shc promotes B-cell apoptosis by uncoupling the BCR from the survival pathways mediated by Akt and Erk. On the basis of these findings, we have investigated p66Shc expression and function in B cells from patients with CLL. The results identify a role for p66Shc in the imbalance among proapoptotic and antiapoptotic Bcl-2 family members in CLL and An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains...

-/-mice, a model of muscular dystrophies due to defects of collagen VI. Experimental approach: We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca 2+ and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1 -/-mice before after treatment with Debio 025. Key results: Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1 -/-mice. Conclusions and implications:Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1 -/-myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.