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.

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...

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...

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ShcA is an important mediator of Ras/ MAPK activation in PTK-regulated pathways triggered by surface receptors. This function is subserved by the constitutively expressed p52-kDa isoform. Besides activating Ras, p52Shc couples the TCR to Rho GTPases, and thereby participates in actin cytoskeleton remodeling in T cells. Here we have addressed the potential involvement of p52Shc in T-cell chemotaxis and the role of the phosphorylatable tyrosine residues, YY239/240 and Y317, in this process. We show that CXCR4 engagement by the homeostatic chemokine, SDF-1␣, results in p52Shc phosphorylation and its assembly into a complex that includes Lck, ZAP-70, and Vav. This process was found to be both Lck and Gi dependent. Expression of p52Shc mutants lacking YY239/240 or Y317, or p52Shc deficiency, resulted in a profound impairment in CXCR4 signaling and SDF-1␣-dependent chemotaxis, underscoring a crucial role of p52Shc as an early component of the CXCR4 signaling cascade. p52Shc was also found to be required for ligand-dependent CXCR4 internalization independently of tyrosine phosphorylation. Remarkably, CXCR4 engagement promoted phosphorylation of the chain of the TCR/CD3 complex, which was found to be essential for CXCR4 signaling, as well as for SDF-1␣-dependent receptor endocytosis and che IntroductionBy controlling leukocyte trafficking and homing to specific microenvironments, chemokines and their receptors represent a strategic element in the regulation of immune responses. 1 Initially implicated in directing transmigration of neutrophils and other cellular components of the innate immune system to inflamed tissues, chemokines have been found to coordinate temporally and topologically the directional migration and positioning of leukocytes within lymphoid organs and tissues not only in inflammation, but also in homeostatic conditions, thereby participating both in hematopoietic cell differentiation in central lymphoid organs and in the initiation of adaptive immune responses in peripheral lymphoid organs. Although the distinction has become less clear-cut, chemokines have been classified on the basis of their primary function as inflammatory and homeostatic. Among the latter is SDF-1␣/ CXCL12, which interacts with CXCR4. In the hematopoietic system, CXCR4 controls stem-cell migration to the bone marrow and retention of B-cell and granulocytic precursors within this microenvironment, thereby permitting their differentiation. Furthermore, SDF-1␣ is a potent chemoattractant for mature T cells, monocytes, and neutrophils. 2 CXCR4 has also been implicated in brain and heart development and angiogenesis, as well as in some pathological conditions, including tumor metastasis and joint infiltration by inflammatory CD4 ϩ cells in rheumatoid arthritis. 3 Like other chemokine receptors, CXCR4 is a 7-spanning transmembrane receptor coupled to a pertussis toxin (PTX)-sensitive heterotrimeric Gi protein, which, by inhibiting the activity of adenylate cyclase, modulates the levels of intracellular cAMP. 4 This second messenger re...