Identification of the Axis ß-Catenin-BTK in the Dynamic Adhesion of Chronic Lymphocytic Leukemia Cells to Their Microenvironment.

In the microenvironment, cell interactions are established between different cell types to regulate their migration, survival and activation. ß-Catenin is a multifunctional protein that stabilizes cell-cell interactions and regulates cell survival through its transcriptional activity. We used chronic lymphocytic leukemia (CLL) cells as a cellular model to study the role of ß-catenin in regulating the adhesion of tumor cells to their microenvironment, which is necessary for tumor cell survival and accumulation. When co-cultured with a stromal cell line (HS-5), a fraction of the CLL cells adhere to stromal cells in a dynamic fashion regulated by the different levels of ß-catenin expression. In non-adherent cells, ß-catenin is stabilized in the cytosol and translocates into the nucleus, increasing the expression of cyclin D1. In adherent cells, the level of cytosolic ß-catenin is low but membrane ß-catenin helps to stabilize the adhesion of CLL to stromal cells. Indeed, the overexpression of ß-catenin enhances the interaction of CLL with HS-5 cells, suggesting that this protein behaves as a regulator of cell adhesion to the stromal component and of the transcriptional regulation of cell survival. Inhibitors that block the stabilization of ß-catenin alter this equilibrium and effectively disrupt the support that CLL cells receive from the cross-talk with the stroma.

Immuno-regulatory malignant B cells contribute to Chronic Lymphocytic Leukemia progression.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFß1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFß signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFß1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.

Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21.

Tumor microenvironment exerts a critical role in sustaining homing, retention, and survival of chronic lymphocytic leukemia (CLL) cells in secondary lymphoid organs. Such conditions foster immune surveillance escape and resistance to therapies. The physiological microenvironment is rendered tumor permissive by an interplay of chemokines, chemokine receptors, and adhesion molecules as well as by direct interactions between malignant lymphocytes and stromal cells, T cells, and specialized macrophages referred to as nurselike cells (NLCs). To characterize this complex interplay, we investigated the altered architecture on CLL lymph nodes biopsies and observed a dramatic loss of tissue subcompartments and stromal cell networks as compared with nonmalignant lymph nodes. A supplemental high density of CD68+ cells expressing the homeostatic chemokine CCL21 was randomly distributed. Using an imaging flow cytometry approach, CCL21 mRNA and the corresponding protein were observed in single CD68+ NLCs differentiated in vitro from CLL peripheral blood mononuclear cells. The chemokine was sequestered at the NLC membrane, helping capture of CCR7-high-expressing CLL B cells. Inhibiting the CCL21/CCR7 interaction by blocking antibodies or using therapeutic ibrutinib altered the adhesion of leukemic cells. Our results indicate NLCs as providers of an alternative source of CCL21, taking over the physiological task of follicular reticular cells, whose network is deeply altered in CLL lymph nodes. By retaining malignant B cells, CCL21 provides a protective environment for their niching and survival, thus allowing tumor evasion and resistance to treatment. These findings argue for a specific targeting or reeducation of NLCs as a new immunotherapy strategy for this disease.

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Regulatory interplay between Vav1, Syk and ß-catenin occurs in lung cancer cells.

Vav1 exhibits two signal transducing properties as an adaptor protein and a regulator of cytoskeleton organization through its Guanine nucleotide Exchange Factor module. Although the expression of Vav1 is restricted to the hematopoietic lineage, its ectopic expression has been unraveled in a number of solid tumors. In this study, we show that in lung cancer cells, as such in hematopoietic cells, Vav1 interacts with the Spleen Tyrosine Kinase, Syk. Likewise, Syk interacts with ß-catenin and, together with Vav1, regulates the phosphorylation status of ß-catenin. Depletion of Vav1, Syk or ß-catenin inhibits Rac1 activity and decreases cell migration suggesting the interplay of the three effectors to a common signaling pathway. This model is further supported by the finding that in turn, ß-catenin regulates the transcription of Syk gene expression. This study highlights the elaborated connection between Vav1, Syk and ß-catenin and the contribution of the trio to cell migration.

The adaptor protein APS modulates BCR signalling in mature B cells.

Activation process of mature B cell is predominantly driven by specific BCR-mediated pathways, switched on and off all through late B cell differentiation stages. Mice deficient for APS, a member of the Lnk/SH2B family of adaptor proteins, showed that this adaptor plays a BCR-mediated regulatory role in mature B cells. However, the intermediates involved in this adaptor modulating functions in B cells are still unknown. In the present study, we investigated the role of APS in regulating BCR signalling notably through cytoskeleton remodeling in mature B cells. Herein, we showed that APS function is stage specific, as it exclusively intervenes in mature B cells. Upon activation, APS colocalizes with the BCR and associates with important regulators of BCR signalling, such as Syk and Cbl kinase. Importantly, APS interferes, as a scaffold protein, with the stability of Syk kinase by recruiting Cbl. This function is mainly mediated by APS SH2 domain, which regulates BCR-evoked cell dynamics. Our findings thus reveal that APS plays a regulatory role in BCR-induced responses by specifically modulating its interacting partners, which positions APS as a relevant modulator of BCR signalling in mature B cells.

Stabilization of ß-catenin upon B-cell receptor signaling promotes NF-kB target genes transcription in mantle cell lymphoma.

B-cell receptor (BCR) signaling pathways and interactions with the tumor microenvironment account for mantle cell lymphoma (MCL) cells survival in lymphoid organs. In several MCL cases, the WNT/ß-catenin canonical pathway is activated and ß-catenin accumulates into the nucleus. As both BCR and ß-catenin are important mediators of cell survival and interaction with the microenvironment, we investigated the crosstalk between BCR and WNT/ß-catenin signaling and analyzed their impact on cellular homeostasis as well as their targeting by specific inhibitors. ß-catenin was detected in all leukemic MCL samples and its level of expression rapidly increased upon BCR stimulation. This stabilization was hampered by the BCR-pathway inhibitor Ibrutinib, supporting ß-catenin as an effector of the BCR signaling. In parallel, MCL cells as compared with normal B cells expressed elevated levels of WNT16, a NF-κB target gene. Its expression increased further upon BCR stimulation to participate to the stabilization of ß-catenin. Upon BCR stimulation, ß-catenin translocated into the nucleus but did not induce a Wnt-like transcriptional response, i.e., TCF/LEF dependent. ß-catenin rather participated to the regulation of NF-κB transcriptional targets, such as IL6, IL8, and IL1. Oligo pull down and chromatin immunoprecipitation experiments demonstrated that ß-catenin is part of a protein complex that binds the NF-κB DNA consensus sequence, strengthening the idea of an association between the two proteins. An inhibitor targeting ß-catenin transcriptional interactions hindered both NF-κB DNA recruitment and induced primary MCL cells apoptosis. Thus, ß-catenin likely represents another player through which BCR signaling impacts on MCL cell survival.

Mesenchymal stem cells reduce hypoxia-induced apoptosis in alveolar epithelial cells by modulating HIF and ROS hypoxic signaling.

Distal lung diseases, such as pulmonary fibrosis or acute lung injury, are commonly associated with local alveolar hypoxia that may be deleterious through the stimulation of alveolar epithelial cell (AEC) apoptosis. In various murine models of alveolar injury, administration of allogenic human mesenchymal stem cells (hMSCs) exerts an overall protective paracrine effect, limiting lung inflammation and fibrosis. However, the precise mechanisms on lung cells themselves remain poorly understood. Here, we investigated whether hMSC-conditioned medium (hMSC-CM) would protect AECs from hypoxia-induced apoptosis and explored the mechanisms involved in this cytoprotective effect. Exposure of rat primary AECs to hypoxia (1.5% O2 for 24 h) resulted in hypoxia-inducible factor (HIF)-1α protein stabilization, partly dependent on reactive oxygen species (ROS) accumulation, and in a twofold increase in AEC apoptosis that was prevented by the HIF inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole and the antioxidant drug N-acetyl cysteine. Incubation of AECs with hMSC-CM significantly reduced hypoxia-induced apoptosis. hMSC-CM decreased HIF-1α protein expression, as well as ROS accumulation through an increase in antioxidant enzyme activities. Expression of Bnip3 and CHOP, two proapoptotic targets of HIF-1α and ROS pathways, respectively, was suppressed by hMSC-CM, while Bcl-2 expression was restored. The paracrine protective effect of hMSC was partly dependent on keratinocyte growth factor and hepatocyte growth factor secretion, preventing ROS and HIF-1α accumulation.

Mutation of Vav1 adaptor region reveals a new oncogenic activation.

Vav family members function as remarkable scaffold proteins that exhibit both GDP/GTP exchange activity for Rho/Rac GTPases and numerous protein-protein interactions via three adaptor Src-homology domains. The exchange activity is under the unique regulation by phosphorylation of tyrosine residues hidden by intra-molecular interactions. Deletion of the autoinhibitory N-terminal region results in an oncogenic protein, onco-Vav, leading to a potent activation of Rac GTPases whereas the proto-oncogene barely leads to transformation. Substitution of conserved residues of the SH2-SH3 adaptor region in onco-Vav reverses oncogenicity. While a unique substitution D797N did not affect transformation induced by onco-Vav, we demonstrate that this single substitution leads to transformation in the Vav1 proto-oncogene highlighting the pivotal role of the adaptor region. Moreover, we identified the cell junction protein ß-catenin as a new Vav1 interacting partner. We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of ß-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts. In addition, a similar interaction is evidenced in epithelial lung cancer cells expressing ectopically Vav1. In these cells, Vav1 is also involved in the modulation of ß-catenin phosphorylation. Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity.

Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells.

Stem cell factor (SCF) plays critical roles in proliferation, survival, migration, and function of hematopoietic progenitor and mast cells through binding to Kit receptor. Previous studies have implicated the adaptor protein Lnk as an important negative regulator of SCF signaling. However, the molecular mechanism underlying this regulation is unclear. Here, we showed that the Src homology 2 domain (SH2) of Lnk binds directly and preferentially to phosphorylated tyrosine 567 in Kit juxtamembrane domain. Using Lnk(-/-) bone marrow mast cells (BMMCs) transduced with different Lnk proteins, we demonstrated that Lnk down-regulates SCF-induced proliferation with attenuation of mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase signaling. Furthermore, we showed that Lnk(-/-) BMMCs displayed increased SCF-dependent migration compared with wild-type cells, revealing a novel Lnk-mediated inhibitory function. This correlated with enhanced Rac and p38 MAPK activation. Finally, we found that Lnk domains and carboxy-terminal tyrosine contribute differently to inhibition of in vitro expansion of hematopoietic progenitors. Altogether, our results demonstrate that Lnk, through its binding to Kit tyrosine 567, negatively modulates specific SCF-dependent signaling pathways involved in the proliferation and migration of primary hematopoietic cells.

A dual role of IFN-alpha in the balance between proliferation and death of human CD4+ T lymphocytes during primary response.

Type I IFNs (IFN-alphabeta) enhance immune responses, notably T cell-mediated responses, in part by promoting the functional activities of dendritic cells. In this study, we analyzed the direct impact of IFN-alpha on proliferative and apoptotic signals upon in vitro activation of human naive CD4+ T lymphocytes. We demonstrate that IFN-alpha protects T cells from the intrinsic mitochondrial-dependent apoptosis early upon TCR/CD28 activation. IFN-alpha acts by delaying entry of cells into the G1 phase of the cell cycle, as well as by increasing Bcl-2 and limiting Bax activation. Later, upon activation, T cells that were exposed to IFN-alpha showed increased levels of surface Fas associated with partially processed caspase-8, a key component of the extrinsic apoptotic pathway. Caspase-8 processing was augmented furthermore by Fas ligation. Overall, these findings support a model whereby IFN-alpha favors an enhanced clonal expansion, yet it sensitizes cells to the Ag-induced cell death occurring at the end of an immune response. These observations point to a complex role of type I IFN in regulating the magnitude of proliferation and survival of naive CD4+ T cells during primary response and underline how crucial could be the timing of exposure to this cytokine.

The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression.

The four mammalian Jak tyrosine kinases are non-covalently associated with cell surface receptors binding helical bundled cytokines. In the type I interferon receptor, Tyk2 associates with the IFNAR1 receptor subunit and positively influences ligand binding to the receptor complex. Here, we report that Tyk2 is essential for stable cell surface expression of IFNAR1. In the absence of Tyk2, mature IFNAR1 is weakly expressed on the cell surface. Rather, it is localized into a perinuclear endosomal compartment which overlaps with that of recycling transferrin receptors and with early endosomal antigen-1 (EEA1) positive vesicles. Conversely, co-expressed Tyk2 greatly enhances surface IFNAR1 expression. Importantly, we demonstrate that Tyk2 slows down IFNAR1 degradation and that this is due, at least in part, to inhibition of IFNAR1 endocytosis. In addition, Tyk2 induces plasma membrane relocalization of the R2 subunit of the interleukin-10 receptor. These results reveal a novel function of a Jak protein on internalization of a correctly processed cytokine receptor. This function is distinct from the previously reported effect of other Jak proteins on receptor exit from the endoplasmic reticulum.

Down-modulation of responses to type I IFN upon T cell activation.

The immunomodulatory role of type I IFNs (IFN-alpha/beta) in shaping T cell responses has been demonstrated, but the direct effects of IFN on T cells are still poorly characterized. Particularly, because IFN exert an antiproliferative activity, it remains elusive how the clonal expansion of effector T cells can paradoxically occur in the event of an infection when large amounts of IFN are produced. To address this issue, we have studied the effects of type I IFN in an in vitro differentiation model of human primary CD4(+) T cells. We found that IFN-alpha treatment of resting naive T cells delayed their entry into the cell cycle after TCR triggering. Conversely, the ongoing expansion of effector T cells was not inhibited by the presence of IFN. Moreover, activated T cells showed a significantly reduced induction of IFN-sensitive genes, as compared with naive precursors, and this decline occurred independently of subset-specific polarization. The residual type I IFN response measured in activated T cells was found sufficient to inhibit replication of the vesicular stomatitis virus. Our data suggest that the activation of T lymphocytes includes regulatory processes that restrain the transcriptional response to IFN and allow the proliferation of effector cells in the presence of this cytokine.

Selective expression of type I IFN genes in human dendritic cells infected with Mycobacterium tuberculosis.

Type I IFN regulates different aspects of the immune response, inducing a cell-mediated immunity. We have recently shown that the infection of dendritic cells (DC) with Mycobacterium tuberculosis (Mtb) induces IFN-alpha. In this work we have monitored a rapid induction of IFN-beta followed by the delayed production of the IFN-alpha1 and/or -alpha13 subtypes. The Mtb infection rapidly activates the NF-kappaB complex and stimulates the phosphorylation of IFN regulatory factor (IRF)-3, events known to induce IFN-beta expression in viral infection. In turn, the autocrine production of IFN-beta induces the IFN-stimulated genes that contain binding sites for activated STATs in their promoters. Among the IFN-stimulated genes induced in DC through STAT activation are IRF-1 and IRF-7. The expression of IRF-1 appears to be dependent on the sequential activation of NF-kappaB and STAT-1. Once expressed, IRF-1 may further stimulate the transcription of IFN-beta. Induction of IRF-7 is also regulated at the transcriptional level through the binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears to be required for the delayed induction of the IFN-alpha1/13 genes. Although correlative, our results strongly support the existence of a cascade of molecular events in Mtb-infected DC. Upon infection, constitutively expressed NF-kappaB and IRF-3 are activated and likely contribute to the rapid IFN-beta expression. In turn, IFN-beta-induced IRF-1 and IRF-7 may cooperate toward induction of IFN-alpha1/13 if infection persists and these factors are activated.

Do reduced levels of steroid 21-hydroxylase confer a survival advantage in fetuses affected by sex chromosome aberrations?

We investigated whether molecular defects in the CYP21 gene were detectable in two common sex chromosome aberrations, the Turner and the Klinefelter syndromes. We found abnormal 17-hydroxyprogesterone levels after adrenal stimulation in 26/60 (43.3%) patients affected by these chromosome aberrations, as compared with only 11/68 (16.2%) normal controls (P=0.0014, odds ratio 4.0). Screening of the CYP21 gene identified a single Val281Leu missense mutation in exon 7 in 9/63 (14.3%) of the patients, all nine of whom were heterozygote carriers; the mutation frequency was significantly higher than in the general population (P=0.007, odds ratio=3.5). The hormonal and molecular data indicate that these common sex chromosome aberrations are associated with a remarkably high frequency of steroidogenic defects. It may be hypothesised that reduced levels of steroid 21-hydroxylase could confer a survival advantage, leading to a successful pregnancy.