Proteomic and phospholipidomic characterization of extracellular vesicles inducing tumor microenvironment in Epstein-Barr virus-associated lymphomas.

Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLß2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLß2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Spatiotemporal basis of CTLA-4 costimulatory molecule-mediated negative regulation of T cell activation.

T cell activation is positively and negatively regulated by a pair of costimulatory receptors, CD28 and CTLA-4, respectively. Because these receptors share common ligands, CD80 and CD86, the expression and behavior of CTLA-4 is critical for T cell costimulation regulation. However, in vivo blocking of CD28-mediated costimulation by CTLA-4 and its mechanisms still remain elusive. Here, we demonstrate the dynamic behavior of CTLA-4 in its real-time competition with CD28 at the central-supramolecular activation cluster (cSMAC), resulting in the dislocalization of protein kinase C-θ and CARMA1 scaffolding protein. CTLA-4 translocation to the T cell receptor microclusters and the cSMAC is tightly regulated by its ectodomain size, and its accumulation at the cSMAC is required for its inhibitory function. The CTLA-4-mediated suppression was demonstrated by the in vitro anergy induction in regulatory T cells constitutively expressing CTLA-4. These results show the dynamic mechanism of CTLA-4-mediated T cell suppression at the cSMAC.

Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation.

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.

Novel functional small RNAs are selectively loaded onto mammalian Ago1.

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1-4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein-Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1-4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

Overexpression of miR-669m inhibits erythroblast differentiation.

MicroRNAs (miRNAs), one of small non-coding RNAs, regulate many cell functions through their post-transcriptionally downregulation of target genes. Accumulated studies have revealed that miRNAs are involved in hematopoiesis. In the present study, we investigated effects of miR-669m overexpression on hematopoiesis in mouse in vivo, and found that erythroid differentiation was inhibited by the overexpression. Our bioinformatic analyses showed that candidate targets of miR-669m which are involved in the erythropoiesis inhibition are A-kinase anchoring protein 7 (Akap7) and X-linked Kx blood group (Xk) genes. These two genes were predicted as targets of miR-669m by two different in silico methods and were upregulated in late erythroblasts in a public RNA-seq data, which was confirmed with qPCR. Further, miR-669m suppressed luciferase reporters for 3' untranslated regions of Akap7 and Xk genes, which supports these genes are direct targets of miR-669m. Physiologically, miR-669m was not expressed in the erythroblast. In conclusion, using miR-669m, we found Akap7 and Xk, which may be involved in erythroid differentiation, implying that manipulating these genes could be a therapeutic way for diseases associated with erythropoiesis dysfunction.

Dasatinib exacerbates splenomegaly of mice inoculated with Epstein-Barr virus-infected lymphoblastoid cell lines.

Latent infection of Epstein-Barr virus (EBV) is associated with a poor prognosis in patients with B cell malignancy. We examined whether dasatinib, a multi kinase inhibitor, which is broadly used for chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia is effective on EBV-positive B cell malignancies, using lymphoblastoid cell lines (LCLs) in vitro and in vivo. As a result, in vitro experiments showed that dasatinib induced cell death of the EBV-LCLs which was not accompanied with a lytic reactivation of EBVs. To evaluate the effectiveness in EBV latency type III represented by immunodeficiency lymphoma, LCL-inoculated immunodeficient NOD/shi-scid/Il2rgnul (NOG) mice were treated with dasatinib. However, in vivo experiments revealed that dasatinib treatment exacerbated tumor cell infiltration into the spleen of LCL-inoculated NOG mice, whereas tumor size at the inoculated site was not affected by the treatment. These results suggest that dasatinib exacerbates the pathogenesis at least in some situations although the drug is effective in vitro. Hence, we should carefully examine a possibility of dasatinib repositioning for EBV+ B cell malignancies.

Lck couples Shc to TCR signaling.

Recent genetic evidence demonstrated that Shc is a critical molecule for T cell activation and differentiation. However, how Shc is coupled to the T cell antigen receptor (TCR) has not been clearly characterized. Here we report that the tyrosine kinase Lck functions as a connecting molecule for TCR and Shc. Lck plays a critical role in TCR signal transduction by phosphorylating the immuno-receptor tyrosine based activation motif (ITAM). Our data shows that the PTB domain of Shc binds the SH2/3 domains of Lck in a phosphotyrosine-independent manner. Inhibition of the Lck/Shc interaction led to the loss of IL-2 promoter activation, confirming that the role of Shc in IL-2 production requires its interaction with Lck. Together, the data show that Shc is connected to the activated TCR via direct interaction with Lck.

Laminar patterning in the developing neocortex by temporally coordinated fibroblast growth factor signaling.

Laminar organization, a fundamental neural architecture in the CNS, is a prominent feature of the neocortex, where the cortical neurons in spatially distinct layers are generated from the common progenitors in a temporally distinct manner during development. Despite many advances in the characterization of the molecular mechanisms of the radial migration of cortical neurons, the way in which the early-late temporal sequence of cortical neuron generation is linked with the deep-superficial spatial sequence of cell body positioning remains obscure. Using in vivo electroporation-mediated gene transfer, we show here that the activities mediated by fibroblast growth factor receptors (FGFRs) in cortical progenitors are critical for conferring proper migratory properties on nascent neuronal progeny. Furthermore, we provide supportive evidence that Pea3 subfamily members of Ets (Pea3-Ets) transcription factors mediate the activities of FGFR at the mid to late phase of neocortical development. In addition, using FGF18 knock-out mice, we demonstrate that FGF18 expressed by early-generated cortical neurons in the cortical plate is critical for the expression of Pea3-Ets transcription factors and that FGF18 is sufficient to induce their expressions. Our results thus imply that a feedback mechanism mediated by FGF signaling is involved in setting up the proper laminar positioning of cortical neurons; FGF18 derived from early-generated cortical neurons acts on the cortical progenitors expressing FGFRs and induces the expression of Pea3-Ets transcription factors that, in turn, confer proper migratory behaviors on nascent cortical progeny during the mid to late stages of neocortical development.

Generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles.

An early and fundamental step of the laminar organization of developing neocortex is controlled by the developmental programs that critically depend on the activities of reelin-positive cells in the marginal zone. However, the ontogeny of reelin-positive cells remained elusive. To gain insights into the spatial and temporal regulation of reelin-positive marginal zone cell development, we used a transgenic mouse line in which we defined the green fluorescent protein (GFP) transgene as a novel reliable molecular marker of reelin-positive marginal zone cells from the early stages of their development. We further used exo utero electroporation-mediated gene transfer that allows us to mark progenitor cells and monitor the descendants in the telencephalon in vivo. We show here the generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles, including the cortical hem, where the prominent expression of GFP is initially detected. These neurons tangentially migrate at the cortical marginal zone and are distributed throughout the entire neocortex in a caudomedial-high to rostrolateral-low gradient during the dynamic developmental period of corticogenesis. Therefore, our findings on reelin-positive marginal zone cells, in addition to the cortical interneurons, add to the emerging view that the neocortex consists of neuronal subtypes that originate from a focal source extrinsic to the neocortex, migrate tangentially into the neocortex, and thereby underlie neural organization of the neocortex.

Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2.

Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1-mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1-TCR colocalization within microclusters is required for efficient PD-1-mediated suppression. This inhibitory mechanism also functions in PD-1(hi) T cells generated in vivo and can be overridden by a neutralizing anti-PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.

Distinct ontogenic and regional expressions of newly identified Cajal-Retzius cell-specific genes during neocorticogenesis.

Cajal-Retzius (CR) cells are early-generated transient neurons and are important in the regulation of cortical neuronal migration and cortical laminar formation. Molecular entities characterizing the CR cell identity, however, remain largely elusive. We purified mouse cortical CR cells expressing GFP to homogeneity by fluorescence-activated cell sorting and examined a genome-wide expression profile of cortical CR cells at embryonic and postnatal periods. We identified 49 genes that exceeded hybridization signals by >10-fold in CR cells compared with non-CR cells at embryonic day 13.5, postnatal day 2, or both. Among these CR cell-specific genes, 25 genes, including the CR cell marker genes such as the reelin and calretinin genes, are selectively and highly expressed in both embryonic and postnatal CR cells. These genes, which encode generic properties of CR cell specificity, are eminently characterized as modulatory composites of voltage-dependent calcium channels and sets of functionally related cellular components involved in cell migration, adhesion, and neurite extension. Five genes are highly expressed in CR cells at the early embryonic period and are rapidly down-regulated thereafter. Furthermore, some of these genes have been shown to mark two distinctly different focal regions corresponding to the CR cell origins. At the late prenatal and postnatal periods, 19 genes are selectively up-regulated in CR cells. These genes include functional molecules implicated in synaptic transmission and modulation. CR cells thus strikingly change their cellular phenotypes during cortical development and play a pivotal role in both corticogenesis and cortical circuit maturation.

Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression.

Shc, a prototypic adapter molecule, has been implicated in T cell receptor (TCR) signal transduction, but its role has not been identified clearly. Here we report that Shc is essential for TCR-induced IL-2 production but is dispensable for CD69 or CD25 expression. Engagement of TCR in mutant Jurkat T cells lacking Shc fails to produce IL-2 because of impaired mitogen-activated protein kinase activation. Activation of c-Rel, a transcription factor essential for IL-2 expression, was impaired also. In contrast, activation of nuclear factor of activated T cell and expression of CD69/CD25 were comparable between the mutant and wild-type Jurkat cells. These defects were rescued by expression of exogenous Shc. Activation of c-Rel using the estrogen receptor fusion protein restored the activation of the IL-2 promoter in an estrogen-dependent manner. These results show that Shc plays an essential role in the TCR-induced activation of c-Rel and the IL-2 promoter.