It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (+o120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

The transmembrane protein LAT (linker for activation of T cells) couples the T cell receptor (TCR) to downstream signaling effectors. Mice homozygous for a mutation of a single LAT tyrosine residue showed impeded T cell development. However, later they accumulated polyclonal helper T (TH) cells that chronically produced type 2 cytokines in large amounts. This exaggerated TH2 differentiation caused tissue eosinophilia and massive maturation of plasma cells secreting to immunoglobulins of the E and G1 isotypes. This paradoxical phenotype establishes an unanticipated inhibitory function for LAT that is critical for the differentiation and homeostasis of TH cells.

P.Drevot and C.Langlet contributed equally to this workRecent studies suggest that rafts are involved in numerous cell functions, including membrane traf®c and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent-insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37°C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of~70 nm. These ®ndings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR±CD3 complex, Lck and ZAP-70 kinases, and CD4 co-receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for ef®cient TCR signal initiation upon stimulation. Keywords: lipid raft/membrane domain/signal transduction/T cell receptor IntroductionMembrane rafts are found in all mammalian cell types as well as in Drosophila, Dictyostelium and yeast (Simons and Ikonen, 1997;Brown and London, 1998;Simons and Toomre, 2000). Not only are rafts enriched in sphingolipids (sphingomyelins and glycosphingolipids) and cholesterol, but these constituents are essential for the formation of rafts (Simons and Ikonen, 1997;Brown and London, 2000). An increasing amount of data suggest that rafts play fundamental roles in diverse cellular functions, particularly in signal transduction, by promoting a segregated arrangement of membrane proteins and lipids (Brown and London, 2000;Simons and Toomre, 2000).Studies in model membranes indicate that rafts correspond to a phase of the lipid bilayer, namely the liquidordered (lo) phase (Brown and London, 2000;Simons and Toomre, 2000). The formation of this lo phase is promoted by sphingolipids, the long saturated acyl chains of which allow tight molecular packing (Brown and London, 2000), and further facilitated by the presence of cholesterol (Simons and Ikonen, 1997;Brown and London, 2000). The use of GPI-anchored proteins and other raft markers to investigate the existence of rafts in living cells has revealed that they are usually very small in size (Simons and Toomre, 2000).Engagement of the T cell receptor (TCR) by its speci®c peptide-MHC (pMHC) ligand triggers intracellular signaling cascades that are required for T lymphocyte development and functions (for a review see Weiss and Littman, 1994). Such cascades are initiated by the activation of a signal transduction machinery, the main components of which include the TCRab heterodimer and the tightly associated CD3 e, g, d and z polypeptides, Lck and ZAP-70...

Objective: Natural killer (NK) cells play a critical role in the innate antitumor immune response. Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC). However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure. Methods:The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR. Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting. The role of circUHRF1 in NK cell dysfunction was assessed by ELISA. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells. In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues.Results: Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC. In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion. A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration. Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p. Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients.Conclusions: Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC. CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC.

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

Population genetic structure and approximate Bayesian computation analyses reveal the southern origin and northward dispersal of the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae) in its native range AbstractThe oriental fruit moth (OFM) Grapholita molesta is one of the most destructive orchard pests. Assumed to be native to China, the moth is now distributed throughout the world. However, the evolutionary history of this moth in its native range remains unknown. In this study, we explored the population genetic structure, dispersal routes and demographic history of the OFM in China and South Korea based on mitochondrial genes and microsatellite loci. The Mantel test indicated a significant correlation between genetic distance and geographical distance in the populations. Bayesian analysis of population genetic structure (BAPS) identified four nested clusters, while the GENELAND analysis inferred five genetic groups with spatial discontinuities. Based on the approximate Bayesian computation approach, we found that the OFM was originated from southern China near the Shilin area of Yunnan Province. The early divergence and dispersal of this moth was dated to the Penultimate glaciation of Pleistocene. Further dispersal from southern to northern region of China occurred before the last glacial maximum, while the expansion of population size in the derived populations in northern region of China occurred after the last glacial maximum. Our results indicated that the current distribution and structure of the OFM were complicatedly influenced by climatic and geological events and human activities of cultivation and wide dissemination of peach in ancient China. We provide an example on revealing the origin and dispersal history of an agricultural pest insect in its native range as well as the underlying factors.

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.

Purpose: CD19-specific chimeric antigen receptor (CAR) T-cell therapy is effective against refractory or relapsed (R/R) B-cell lymphoma, but the efficacy is hindered by the existence of PD-1/PD-L1 pathway. Patients and Methods: Here, we generated a novel anti-CD19 CAR-expressing PD-1/CD28 chimeric switch-receptor (CD19-PD-1/CD28-CAR). We then conducted a phase Ib study to evaluate safety and efficacy of CD19-PD-1/CD28-CAR T cells in the treatment of PD-L1+ B-cell lymphoma. Results: We found that CD19-PD-1/CD28-CAR T cells had superior T-cell proliferation, cytokine production, and sequentially capability of killing PD-L1+ B-cell lymphoma cells in vitro and in vivo relative to the prototype, CD19-CAR T cells. Among 17 adult patients with R/R lymphoma who received the CAR T therapy, 10 patients had objective response (58.8%), including seven patients with complete remission (41.2%). At a median follow-up 15 months, median overall survival for all patients was not reached. Remarkably, no severe neurologic toxicity or cytokine release syndrome was observed. Conclusions: This first-in-human study demonstrates the tolerability, safety, and encouraging efficacy of CD19-PD-1/CD28-CART in PD-L1+ large B-cell lymphoma.