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Lymphocytes can be functionally partitioned into subsets belonging to the innate or adaptive arms of the immune system. Subsets of innate and innate-like lymphocytes may or may not express Ag-specific receptors of the adaptive immune system, yet they are poised to respond with innate-like speed to pathogenic insults but lack the capacity to develop classical immunological memory. These lymphocyte subsets display a number of common properties that permit them to integrate danger and stress signals dispatched by innate sensor cells to facilitate the generation of specialized effector immune responses tailored toward specific pathogens or other insults. In this review, we discuss the functions of distinct subsets of innate and innate-like lymphocytes. A better understanding of the mechanisms by which these cells are activated in different contexts, their interactions with other immune cells, and their role in health and disease may inform the development of new or improved immunotherapies.

Supplemental Figure 1. Densitometry analysis of p-PLCγ1and p-EphA2 inhibition by ALWand human lung TMA analysis. Supplemental Figure 2. sgPLCG1 mismatch detection.

Supplemental Figure S2: Inhibiting Slit activity rescues VEGF-induced vascular assembly and migration in EphA2 knockdown in human endothelial cells.

Supplemental Figure Legends

Oligonucleotides used in study

mLST8-mTOR uncoupling mutations inhibit PTEN-null tumor growth

mTOR is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. Here we assessed mLST8 loss in a panel of normal and cancer cells and observed little to no impact on assembly or activity of mTORC1. However, mLST8 loss blocked mTOR association with mTORC2 cofactors RICTOR and SIN1, thus abrogating mTORC2 activity. Similarly, a single pair of mutations on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly and activity without affecting mTORC1. We also discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor SIN1. In PTEN-null prostate cancer xenografts, mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation and decreased tumor cell proliferation and tumor growth in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation that could enable therapeutic mTORC2-selective inhibition as a therapeutic strategy.Significance:These findings show that mLST8 functions as a scaffold to maintain mTORC2 integrity and kinase activity, unveiling a new avenue for development of mTORC2-specific inhibitors.

Supplementary Materials and Methods

PDF file - 612K, Knockdown of EPHA2 inhibits cell viability and tumor cell migration in NSCLC cells (S1); Effects of EPHA2 on JNK and c-JUN expression and activities in NSCLCs (S2); SP600125 inhibits phosphorylation of JNK and c-JUN and cell viability (S3); Knockdown of EPHA2 inhibits tumor formation in vivo (S4); Knockdown of EPHA2 in H1650 and H23 cells decreases ALDH-positive population (S5); Sorted ALDH-positive cells are enriched with cancer stem-like cells (S6).

Legends for Supplemental Figures S1-S6

Description of additional methods and procedures used in the study.

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non–small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here, we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a c-JUN-NH2-kinase (JNK) inhibitor, recapitulated defects in EPHA2-deficient tumor cells, whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem–like cells (CSC). RNA interference-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH+ cancer stem–like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH+ populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung CSCs. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in CSC function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. Cancer Res; 74(9); 2444–54. ©2014 AACR.

Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFRT790M mutations in vitro and inhibited tumor growth and progression in an inducible EGFRL858R+T790M-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors. Cancer Res; 76(2); 305–18. ©2016 AACR.

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Localization of EPHA2 in erlotinib resistant cells (S1); Validation of EPHA2 antibody specificity in patient tumor specimens (S2); EPHA2 kinase activity is required in maintaining cell viability of erlotinib resistant lung cancer (S3); Biochemical analysis of TKI-­�resistant tumors from EGFRL858R+T790MEphA2-­�/-­� and control mice (S4); Signaling analysis in EPHA2 knockdown cells by siRNA-­�mediated gene silencing (S5); Localization of EPHA2 upon ligand or EPHA2 inhibitor treatment (S6).

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired “browning” of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

The development of a broad repertoire of T cells in the immune system requires interaction of T cell receptors expressed by immature T cells with peptide/major histocompatibility complexes (MHCs) displayed by specialized epithelial cells in the thymus, in a process called T cell positive selection. Thymic epithelial cells (TECs) display unique antigen processing machinery which shapes the collection of self-peptides that drive positive selection. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to the generation of positively selecting peptides in TECs. We found that TEC-specific PIK3C3/VPS34 facilitates the positive selection of CD4 but not CD8 T lineage cells, in a mechanism independent of its role in canonical macroautophagy/autophagy. Instead, we propose that PIK3C3/VPS34 alters vesicle trafficking in TECs that modulates lysosomal protease activity which, in turn, controls the generation of MHC class II-presented peptides optimized for positive selection of CD4 T cells.

In advanced charged device model (CDM) protection engineering, it is necessary to provide dedicated dual-directional electrostatic discharge (ESD) protection between input/output (I/O) and ground (GND) to discharge the large amount of charge stored in the silicon substrate efficiently. This letter presents two improved bidirectional and low-voltage silicon-controlled rectifiers (BLVSCR-type1 and BLVSCR-type2), which are composed of two diode-triggered SCRs of opposite polarity in parallel. By improving the device structure and metal connection, the BLVSCR-type1 and BLVSCR-type2 can offer robust ESD capabilities. Compared with the conventional bidirectional direct-connected SCR (BDCSCR), experimental results show that the proposed BLVSCR-type1 can render a three order of magnitude reduction in the leakage current at a 1.2V I/O port and more stable parasitic capacitance characteristic. Moreover, the BLVSCR-type2 can possess a compact layout area reduced by as much as 35% when comparing to BLVSCR-type1.

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex plays a role in both canonical and noncanonical autophagy, key processes that control immune-cell responsiveness to a variety of stimuli. Our previous studies found that PIK3C3 is a critical regulator that controls the development, homeostasis, and function of dendritic and T cells. In this study, we investigated the role of PIK3C3 in myeloid cell biology using myeloid cell-specific Pik3c3-deficient mice. We found that Pik3c3-deficient macrophages express increased surface levels of major histocompatibility complex (MHC) class I and class II molecules. In addition, myeloid cell-specific Pik3c3 ablation in mice caused a partial impairment in the homeostatic maintenance of macrophages expressing the apoptotic cell uptake receptor TIM-4. Pik3c3 deficiency caused phenotypic changes in myeloid cells that were dependent on the early machinery (initiation/nucleation) of the classical autophagy pathway. Consequently, myeloid cell-specific Pik3c3-deficient animals showed significantly reduced severity of experimental autoimmune encephalomyelitis (EAE), a primarily CD4(+) T-cell-mediated mouse model of multiple sclerosis (MS). This disease protection was associated with reduced accumulation of myelin-specific CD4(+) T cells in the central nervous system and decreased myeloid cell IL-1β production. Further, administration of SAR405, a selective PIK3C3 inhibitor, delayed disease progression. Collectively, our studies establish PIK3C3 as an important regulator of macrophage functions and myeloid cell-mediated regulation of EAE. Our findings also have important implications for the development of small-molecule inhibitors of PIK3C3 as therapeutic modulators of MS and other autoimmune diseases.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key early player in macroautophagy/autophagy. In this study, we assessed the contribution of PIK3C3 to T cell metabolism and function. We found that Pik3c3-deficient T cells exhibited impaired cellular metabolism, and Pik3c3-deficient CD4+ T cells failed to differentiate into T helper 1 cells. These alterations were associated with reduced levels of active mitochondria upon T cell activation. In addition, conditional Pik3c3-deficient animals failed to mount autoreactive T cell responses and were resistant to experimental autoimmune encephalomyelitis (EAE). Interestingly, the deletion of Pik3c3 had little effect on the capacity of animals to clear tumor metastases. Collectively, our studies have revealed a critical role of PIK3C3 in T cell metabolism and the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat multiple sclerosis and other inflammatory diseases by targeting PIK3C3.Abbreviations: CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; ECAR: extracellular acidification rate; iNKT: invariant natural killer T; LAP: LC3-associated phagocytosis; LLC: Lewis lung carcinoma; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDSC: myeloid-derived suppressor cell; MOG: myelin oligodendrocyte glycoprotein; NK: natural killer; OCR: oxygen consumption rate; PI: propidium iodide; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RNA-seq: RNA-sequencing; TCR: T cell receptor; TMRE: tetramethylrhodamine ethyl ester perchlorate.

In this brief, two novel diode-triggered silicon-controlled rectifiers (DTSCRs) with fast turn-on speed have been presented. By embedding current gain amplifier modules (i.e., Sziklai pair and Darlington pair) into the conventional DTSCR, the current gain of parasitic bipolar junction transistors (BJTs) on the silicon-controlled rectifier (SCR) path in the new devices becomes much larger than that of the conventional counterpart, thus resulting in a faster turn-on speed and better quasi-static ${I}$ – ${V}$ characteristics. As such, the quasi-static triggering characteristic of the new DTSCR improves by 80%, and the turn-on speed improves by 42%–93% for different current levels.

An improved lateral double-diffused MOS silicon-controlled rectifier (ILDMOS-SCR) for high-voltage electrostatic discharge (ESD) protection applications has been proposed and verified in a 0.18 µm 5 V/24 V BCD process. The proposed improved lateral double-diffused MOS silicon-controlled rectifier (LDMOS-SCR) is constructed by adding a gated P-type/intrinsic/N-type (PIN) diode into a conventional LDMOS-SCR. With part of the ESD current discharges from the surface gated PIN diode path, the proposed ILDMOS-SCR achieves a high holding voltage of 30 V as well as a high failure current of 10.04 A, which is evaluated to pass 15 KV Human Body Model (HBM) ESD stress.

In this article, an improved silicon-controlled rectifier (SCR) for low-voltage (LV) electrostatic discharge (ESD) applications has been presented. By employing an N+/P-ESD diode and optimizing the SCR layout, both the triggering and current-discharging paths of the new SCR become much shorter than the prior arts, thus generating better clamping ability, faster turn-on speed, and lower overshoot voltage. As a result, the human body model (HBM) robustness increases by 19%, 46%, and 267% for the proposed device with one, two, and three stacking units, respectively, and the charged-device model (CDM) robustness and turn-on speed of the improved device increase by 80% and 27%, respectively, over its conventional counterpart. Furthermore, the contradiction between quasi-static triggering characteristics and transient overshoot voltage frequent in current-assisted triggering of ESD devices has been investigated, where the common method to optimize the trigger voltage by increasing the resistance of triggering path will deteriorate the overshoot characteristics hugely. To make the ESD devices satisfy the emerging higher CDM protection requirement, a tradeoff between the quasi-static trigger voltage and the transient overshoot voltage should be made elaborately.

Gate-grounded n-channel MOSFET (GGNMOS) has been widely used in electrostatic discharge (ESD) protection applications. In this letter, an enhanced GGNMOS, called the EGGNMOS, is proposed and demonstrated. The new device has the same topology as the conventional GGNMOS, except that a few P+ regions are being added to the N+ guard ring (NGR). This novel approach enables the formation of parasitic silicon-controlled rectifiers (SCR’s), thus, resulting in a much higher current handling capability in the EGGNMOS than the GGNMOS under the same silicon footprint. Our experimental results indicate that the proposed EGGNMOS, with an ESD design window suitable for 5V-CMOS technologies, could achieve an ESD failure current density eight time higher that the GGNMOS. Moreover, the transient overshoot characteristic of EGGNMOS is comparable with that of the GGNMOS. Three-dimensional TCAD simulation has also been carried out to verify that the NGR isolation functionality of EGGNMOS is not being comprised.

mTOR is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. Here we assessed mLST8 loss in a panel of normal and cancer cells and observed little to no impact on assembly or activity of mTORC1. However, mLST8 loss blocked mTOR association with mTORC2 cofactors RICTOR and SIN1, thus abrogating mTORC2 activity. Similarly, a single pair of mutations on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly and activity without affecting mTORC1. We also discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor SIN1. In PTEN-null prostate cancer xenografts, mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation and decreased tumor cell proliferation and tumor growth in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation that could enable therapeutic mTORC2-selective inhibition as a therapeutic strategy. Significance: These findings show that mLST8 functions as a scaffold to maintain mTORC2 integrity and kinase activity, unveiling a new avenue for development of mTORC2-specific inhibitors.

In this paper, an enhanced modified lateral silicon-controlled rectifier (EMLSCR) has been proposed and demonstrated. Compared with the traditional MLSCR, the EMLSCR possesses a lower trigger voltage of 7.0 V as well as a higher robustness, making it very suitable for the electrostatic discharge (ESD) protection for 2.5-V/3.3-V/5-V IOs in the advanced 65-nm epitaxial CMOS technology. Additionally, an improved transmission line pulsing (TLP) measurement strategy by adopting TLP pulses with various rise time has been put forward to provide a convenient and economical approach to explore the conduction uniformity of the ESD device for the first time. Furthermore, two optimization methods are proposed and evaluated to overcome the prefailure issue due to the small well resistance inherited in the EPI process as well. The method of well pickup segmentation is demonstrated to be more efficient since it could increase the well resistance and augment the effective emitter area of parasitic bipolar junction transistors (BJTs) under constant device area.

In this paper, a novel low-voltage triggered silicon-controlled rectifier (NLVTSCR) with low trigger voltage and higher holding voltage is proposed and implemented in a 28nm CMOS process. The proposed NLVTSCR in the TLP test has a low trigger voltage and an adjustable high holding voltage from 3.44Vto 4.93V. In addition, it does not require any additional masks, making it an excellent candidate for 3.3V ESD protection. Compared to conventional low triggered voltage silicon-controlled rectifier (LVTSCR), the proposed NLVTSCR device provides a higher holding voltage than its conventional counterpart.

Faraday instability has great application value in the fields of controlling polymer processing, micromolding colloidal lattices on structured suspensions, organizing particle layers, and conducting cell culture. To regulate Faraday instability, in this article, we attempt to introduce an elastic polymer film covering the surface of a viscous fluid layer and theoretically study the behaviors of the Faraday instability phenomenon and the effect of the elastic polymer film. Based on hydrodynamic theory, the Floquet theory is utilized to formulate its stability criterion, and the critical acceleration amplitude and critical wave number are calculated numerically. The results show that the critical acceleration amplitude for Faraday instability increases with three increasing bending stiffness of the elastic polymer film, and the critical wave number decreases with increasing bending stiffness. In addition, surface tension and viscosity also have important effects on the critical acceleration amplitude and critical wave number. The strategy of controlling Faraday instability by covering an elastic polymer film proposed in this paper has great application potential in new photonic devices, metamaterials, alternative energy, biology, and other fields.