Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells

Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies, and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (∼0.01–1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T-cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.Invariant natural killer T (iNKT) cells are a small population of αβ T lymphocytes highly conserved from mice to humans. Like conventional αβ T cells, iNKT cells are derived from hematopoietic stem cells (HSCs) and develop in the thymus. However, they differ from conventional T cells in several important aspects, including their display of NK cell markers, their recognition of glycolipid antigens presented by the nonclassical monomorphic major histocompatibility complex (MHC) molecule CD1d, and their expression of semi-invariant T-cell receptors (identical α chains paired with a limited selection of β chains) (1, 2). Despite their small numbers in vivo (∼0.1–1% in mouse blood and ∼0.01–1% in human blood), iNKT cells have been suggested to play important roles in regulating many diseases, including cancer, infections, allergies, and autoimmunity (3). When stimulated, iNKT cells rapidly release a large amount of effector cytokines like IFN-γ and IL-4, both as a cell population and at the single-cell level. These cytokines then activate various immune effector cells, such as natural killer (NK) cells and dendritic cells (DCs) of the innate immune system, as well as CD4 helper and CD8 cytotoxic conventional αβ T cells of the adaptive immune system via activated DCs (3, 4). Because of their unique activation mechanism, iNKT cells can attack multiple diseases independent of antigen and MHC restrictions, making them attractive universal therapeutic agents (3, 4). Notably, because of the capacity of effector NK cells and conventional αβ T cells to specifically recognize diseased tissue cells, iNKT cell-induced immune reactions result in limited off-target side effects (3, 4).Restricted by their extremely low numbers, both the study of iNKT cells and their clinical applications have been challenging. iNKT T-cell receptor (TCR) transgenic mice (5, 6) and iNKT induced pluripotent stem (iPS) cell-derived transgenic mice (7) provide valuable tools to study iNKT cell biology in mice, but these methods are both costly and time-consuming. In addition, approaches using transgenic mice have no direct clinical application. As an alternative, a TCR-engineered HSC adoptive transfer strategy could overcome these limitations and become clinically applicable. Since its demonstration in mice in the early 2000s, this HSC-engineered T-cell strategy has been widely used to successfully generate both mouse and human antigen-specific conventional αβ T cells in multiple mouse and humanized mouse models (8–13). Human clinical trials testing this strategy for treating melanoma are also ongoing (14). Based on these previous works and the scientific principle that iNKT cells follow a “TCR instruction” development path similar to that of conventional αβ T cells (15), we hypothesized that HSCs could be engineered to express iNKT TCR genes and be programmed to develop into clonal iNKT cells. In the present report, we demonstrated the feasibility of this new HSC-engineered iNKT cell approach in mice and provided evidence to support its therapeutic potential in a mouse melanoma lung metastasis model.     

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.It is well-established that different populations of T lymphocytes can recognize not only peptides in the context of major histocompatibility complex (MHC) class I (MHCI) and MHCII molecules but also, foreign and self-lipids in association with CD1 proteins (1), antigen-presenting molecules that share structural similarities with MHCI molecules. Of five CD1 isoforms, CD1d restricts the activity of a family of cells known as invariant natural killer T (iNKT) cells because of their semiinvariant T-cell receptor (TCR) use (1). To date, the exogenous glycolipid α-GalactosylCeramide (α-GalCer) represents the best characterized CD1d-restricted agonist for iNKT cells (2). Unlike conventional peptide-specific T cells, iNKT cells react against CD1d⁺ antigen-presenting cells (APCs) in the absence of exogenous antigens, a feature defined as autoreactivity (3). iNKT cell autoreactivity underpins the constitutive memory phenotype of iNKT cells and their ability to be activated during a variety of immune responses from infections to cancer and autoimmunity (1). Some of the endogenous antigens known to elicit iNKT cell autoreactivity belong to glycosphingolipid families, with a mix of α- and β-anomeric configurations (4–7). How iNKT cell autoreactivity is fine-tuned to prevent autoimmunity is subject of much investigation. Previous results have shown that exposure of APCs to Toll-like receptor (TLR) agonists enhances iNKT cell autoreactivity (8, 9), consistent with the proposed mechanism by which ligand availability is regulated by lysosomal glycosidases (4, 6).The recent application of advanced optical techniques (10–13) in combination with substrate patterning and functionalization (14, 15) is providing detailed information on how the lateral organization of a variety of molecules located on both sides of the immunological synapse contributes to controlling T-cell activation. Specifically, single-molecule dynamic approaches and superresolution optical nanoscopy experiments have provided indisputable proof that many receptors on the cell membrane organize in small nanoclusters before ligand activation (16). Membrane nanodomains enriched in cholesterol and sphingolipids (17), protein–protein interactions (18), and interactions between transmembrane proteins and the cytoskeleton (19, 20) have been all implicated in regulating receptor dynamics and nanoclustering. An emerging concept attributes the actin cytoskeleton the ability of imposing barriers or fences on the cell membrane, restricting the lateral mobility of transmembrane proteins (19–21). This transient restriction would, in turn, increase the local concentration of transmembrane proteins, leading to protein nanoclusters. For instance, it has been shown that the actin cytoskeleton promotes the dimerization rate of EGF receptors and facilitates ligand binding and signaling activation (18, 22). Confinement of CD36 has also been observed as a result of its diffusion along linear channels dependent on the integrity of the cortical cytoskeleton (23). This constrained diffusion promotes CD36 clustering, influencing CD36-mediated signaling and internalization. A similar mechanism has been proposed for the maintenance of MHCI clusters on the cell membrane by the actin cytoskeleton, with loss of MHCI clustering resulting in a decreased CD8 T-cell activation (24, 25).Recent confocal microscopy studies have revealed that the association between agonist-loaded CD1d molecules and lipid rafts might contribute to the regulation of iNKT cell activation (26). This elegant study for the first time, to our knowledge, linked the spatial organization of CD1d molecules on the cell membrane of APCs with the activation profile of iNKT cells. However, it remains unclear whether the results of these experiments obtained using mouse cells can be extended to human cells and whether additional insights can be obtained by using higher-resolution microscopy. Indeed, it is not yet known whether surface-expressed CD1d molecules exist as monomers or nanoclusters and whether the actin cytoskeleton might regulate CD1d lateral organization and iNKT cell activation. Interestingly, it has been recently reported that the actin cytoskeleton impairs antigen presentation by CD1d and that disruption of F actin or inhibition of the ρ-associated protein kinase enhances CD1d-mediated antigen presentation (27). These results suggest that the actin cytoskeleton might regulate, in a not yet known manner, antigen presentation by CD1d molecules.Here, we combined dual-color single-molecule dynamic approaches with superresolution optical nanoscopy to characterize for the first time, to our knowledge, the spatiotemporal behavior of CD1d on living human myeloid cells. We find that α-GalCer–loaded human CD1d (hCD1d) molecules are organized in nanoclusters on the cell membrane of APCs. We report that the actin cytoskeleton prevents enhanced hCD1d nanoclustering by hindering physical encountering between hCD1d diffusing nanoclusters, thus reducing basal iNKT cell activation. Furthermore, we observed an increase in nanocluster density on activation of APCs with inflammatory stimuli, such as TLR stimulation, mirroring the increased iNKT cell stimulation. Notably, even during inflammation, the actin cytoskeleton retains an important role to limit hCD1d cluster size and iNKT cell activation. Overall, our results suggest that regulation of CD1d nanoclustering through the actin cytoskeleton represents a previously unidentified mechanism to fine-tune peripheral iNKT cell autoreactivity.     

KLRG+ invariant natural killer T cells are long-lived effectors

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)–mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d⁺ cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ–producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1⁺ (Killer cell lectin-like receptor subfamily G, member 1–positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3β by RNA deep sequencing demonstrated that some particular KLRG1⁺ iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1⁺ iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.Invariant natural killer T cells (iNKT cells) express an invariant T-cell antigen receptor (TCR) α-chain and recognize a complex of the antigen-presenting the MHC-like molecule CD1d and a glycolipid (1–3). The activation of iNKT cells depends on the ligand delivery system. α-Galactosylceramide (α-GalCer), a well-known potent synthetic iNKT ligand, can be loaded onto CD1d-expressing cells, such as dendritic cells (DCs), CD1d-transfected tumor cells, and fibroblasts (4, 5). When activated by these α-GalCer–loaded CD1d⁺ cells in vivo, iNKT cells are capable of releasing large quantities of IFN-γ (4, 5). Clinical studies using α-GalCer–loaded monocyte-derived DCs have demonstrated the safety and efficacy of iNKT cell therapy (6, 7). On the other hand, we and others have shown that iNKT cells become contracted after activation and become anergic when unbound α-GalCer is administered (4, 8, 9). The different fates of iNKT cells’ response in the two models (i.e., one group of mice treated with unbounded α-GalCer and the other group of mice treated with α-GalCer–loaded CD1d⁺ cells), particularly the fate of iNKT cells after the vaccination with CD1d⁺ cells loaded with α-GalCer, have not been fully characterized.Innate lymphocytes, which mount rapid effector responses, have been thought to be short-lived. However, NK cells (10) and γδT cells (11) with memory-type characteristics have been identified recently. In this study, we demonstrate an effector memory-like KLRG1 (Killer cell lectin-like receptor subfamily G, member 1)-expressing iNKT cell response in mice immunized with CD1d⁺ cells loaded with the iNKT-cell ligand. KLRG1 was used as a surrogate marker of terminally differentiated short-lived effector CD8⁺ T cells or effector memory-type CD8⁺ T cells (12). The contribution of the KLRG1⁺ T-cell subset to immunity has been defined recently: KLRG1⁺CD4⁺ and CD8⁺ T cells exhibited strong antitumor effects compared with KLRG1⁻ T cells (13, 14). In addition, KLRG1⁺CD8⁺ T cells isolated from influenza virus-infected mice survive for a long period, proliferate well, and participate in a recall response (15). KLRG1 also has been identified as a surface marker on mature NK (16) and memory NK cells (10). Memory NK cells express KLRG1^hi and CD62L^low and produce high IFN-γ after stimulation, much like the effector memory CD8⁺ T cells. In view of these findings, our current study investigating the fate of KLRG1⁺ iNKT cells induced by CD1d⁺ cells loaded with an iNKT cell ligand would have potential implications for future iNKT-cell–based therapies.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

Being activated by depolarizing voltages and increases in cytoplasmic Ca²⁺, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.High-conductance voltage- and calcium-activated potassium (BK) channels are homotetrameric proteins of α-subunits encoded by the slo1 gene (1). These channels are expressed in virtually all mammalian tissues, where they detect and integrate membrane voltage and calcium concentration changes dampening the responsiveness of cells when confronted with excitatory stimuli. They are abundant in the CNS and nonneuronal tissues, such as smooth muscle or hair cells. This wide distribution is associated with an outstandingly large functional diversity, in which BK channel activity appears optimally adapted to the particular physiological demands of each cell type (2). On the other hand, small alterations in BK channel function may contribute to the pathophysiology of hypertension, asthma, cancer, epilepsy, diabetes, and other conditions in humans (3–8). Alternative splicing, posttranslational modifications, and regulation by auxiliary proteins have been proposed to contribute to this functional diversity (1, 2, 9–16).The BK channel α-subunit is formed by a single polypeptide of about 1,200 amino acids that contains all of the key structural elements for ion permeation, gating, and modulation by ions and other proteins. Tetramers of α-subunits form functional BK channels. Each subunit has seven hydrophobic transmembrane segments (S0–S6), where the voltage-sensor domain (VSD) and pore domain (PD) reside (2). The N terminus faces the extracellular side of the membrane, whereas the C terminus is intracellular. The latter contains four hydrophobic α-helices (S7–S10) and the main Ca²⁺ binding sites (2). VSDs formed by segments S1–S4 harbor a series of charged residues across the membrane that contributes to voltage sensing (2). Upon membrane depolarization, each VSD undergoes a rearrangement (17) that prompts the opening of a highly K⁺-selective pore formed by the four PDs that come together at the symmetry center of the tetramer.Although BK channel expression is ubiquitous, in most physiological scenarios their functioning is provided by their coassembly with auxiliary proteins, such as β-subunits. This coassembly brings channel activity into the proper cell/tissue context (11, 13). Four different β-subunits have been cloned (β1–β4) (18–24), all of which have been observed to modify BK channel function. Albeit to a different extent, all β-subunits modify the Ca²⁺ sensitivity, voltage dependence, and gating properties of BK channels, hence modifying plasma membrane excitability balance. Regarding auxiliary β-subunits, β1- and β2-subunits increase apparent Ca²⁺ sensitivity and decelerate macroscopic current kinetics (14, 20, 21, 25–30); β2 and β3 induce fast inactivation as well as an instantaneous outward rectification (20, 21, 24, 31, 32); and β4 slows down activation and deactivation kinetics (12, 23) and modifies Ca²⁺ sensitivity (12, 33, 34).It should be kept in mind that β-subunits are potential targets for different molecules that modulate channel function, such as alcohol (35), estrogens (15), hormones (36), and fatty acids (37, 38). Additionally, scorpion toxin affinity in BK channels would tend to increase when β1 is coexpressed with the α-subunit (22).To identify the molecular elements that give β1 the ability to modulate the voltage sensor of BK channels, we constructed chimeric proteins of β1/β2- and β1/β3-subunits by swapping their N and C termini, the transmembrane (TM) segments, and the extracellular loops and recorded their gating currents. Two lysine residues that are unique to the N terminus of β1 were identified to be sufficient for BK voltage-sensor modulation.     

Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer

The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.Despite decades of research, the etiology of breast cancer remains unclear. It is currently thought that most breast cancers occur in the normal terminal duct lobular unit and progress in a stepwise fashion over time (1). Ductal carcinoma in situ (DCIS) means the cancer has not spread beyond the duct into any normal surrounding breast tissue and is thought by some to be the direct precursor of invasive ductal carcinoma (IDC).Estrogens play an important role in normal breast development as well as breast cancer progression (2). Most of the effects of estrogen are mediated through its two receptors: estrogen receptor α (ERα) and β (ERβ) (3). ERα is expressed in 50–80% of breast tumors, and its presence is the main indicator for antihormonal therapy (4). ERβ was first discovered in 1996, and its role in breast cancer is still being explored (5–7).The first step in understanding the role of ERβ in breast cancer was to define the expression pattern of ERβ in the normal human breast and in various stages of cancer. Since its discovery, several laboratories have reported ERβ expression in clinical samples (8–28). Most of these studies investigated the expression of ERβ in invasive breast cancer samples (12–15, 17, 19, 21–23). Some studies have reported ERβ expression in invasive breast cancer and normal breast tissue (11, 18, 26–28), but few have compared the expression of ERβ in the normal tissue, DCIS, and IDC within the same sample. Usually tumor samples are taken from one patient and normal tissue from another patient (8–10). Samples taken from different patients have intrinsic limitation (i.e., they cannot account for variations between different patients). In addition, because tumors are heterogeneous, core biopsies do not fully reflect the histological and biological diversity of breast tumors (29).The roles of ERβ1 and its splice variant ERβ2 in breast cancer are still unclear. As reviewed by Murphy and Leygue (30), some studies show a loss of ERβ1 as ductal cancer progresses, but others do not. Some studies show ERβ2 as a marker of bad prognosis (31), and others not (19). Some of these differences may be due to differences in antibody use and differences in tissue fixation and handling.When ERα and ERβ are coexpressed in breast cancer it is unclear whether tamoxifen treatment will be successful. This is because tamoxifen acts as an agonist of ERβ at activator protein 1 (AP-1) sites (32) and thus should oppose the antiproliferative effects of the tamoxifen–ERα complex. Yan et al. (33) have found that expression of ERβ predicts tamoxifen benefit in patients with ERα-negative early breast cancer, whereas Esslimani-Sahla et al (23) have found that low ERβ level is an independent marker, better than ERα level, to predict tamoxifen resistance. Although apparently saying different things, these two results actually agree with each other: in ERα-negative breast cancer, estrogen is not driving proliferation, so tamoxifen via ERβ may interfere with another growth signaling pathway. In ERα-positive cancers whose proliferation is driven by E2, tamoxifen with ERβ would oppose the antiproliferative effects of the ERα–tamoxifen complex.Investigation of the expression pattern of ERβ in normal tissue, DCIS, and IDC is important to understand the function of this receptor in the progression of breast cancer. We have a set of samples obtained from surgical excision of breast tumors from women before pharmacological intervention. The cohorts include lobular cancer, which has not yet been thoroughly studied for ERβ expression. Lobular cancer is an ERα-positive form of breast cancer characterized by loss of E-cadherin and relatively low proliferation rate. It is accompanied by a resistance to anoikis (34). It accounts for 10–15% of diagnosed breast cancer, and there are still many questions about the optimal therapeutic approach to this cancer. We have explored the changes in expression of the two ERs using identical protocols and reagents in different developmental stages of breast cancer within each patient.     

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants.Next-generation sequencing is transforming our understanding of human genetic variation (1) and providing profound insights into the impact of both inherited and de novo variants on human health (2, 3). At the same time, the data from these studies present serious challenges in providing information to individuals who are found to have variant forms of different proteins. To highlight these challenges, in this report we describe our experience in analyzing missense variants of the platelet αIIbβ3 integrin receptor from The Human Genome Mutation Database (HGMD), the 1000 Genomes project (1000G), the United Kingdom 10K Whole Exome Sequencing project (U.K.10KWES), the United Kingdom 10K Whole Genome Sequencing project (U.K.10KWGS), and The National Heart, Lung and Blood Institute Exome Sequencing Project (ESP); the latter four sources encompass ∼32,000 alleles derived from 16,108 individuals.The αIIbβ3 receptor has a number of virtues as a model system. First, it is required for hemostasis because platelet aggregation requires cross-linking of the activated form of αIIbβ3 by macromolecular ligands (4). Thus, defects in its biogenesis, activation, or ligand binding lead to the rare bleeding diathesis Glanzmann thrombasthenia (GT), an autosomal recessive disorder (5, 6). Patients with GT come to medical attention because of their hemorrhagic symptoms, and thus have been carefully analyzed clinically and with tests of platelet function for nearly 50 y (5, 7). The biochemical and molecular abnormalities in GT have been studied for nearly 40 y (4, 6, 8–10). In the past 10 y, high-resolution crystallography, electron microscopy, and computational studies of the αIIbβ3 receptor have provided atomic-level information on the correlation between receptor structure and function (11–21). In addition, ethnic groups with relatively high prevalence of GT have been defined that share the same genetic abnormality based on founder mutations, and the dates that some of the mutations entered the population have been estimated (22–28). An on-line registry of GT abnormalities, including patient phenotypes was developed in 1997 (29) and currently contains 51 αIIb and 43 β3 missense variants linked to the disorder (sinaicentral.mssm.edu/intranet/research/glanzmann). The frequency of GT in the general population has not been established but it has a world-wide distribution, and based on data from hematologic practices, it is rare except in areas with a high rate of consanguineous mating (30).Second, alloimmune disorders, including neonatal thrombocytopenia and posttransfusion purpura, due to amino acid substitutions in either αIIb or β3, have been characterized at the molecular biological level and correlated with mechanisms of immunologic recognition (31).Third, inherited macrothrombocytopenia and anisothrombocytopenia have been associated with heterozygous missense variants or deletions in αIIb or β3. All of these appear to induce constitutive activation of the receptor and impair proplatelet formation (32–38).Fourth, αIIbβ3 contributes to pathological platelet thrombus formation in human ischemic cardiovascular disease and αIIbβ3 is a validated target for antithrombotic therapy (39–41).Fifth, αIIbβ3 is a member of the large integrin family of receptors, which includes 24 receptors derived from 18 α- and 8 β-subunits (41, 42). These receptors are involved in important biologic processes, including development, cell migration, homing, cell survival, and adaptive immunity (41–43). More is known about the structure–function relationships of αIIbβ3 than the other members of the group, and so it serves as the paradigmatic integrin receptor (44, 45).Sixth, 3D molecular models have been built based on crystallographic and NMR data to analyze the effects of novel amino acid substitutions on receptor structure and function and the generation of alloantigens (15, 46–53). The data from these models and assessments of the severity of the amino acid change in the variants have the potential to aid in predicting whether a novel variant is likely to affect receptor function and immunogenicity (54–59).     

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.Metazoan life is dependent upon the use of molecular oxygen for an array of metabolic processes. Tissue hypoxia occurs during periods of imbalance between oxygen supply and consumption. One of the primary cellular responses to hypoxia is the activation of the hypoxia-inducible factor (HIF) program (1–4). HIF consists of oxygen-regulated α-subunits HIF-1α and HIF-2α and a constitutively expressed β-subunit (HIF-β). In the presence of oxygen, a series of nonheme Fe(II)- and 2-oxoglutarate–dependent dioxygenase oxygen sensors, referred to as HIF prolylhydroxylases (HIF PHDs), promote the hydroxylation of key proline residues on the HIF-α subunits (5, 6). This serves as a recognition site for the von Hippel-Lindau (VHL) tumor-suppressor protein, which mediates ubiquitination and proteasomal degradation of HIF-1α and HIF-2α (7–9). Hypoxia inhibits HIF PHDs, allowing HIF-1α and HIF-2α to evade VHL recognition and assemble with HIF-β to produce the active heterodimeric HIF factor. Once activated, HIF-1α and HIF-2α cooperate through common and distinct pathways to regulate hypoxic gene expression and cellular adaptation to hypoxia (10).A notable feature of the HIF response is the differential expression pattern of HIF-1α and HIF-2α in normal tissues. HIF-1α mRNA is ubiquitous and constitutively expressed in adult cells. In stark contrast, HIF-2α mRNA is detected in a few cell types of adult tissues and is typically not expressed by epithelia (11). This suggests a physiological necessity to fine-tune the HIF program depending upon the cellular settings by negatively regulating the HIF-2α gene (EPAS1) upstream of the HIF oxygen-sensing enzymes. The negative regulation of EPAS1 is often compromised in cancers, as HIF-2α mRNA is observed in the vast majority of overt tumors (11–13). This is particularly evident in renal cancer. Elegant studies by the Maxwell group (13) and others (14) revealed that HIF-2α mRNA is absent in human kidney tubule epithelia but present in dysplastic foci of the nephron. In these incipient renal tumor cells, HIF-2α may function as an oncoprotein (15), collaborating with, or activating, multiple growth-promoting pathways including cancer stewards c-myc (16), ras (17), and EGFR (18, 19). Silencing of HIF-2α suppresses tumorigenesis of various genetically diverse cancers, further highlighting its central role in malignancy (16, 17, 20, 21), although this depends on the experimental context (22). Therefore, EPAS1 is silent in adult epithelia but undergoes unscheduled activation in several malignancies, driving proliferation in the hypoxic tumor microenvironment (23).A clue to the mechanisms involved in the unscheduled activation of EPAS1 during early tumorigenesis may reside in its promoter, which harbors an enrichment of cytosine and guanine bases that often serve as sites of DNA methylation and epigenetic gene silencing (24–27). Cytosine methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a, and DNMT3b. DNMT1 maintains the methylation pattern from the template strand to the newly synthesized strand during DNA replication (28). DNMT3a and DNMT3b are de novo methyltransferases that establish postreplicative methylation patterns (29). Alterations in DNA methylation patterns are common in tumors and likely play a central role in aberrant gene expression that characterizes the malignant phenotype (26, 30, 31). This is particularly evident for DNMT3a, as recent studies have identified mutations in DNMT3a in patients with acute myeloid leukemia (32, 33) or down-regulation of DNMT3a mRNA in a variety of solid tumors (34). It is suggested that DNMT3a is a tumor-suppressor gene and that its mutation, or mRNA down-regulation, contributes to reducing global DNMT3a methyltransferase activity (35, 36). Currently, a key challenge is to link aberrant methylation profiles commonly observed in malignant lesions, including alterations in the DNMT3a epigenetic program, to genes that directly promote the tumorigenic phenotype.Here we show that DNMT3a methylates and silences EPAS1 in normal cells. Loss of DNMT3a observed in primary tumors and malignant cells causes unscheduled EPAS1 activation. This allows emerging cancer cells to exploit the HIF-2α program that facilitates cancer cell traverse of the hypoxic barrier and formation of tumors larger than the diffusion limit of oxygen. We suggest that the DNMT3a epigenetic program is a gatekeeper of the hypoxic cancer cell phenotype.     

TNF-α–stimulated fibroblasts secrete lumican to promote fibrocyte differentiation

In healing wounds and fibrotic lesions, fibroblasts and monocyte-derived fibroblast-like cells called fibrocytes help to form scar tissue. Although fibrocytes promote collagen production by fibroblasts, little is known about signaling from fibroblasts to fibrocytes. In this report, we show that fibroblasts stimulated with the fibrocyte-secreted inflammatory signal tumor necrosis factor-α secrete the small leucine-rich proteoglycan lumican, and that lumican, but not the related proteoglycan decorin, promotes human fibrocyte differentiation. Lumican competes with the serum fibrocyte differentiation inhibitor serum amyloid P, but dominates over the fibroblast-secreted fibrocyte inhibitor Slit2. Lumican acts directly on monocytes, and unlike other factors that affect fibrocyte differentiation, lumican has no detectable effect on macrophage differentiation or polarization. α2β1, αMβ2, and αXβ2 integrins are needed for lumican-induced fibrocyte differentiation. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, lumican is present at low levels throughout the tissue, whereas patients with advanced disease have pronounced lumican expression in the fibrotic lesions. These data may explain why fibrocytes are increased in fibrotic tissues, suggest that the levels of lumican in tissues may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and indicate that modulating lumican signaling may be useful as a therapeutic for fibrosis.During wound healing, monocytes leave the circulation, enter the tissue, and differentiate into fibroblast-like cells called fibrocytes (1–6). Fibrocytes are also found in the scar tissue-like lesions associated with fibrotic diseases such as pulmonary fibrosis, congestive heart failure, cirrhosis of the liver, and nephrogenic systemic fibrosis (3, 7–11). Fibrocytes express markers of both hematopoietic cells (CD34, CD45, FcγR, LSP-1, MHC class II) and stromal cells (collagens, fibronectin, and matrix metalloproteases) (2, 3, 12–14). Fibrocytes also promote angiogenesis by secreting VEGF, bFGF, IL-8, and PDGF (15). A key question about fibrocyte differentiation and fibrosis is why fibrocytes are readily observed in fibrotic lesions, but are rarely observed in healthy tissues (3, 10, 16–19).Fibrosis is a dynamic process involving many cells besides fibrocytes (20, 21). In fibrotic lesions, tissue-resident fibroblasts proliferate and produce excessive amounts of extracellular matrix (ECM) that distorts tissue architecture, leading to tissue destruction (21, 22). Fibrocytes secrete a variety of cytokines including IL-13, TGF-β, CTGF, and TNF-α that promote the proliferation, migration, and extracellular matrix production by the local fibroblasts (15, 23–26). Conversely, fibroblasts secrete a variety of factors that promote leukocyte entry, survival, and retention during inflammation (27–30). An intriguing possibility is that a runaway positive feedback loop involving unknown signals from fibrocyte-activated fibroblasts back to fibrocytes may lead to the persistence of fibrotic lesions.In this report, we show that fibroblasts stimulated with TNF-α secrete the small leucine-rich proteoglycan lumican, and that lumican promotes fibrocyte differentiation. In addition, we show that in a mouse pulmonary fibrosis model as well as human patients with pulmonary fibrosis, there appears to be an increase in lumican levels in the lungs, suggesting that pulmonary fibrosis may be in part due to elevated lumican levels. These data suggest that lumican may be one of the unknown signals from fibroblasts to fibrocytes that mediates part of a fibroblast-fibrocyte feedback loop that potentiates fibrosis.     

Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease

Near-infrared fluorescence (NIRF) molecular imaging has been widely applied to monitoring therapy of cancer and other diseases in preclinical studies; however, this technology has not been applied successfully to monitoring therapy for Alzheimer’s disease (AD). Although several NIRF probes for detecting amyloid beta (Aβ) species of AD have been reported, none of these probes has been used to monitor changes of Aβs during therapy. In this article, we demonstrated that CRANAD-3, a curcumin analog, is capable of detecting both soluble and insoluble Aβ species. In vivo imaging showed that the NIRF signal of CRANAD-3 from 4-mo-old transgenic AD (APP/PS1) mice was 2.29-fold higher than that from age-matched wild-type mice, indicating that CRANAD-3 is capable of detecting early molecular pathology. To verify the feasibility of CRANAD-3 for monitoring therapy, we first used the fast Aβ-lowering drug LY2811376, a well-characterized beta-amyloid cleaving enzyme-1 inhibitor, to treat APP/PS1 mice. Imaging data suggested that CRANAD-3 could monitor the decrease in Aβs after drug treatment. To validate the imaging capacity of CRANAD-3 further, we used it to monitor the therapeutic effect of CRANAD-17, a curcumin analog for inhibition of Aβ cross-linking. The imaging data indicated that the fluorescence signal in the CRANAD-17–treated group was significantly lower than that in the control group, and the result correlated with ELISA analysis of brain extraction and Aβ plaque counting. It was the first time, to our knowledge, that NIRF was used to monitor AD therapy, and we believe that our imaging technology has the potential to have a high impact on AD drug development.Alzheimer’s disease (AD) has been considered incurable, because none of the clinically tested drugs have shown significant effectiveness (1–4). Therefore, seeking effective therapeutics and imaging probes capable of assisting drug development is highly desirable. The amyloid hypothesis, in which various Aβ species are believed to be neurotoxic and one of the leading causes of AD, has been considered controversial in recent years because of the failures of amyloid beta (Aβ)-based drug development (1, 3, 5–8). However, no compelling data can prove that this hypothesis is wrong (2, 5), and no other theories indicate a clear path for AD drug development (4). Thus the amyloid hypothesis is still an important framework for AD drug development (1–5, 9–14). Additionally, Kim and colleagues (15) recently reported that a 3D cell-culture model of human neural cells could recapture AD pathology. In this study, their finding that the accumulation of Aβs could drive tau pathology provided strong support for the amyloid hypothesis (15).It is well known that Aβ species, including soluble monomers, dimers, oligomers, and insoluble fibrils/aggregates and plaques, play a central role in the neuropathology of AD (2, 5). Initially, it was thought that insoluble deposits/plaques formed by the Aβ peptides in an AD brain cause neurodegeneration. However, studies have shown that soluble dimeric and oligomeric Aβ species are more neurotoxic than insoluble deposits (16–21). Furthermore, it has been shown that soluble and insoluble species coexist during disease progression. The initial stage of pathology is represented by an excessive accumulation of Aβ monomers resulting from imbalanced Aβ clearance (22, 23). The early predominance of soluble species gradually shifts with the progression of AD to a majority of insoluble species (24, 25). Therefore imaging probes capable of detecting both soluble and insoluble Aβs are needed to monitor the full spectrum of amyloidosis pathology in AD.Thus far, three Aβ PET tracers have been approved by the Food and Drug Administration (FDA) for clinical applications. However, they are not approved for positive diagnosis of AD; rather, they are recommended for excluding the likelihood of AD. The fundamental limitation of these three tracers and others under development is that they bind primarily to insoluble Aβs, not the more toxic soluble Aβs (26–32). Clearly, work remains to be done in developing imaging probes based on Aβs, and imaging probes capable of diagnosing AD positively are undeniably needed.Numerous agents reportedly are capable of inhibiting the generation and aggregation of Aβs in vitro; however, only few have been tested in vivo. Partially, this lack of testing arises from the lack of reliable imaging methods that can monitor the agents’ therapeutic effectiveness in vivo. PET tracers, such as ¹¹C-Pittsburgh compound B (¹¹C-PiB) and ¹⁸F-AV-45, recently have been adapted to evaluate the efficacy of experimental AD drugs in clinical trials (33). However, they are rarely used to monitor drug treatment in small animals (30–32), likely because of the insensitivity of the tracers for Aβ species [particularly for soluble species (34–36)], complicated experimental procedures and data analysis in small animals, the high cost of PET probe synthesis and scanning, and the use of radioactive material. Therefore, a great demand for imaging agents that could be used in preclinical drug development to monitor therapeutic effectiveness in small animals remains unmet.Because of its low cost, simple operation, and easy data analysis, near infrared fluorescence (NIRF) imaging is generally more suitable than PET imaging for animal studies. Several NIRF probes for insoluble Aβs have been reported (37–45). It has been almost 10 y since the first report of NIRF imaging of Aβs by Hintersteiner et al. in 2005 (41). However, to the best of our knowledge, successful application of NIRF probes for monitoring therapeutic efficacy has not yet been reported. Our group recently has designed asymmetrical CRANAD-58 to match the hydrophobic (LVFF) and hydrophilic (HHQK) segments of Aβ peptides and demonstrated its applicability for the detection of both insoluble and soluble Aβs in vitro and in vivo (46). In this report, CRANAD-3 was designed to enhance the interaction with Aβs by replacing the phenyl rings of curcumin with pyridyls to introduce potential hydrogen bonds. Additionally, we demonstrated, for the first time to our knowledge, that the curcumin analog CRANAD-3 could be used as an NIRF imaging probe to monitor the Aβ-lowering effectiveness of therapeutics.