The influence of olfactory experience on the birthrates of olfactory sensory neurons with specific odorant receptor identities.

Olfactory sensory neurons (OSNs) are one of a few neuron types that are generated continuously throughout life in mammals. The persistence of olfactory sensory neurogenesis beyond early development has long been thought to function simply to replace neurons that are lost or damaged through exposure to environmental insults. The possibility that olfactory sensory neurogenesis may also serve an adaptive function has received relatively little consideration, largely due to the assumption that the generation of new OSNs is stochastic with respect to OSN subtype, as defined by the single odorant receptor gene that each neural precursor stochastically chooses for expression out of hundreds of possibilities. Accordingly, the relative birthrates of different OSN subtypes are predicted to be constant and impervious to olfactory experience. This assumption has been called into question, however, by evidence that the birthrates of specific OSN subtypes can be selectively altered by manipulating olfactory experience through olfactory deprivation, enrichment, and conditioning paradigms. Moreover, studies of recovery of the OSN population following injury provide further evidence that olfactory sensory neurogenesis may not be strictly stochastic with respect to subtype. Here we review this evidence and consider mechanistic and functional implications of the prospect that specific olfactory experiences can regulate olfactory sensory neurogenesis rates in a subtype-selective manner.

TGFß attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

Histone variants and cellular plasticity.

The broad diversity of cell types within vertebrates arises from a unique genetic blueprint by combining intrinsic cellular information with developmental and other extrinsic signals. Lying at the interface between cellular signals and the DNA is the chromatin, a dynamic nucleoprotein complex that helps to mediate gene regulation. The most basic subunit of chromatin, the nucleosome, consists of DNA wrapped around histones, a set of proteins that play crucial roles as scaffolding molecules and regulators of gene expression. Growing evidence indicates that canonical histones are commonly replaced by protein variants before and during cellular transitions. We highlight exciting new results suggesting that histone variants are essential players in the control of cellular plasticity during development and in the adult nervous system.

Correction to: Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here, we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a tumor mouse model that has highly endogenous mTEM1 expression in the vasculature. Our data indicated that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

In mice, discrete odors can selectively promote the neurogenesis of sensory neuron subtypes that they stimulate.

In mammals, olfactory sensory neurons (OSNs) are born throughout life, presumably solely to replace neurons lost via turnover or injury. This assumption follows from the hypothesis that olfactory neurogenesis is strictly stochastic with respect to neuron subtype, as defined by the single odorant receptor allele that each neural precursor stochastically chooses out of hundreds of possibilities. This hypothesis is challenged by recent findings that the birthrates of a fraction of subtypes are selectively diminished by olfactory deprivation. These findings raise questions about how, and why, olfactory stimuli are required to promote the neurogenesis of some OSN subtypes, including whether the stimuli are generic (e.g., broadly activating odors or mechanical stimuli) or specific (e.g., discrete odorants). Based on RNA-seq and scRNA-seq analyses, we hypothesized that the neurogenic stimuli are specific odorants that selectively activate the same OSN subtypes whose birthrates are accelerated. In support of this, we have found, using subtype-specific OSN birthdating, that exposure to male and musk odors can accelerate the birthrates of responsive OSNs. Collectively, our findings reveal that certain odor experiences can selectively "amplify" specific OSN subtypes, and that persistent OSN neurogenesis may serve, in part, an adaptive function.

A histological protocol for quantifying the birthrates of specific subtypes of olfactory sensory neurons in mice.

Mammals typically have hundreds of distinct olfactory sensory neuron subtypes, each defined by expression of a specific odorant receptor gene, which undergo neurogenesis throughout life at rates that can depend on olfactory experience. Here, we present a protocol to quantify the birthrates of specific neuron subtypes via the simultaneous detection of corresponding receptor mRNAs and 5-ethynyl-2'-deoxyuridine. For preparation prior to beginning the protocol, we detail procedures for generating odorant receptor-specific riboprobes and experimental mouse olfactory epithelial tissue sections. For complete details on the use and execution of this protocol, please refer to van der Linden et al. (2020).1.

Olfactory Stimulation Regulates the Birth of Neurons That Express Specific Odorant Receptors.

In mammals, olfactory sensory neurons (OSNs) are born throughout life, ostensibly solely to replace damaged OSNs. During differentiation, each OSN precursor "chooses," out of hundreds of possibilities, a single odorant receptor (OR) gene, which defines the identity of the mature OSN. The relative neurogenesis rates of the hundreds of distinct OSN "subtypes" are thought to be constant, as they are determined by a stochastic process in which each OR is chosen with a fixed probability. Here, using histological, single-cell, and targeted affinity purification approaches, we show that closing one nostril in mice selectively reduces the number of newly generated OSNs of specific subtypes. Moreover, these reductions depend on an animal's age and/or environment. Stimulation-dependent changes in the number of new OSNs are not attributable to altered rates of cell survival but rather production. Our findings indicate that the relative birth rates of distinct OSN subtypes depend on olfactory experience.

Gene expression profiling of the olfactory tissues of sex-separated and sex-combined female and male mice.

Olfactory experience can alter the molecular and cellular composition of chemosensory neurons within the olfactory sensory epithelia of mice. We sought to investigate the scope of cellular and molecular changes within a mouse's olfactory system as a function of its exposure to complex and salient sets of odors: those emitted from members of the opposite sex. We housed mice either separated from members of the opposite sex (sex-separated) or together with members of the opposite sex (sex-combined) until six months of age, resulting in the generation of four cohorts of mice. From each mouse, the main olfactory epithelium (MOE), vomeronasal organ (VNO), and olfactory bulb (OB) were removed and RNA-extracted. A total of 36 RNA samples, representing three biological replicates per sex/condition/tissue combination, were analyzed for integrity and used to prepare RNA-seq libraries, which were subsequently analyzed via qPCR for the presence of tissue- or sex-specific markers. Libraries were paired-end sequenced to a depth of ~20 million fragments per replicate and the data were analyzed using the Tuxedo suite.

Sex separation induces differences in the olfactory sensory receptor repertoires of male and female mice.

Within the mammalian olfactory sensory epithelium, experience-dependent changes in the rate of neuronal turnover can alter the relative abundance of neurons expressing specific chemoreceptors. Here we investigate how the mouse olfactory sensory receptor repertoire changes as a function of exposure to odors emitted from members of the opposite sex, which are highly complex and sexually dimorphic. Upon housing mice either sex-separated or sex-combined until six months of age, we find that sex-separated mice exhibit significantly more numerous differentially expressed genes within their olfactory epithelia. A subset of these chemoreceptors exhibit altered expression frequencies following both sex-separation and olfactory deprivation. We show that several of these receptors detect either male- or female-specific odors. We conclude that the distinct odor experiences of sex-separated male and female mice induce sex-specific differences in the abundance of neurons that detect sexually dimorphic odors.

Presenilin Regulates Retinotectal Synapse Formation through EphB2 Receptor Processing.

As the catalytic component of γ-secretase, presenilin (PS) has long been studied in the context of Alzheimer's disease through cleaving the amyloid precursor protein. PS/γ-secretase, however, also cleaves a multitude of single-pass transmembrane proteins that are important during development, including Notch, the netrin receptor DCC, cadherins, drebrin-A, and the EphB2 receptor. Because transgenic PS-KO mice do not survive to birth, studies of this molecule during later embryonic or early postnatal stages of development have been carried out using cell cultures or conditional knock-out mice, respectively. As a result, the function of PS in synapse formation had not been well-addressed. Here, we study the role of PS in the developing Xenopus tadpole retinotectal circuit, an in-vivo model that allows for protein expression to be manipulated specifically during the peak of synapse formation between retinal ganglion cells and tectal neurons. We found that inhibiting PS in the postsynaptic tectal neurons impaired tadpole visual avoidance behavior. Whole cell recordings indicated weaker retinotectal synaptic transmission which was characterized by significant reductions in both NMDA receptor (NMDAR)- and AMPA receptor (AMPAR)-mediated currents. We also found that expression of the C-tail fragment of the EphB2 receptor, which is normally cleaved by PS/γ-secretase and which has been shown to upregulate NMDARs at the synapse, rescued the reduced NMDAR-mediated responses. Our data determine that normal PS function is important for proper formation and strengthening of retinotectal synapses through cleaving the EphB2 receptor.

FAP Delineates Heterogeneous and Functionally Divergent Stromal Cells in Immune-Excluded Breast Tumors.

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN- population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFß signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide-dependent manner, whereas FAP+PDPN- pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

An efficient system for the evolution of aminoacyl-tRNA synthetase specificity.

A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cm(r)) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli.

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.

A specificity switch in selected cre recombinase variants is mediated by macromolecular plasticity and water.

The basis for the altered DNA specificities of two Cre recombinase variants, obtained by mutation and selection, was revealed by their cocrystal structures. The proteins share similar substitutions but differ in their preferences for the natural LoxP substrate and an engineered substrate that is inactive with wild-type Cre, LoxM7. One variant preferentially recombines LoxM7 and contacts the substituted bases through a hydrated network of novel interlocking protein-DNA contacts. The other variant recognizes both LoxP and LoxM7 utilizing the same DNA backbone contact but different base contacts, facilitated by an unexpected DNA shift. Assisted by water, novel interaction networks can arise from few protein substitutions, suggesting how new DNA binding specificities might evolve. The contributions of macromolecular plasticity and water networks in specific DNA recognition observed here present a challenge for predictive schemes.

T cells bearing a chimeric antigen receptor against prostate-specific membrane antigen mediate vascular disruption and result in tumor regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

Quantitative and functional interrogation of parent-of-origin allelic expression biases in the brain.

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased--rather than monoallelic--expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.

Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition.

NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4(+) and CD8(+) NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.

The activity-dependent histone variant H2BE modulates the life span of olfactory neurons.

We have identified a replication-independent histone variant, Hist2h2be (referred to herein as H2be), which is expressed exclusively by olfactory chemosensory neurons. Levels of H2BE are heterogeneous among olfactory neurons, but stereotyped according to the identity of the co-expressed olfactory receptor (OR). Gain- and loss-of-function experiments demonstrate that changes in H2be expression affect olfactory function and OR representation in the adult olfactory epithelium. We show that H2BE expression is reduced by sensory activity and that it promotes neuronal cell death, such that inactive olfactory neurons display higher levels of the variant and shorter life spans. Post-translational modifications (PTMs) of H2BE differ from those of the canonical H2B, consistent with a role for H2BE in altering transcription. We propose a physiological function for H2be in modulating olfactory neuron population dynamics to adapt the OR repertoire to the environment.DOI:http://dx.doi.org/10.7554/eLife.00070.001.