Early generation of a precursor CD8 T cell that can adapt to acute or chronic viral infection.

Virus specific PD-1+ TCF-1+ TOX+ stem-like CD8+ T cells are essential for maintaining T cell responses during chronic infection and are also critical for PD-1 directed immunotherapy. In this study we have used the mouse model of chronic LCMV infection to examine when these virus specific stem-like CD8+ T cells are generated during the course of chronic infection and what is the role of antigen in maintaining the stem-like program. We found that these stem-like CD8+ T cells are generated early (day 5) during chronic infection and that antigen is essential for maintaining their stem-like program. This early generation of stem-like CD8+ T cells suggested that the fate commitment to this cell population was agnostic to the eventual outcome of infection and the immune system prepares a priori for a potential chronic infection. Indeed, we found that an identical virus specific stem-cell like CD8+ T cell population was also generated during acute LCMV infection but these cells were lost once the virus was cleared. To determine the fate of these early PD-1+TCF-1+TOX+ stem-like CD8+ T cells that are generated during both acute and chronic LCMV infection we set up two reciprocal adoptive transfer experiments. In the first experiment we transferred day 5 stem-like CD8+ T cells from chronically infected into acutely infected mice and examined their differentiation after viral clearance. We found that these early stem-like CD8+ T cells downregulated canonical markers of the chronic stem-like CD8+ T cells and expressed markers (CD127 and CD62L) associated with central memory CD8+ T cells. In the second experiment, we transferred day 5 stem-like cells from acutely infected mice into chronically infected mice and found that these CD8+ T cells could function like resource cells after transfer into a chronic environment by generating effector CD8+ T cells in both lymphoid and non-lymphoid tissues while also maintaining the number of stem-like CD8+ T cells. These findings provide insight into the generation and maintenance of virus specific stem-like CD8+ T cells that play a critical role in chronic viral infection. In particular, our study highlights the early generation of stem-like CD8+ T cells and their ability to adapt to either an acute or chronic infection. These findings are of broad significance since these novel stem-like CD8+ T cells play an important role in not only viral infections but also in cancer and autoimmunity.

Titanium Dioxide (E171 Grade) and the Search For Replacement Opacifiers and Colorants: Supplier Readiness Survey, Case Studies and Regulatory Perspective.

Titanium dioxide (TiO2) is used primarily as an opacifier in solid dosage forms and is present in the majority of tablet and capsule dosage forms on the market. The IQ* TiO2 Working Group has previously shown that titanium dioxide has unique properties which are necessary for its function in these formulations and noted that, as the potential replacements lack the semi-conductor properties, high refractive index and whiteness of E171, it might be hard to replicate these properties with alternative materials. In this paper we detail the results of readiness surveys and practical assessments that have been conducted with alternative materials by IQ member companies. A range of technical challenges and regulatory hurdles were identified which mean that, in the short term, it may be difficult to replace titanium dioxide with the currently available alternative materials while readily achieving the same drug product quality attributes, especially for some of the marketed formulations that titanium dioxide is currently used for. We note the higher technical complexity, due to the variability, color fading and identified scale up risk, of E171 free film coatings and the likely impact on development costs and timelines. We also highlight several regulatory hurdles that would have to be overcome if a titanium dioxide replacement was required for some markets but was not mandated by others.

The Tox Gene Encodes Two Proteins with Distinct and Shared Roles in Gene Regulation.

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection-associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

The Role of Titanium Dioxide (E171) and the Requirements for Replacement Materials in Oral Solid Dosage Forms: An IQ Consortium Working Group Review.

Titanium dioxide (in the form of E171) is a ubiquitous excipient in tablets and capsules for oral use. In the coating of a tablet or in the shell of a capsule the material disperses visible and UV light so that the contents are protected from the effects of light, and the patient or caregiver cannot see the contents within. It facilitates elegant methods of identification for oral solid dosage forms, thus aiding in the battle against counterfeit products. Titanium dioxide ensures homogeneity of appearance from batch to batch fostering patient confidence. The ability of commercial titanium dioxide to disperse light is a function of the natural properties of the anatase polymorph of titanium dioxide, and the manufacturing processes used to produce the material utilized in pharmaceuticals. In some jurisdictions E171 is being considered for removal from pharmaceutical products, as a consequence of it being delisted as an approved colorant for foods. At the time of writing, in the view of the authors, no system or material which could address both current and future toxicological concerns of Regulators and the functional needs of the pharmaceutical industry and patients has been identified. This takes into account the assessment of materials such as calcium carbonate, talc, isomalt, starch and calcium phosphates. In this paper an IQ Consortium team outlines the properties of titanium dioxide and criteria to which new replacement materials should be held.

Giant Multilocular Prostatic Cystadenoma in a 14-Year-Old Male: A Case Report of a Pediatric Pelvic Mass.

Giant multilocular prostatic cystadenoma (GMC) is an extremely rare, benign tumor seen in both adult and pediatric males. The neoplasm originates from prostatic tissue and is typically found within the rectovesical pouch, varying in both size and morphology. Microscopically, GMC contains both glandular and cystic prostatic tissue lined by cuboidal and columnar epithelium. Symptoms often arise once the pelvic mass begins to obstruct the surrounding structures and organs, although invasion into surrounding tissue is unlikely. Common symptoms include abdominal pain, urinary retention, and dysuria. The standard treatment for GMC is surgical removal of the mass with good outcomes and only 1 known case of recurrence. Here we present the case of a 14-year-old male with GMC-the youngest patient reported to date-who presented with abdominal pain, difficulty voiding, and hydroureteronephrosis.

Natural experiments and long-term monitoring are critical to understand and predict marine host-microbe ecology and evolution.

Marine multicellular organisms host a diverse collection of bacteria, archaea, microbial eukaryotes, and viruses that form their microbiome. Such host-associated microbes can significantly influence the host's physiological capacities; however, the identity and functional role(s) of key members of the microbiome ("core microbiome") in most marine hosts coexisting in natural settings remain obscure. Also unclear is how dynamic interactions between hosts and the immense standing pool of microbial genetic variation will affect marine ecosystems' capacity to adjust to environmental changes. Here, we argue that significantly advancing our understanding of how host-associated microbes shape marine hosts' plastic and adaptive responses to environmental change requires (i) recognizing that individual host-microbe systems do not exist in an ecological or evolutionary vacuum and (ii) expanding the field toward long-term, multidisciplinary research on entire communities of hosts and microbes. Natural experiments, such as time-calibrated geological events associated with well-characterized environmental gradients, provide unique ecological and evolutionary contexts to address this challenge. We focus here particularly on mutualistic interactions between hosts and microbes, but note that many of the same lessons and approaches would apply to other types of interactions.

Persistence of self-reactive CD8+ T cells in the CNS requires TOX-dependent chromatin remodeling.

Self-reactive CD8+ T cells are important mediators of progressive tissue damage in autoimmune diseases, but the molecular program underlying these cells' functional adaptation is unclear. Here we characterize the transcriptional and epigenetic landscape of self-reactive CD8+ T cells in a mouse model of protracted central nervous system (CNS) autoimmunity and compare it to populations of CNS-resident memory CD8+ T cells emerging from acute viral infection. We find that autoimmune CD8+ T cells persisting at sites of self-antigen exhibit characteristic transcriptional regulation together with distinct epigenetic remodeling. This self-reactive CD8+ T cell fate depends on the transcriptional regulation by the DNA-binding HMG-box protein TOX which remodels more than 400 genomic regions including loci such as Tcf7, which is central to stemness of CD8+ T cells. Continuous exposure to CNS self-antigen sustains TOX levels in self-reactive CD8+ T cells, whereas genetic ablation of TOX in CD8+ T cells results in shortened persistence of self-reactive CD8+ T cells in the inflamed CNS. Our study establishes and characterizes the genetic differentiation program enabling chronic T cell-driven immunopathology in CNS autoimmunity.

Inhibition of the CCL2 receptor, CCR2, enhances tumor response to immune checkpoint therapy.

Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8+ T cell recruitment and activation and a concomitant decrease in CD4+ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies.

Erratum: Describing the dynamic translational science landscape through core voucher utilization - ERRATUM.

[This corrects the article DOI: 10.1017/cts.2019.4.].

Describing the dynamic translational science landscape through Core Voucher utilization.

INTRODUCTION: Core facilities play crucial roles in carrying out the academic research mission by making available to researchers advanced technologies, facilities, or expertise that are unfeasible for most investigators to obtain on their own. To facilitate translational science through support of core services, the University of California, Los Angeles Clinical and Translational Science Institute (UCLA CTSI) created a Core Voucher program. The underlying premise is that by actively promoting interplay between researchers and core facilities, a dynamic feedback loop could be established that could enhance both groups, the productivity of the former and the relevance of the latter. Our primary goal was to give translational investigators what they need to pursue their immediate projects at hand. METHODS: To implement this system across four noncontiguous campuses, open-source web-accessible software applications were created that were scalable and could efficiently administer investigator submissions and subsequent reviews in a multicampus fashion. RESULTS: In the past five years, we have processed over 1400 applications submitted by over 750 individual faculty members across both clinical and nonclinical departments. In total, 1926 core requests were made in conjunction with 1467 submitted proposals. The top 10 most popular cores accounted for 50% of all requests, and the top half of the most popular cores accounted for 90% of all requests. CONCLUSION: Tracking investigator demand provides a unique window into what are the high- and low-priority core services that best support translational research.

TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion.

Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program.

TOX is a critical regulator of tumour-specific T cell differentiation.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

Non-integumentary melanosomes can bias reconstructions of the colours of fossil vertebrates.

The soft tissues of many fossil vertebrates preserve evidence of melanosomes-micron-scale organelles that inform on integumentary coloration and communication strategies. In extant vertebrates, however, melanosomes also occur in internal tissues. Hence, fossil melanosomes may not derive solely from the integument and its appendages. Here, by analyzing extant and fossil frogs, we show that non-integumentary melanosomes have high fossilization potential, vastly outnumber those from the skin, and potentially dominate the melanosome films preserved in some fossil vertebrates. Our decay experiments show that non-integumentary melanosomes usually remain in situ provided that carcasses are undisturbed. Micron-scale study of fossils, however, demonstrates that non-integumentary melanosomes can redistribute through parts of the body if carcasses are disturbed by currents. Collectively, these data indicate that fossil melanosomes do not always relate to integumentary coloration. Integumentary and non-integumentary melanosomes can be discriminated using melanosome geometry and distribution. This is essential to accurate reconstructions of the integumentary colours of fossil vertebrates.

Expression of the DNA-Binding Factor TOX Promotes the Encephalitogenic Potential of Microbe-Induced Autoreactive CD8+ T Cells.

Infections are thought to trigger CD8+ cytotoxic T lymphocyte (CTL) responses during autoimmunity. However, the transcriptional programs governing the tissue-destructive potential of CTLs remain poorly defined. In a model of central nervous system (CNS) inflammation, we found that infection with lymphocytic choriomeningitis virus (LCMV), but not Listeria monocytogenes (Lm), drove autoimmunity. The DNA-binding factor TOX was induced in CTLs during LCMV infection and was essential for their encephalitogenic properties, and its expression was inhibited by interleukin-12 during Lm infection. TOX repressed the activity of several transcription factors (including Id2, TCF-1, and Notch) that are known to drive CTL differentiation. TOX also reduced immune checkpoint sensitivity by restraining the expression of the inhibitory checkpoint receptor CD244 on the surface of CTLs, leading to increased CTL-mediated damage in the CNS. Our results identify TOX as a transcriptional regulator of tissue-destructive CTLs in autoimmunity, offering a potential mechanistic link to microbial triggers.

Development and differentiation of early innate lymphoid progenitors.

Early innate lymphoid progenitors (EILPs) have recently been identified in mouse adult bone marrow as a multipotential progenitor population specified toward innate lymphoid cell (ILC) lineages, but their relationship with other described ILC progenitors is still unclear. In this study, we examine the progenitor-successor relationships between EILPs, all-lymphoid progenitors (ALPs), and ILC precursors (ILCps). Functional, bioinformatic, phenotypical, and genetic approaches collectively establish EILPs as an intermediate progenitor between ALPs and ILCps. Our work additionally provides new candidate regulators of ILC development and clearly defines the stage of requirement of transcription factors key for early ILC development.

Alternative activation generates IL-10 producing type 2 innate lymphoid cells.

Type 2 innate lymphoid cells (ILC2) share cytokine and transcription factor expression with CD4+ Th2 cells, but functional diversity of the ILC2 lineage has yet to be fully explored. Here, we show induction of a molecularly distinct subset of activated lung ILC2, termed ILC210. These cells produce IL-10 and downregulate some pro-inflammatory genes. Signals that generate ILC210 are distinct from those that induce IL-13 production, and gene expression data indicate that an alternative activation pathway leads to the generation of ILC210. In vivo, IL-2 enhances ILC210 generation and is associated with decreased eosinophil recruitment to the lung. Unlike most activated ILC2, the ILC210 population contracts after cessation of stimulation in vivo, with maintenance of a subset that can be recalled by restimulation, analogous to T-cell effector cell and memory cell generation. These data demonstrate the generation of a previously unappreciated IL-10 producing ILC2 effector cell population.

UniEuk: Time to Speak a Common Language in Protistology!

Universal taxonomic frameworks have been critical tools to structure the fields of botany, zoology, mycology, and bacteriology as well as their large research communities. Animals, plants, and fungi have relatively solid, stable morpho-taxonomies built over the last three centuries, while bacteria have been classified for the last three decades under a coherent molecular taxonomic framework. By contrast, no such common language exists for microbial eukaryotes, even though environmental '-omics' surveys suggest that protists make up most of the organismal and genetic complexity of our planet's ecosystems! With the current deluge of eukaryotic meta-omics data, we urgently need to build up a universal eukaryotic taxonomy bridging the protist -omics age to the fragile, centuries-old body of classical knowledge that has effectively linked protist taxa to morphological, physiological, and ecological information. UniEuk is an open, inclusive, community-based and expert-driven international initiative to build a flexible, adaptive universal taxonomic framework for eukaryotes. It unites three complementary modules, EukRef, EukBank, and EukMap, which use phylogenetic markers, environmental metabarcoding surveys, and expert knowledge to inform the taxonomic framework. The UniEuk taxonomy is directly implemented in the European Nucleotide Archive at EMBL-EBI, ensuring its broad use and long-term preservation as a reference taxonomy for eukaryotes.