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Naive CD4+ T lymphocytes initially undergo antigen-specific activation to promote a broad-spectrum response before adopting bespoke cytokine expression profiles shaped by intercellular microenvironmental cues, resulting in pathogen-focused modular cytokine responses. Interleukin (IL)-4-induced Gata3 upregulation is important for the helper type 2 T cell (TH2 cell) polarization associated with anti-helminth immunity and misdirected allergic inflammation. Whether additional microenvironmental factors participate is unclear. Using whole mouse-genome CRISPR-Cas9 screens, we discovered a previously unappreciated role for αvβ3 integrin in TH2 cell differentiation. Low-level αvβ3 expression by naive CD4+ T cells contributed to pan-T cell activation by promoting T-T cell clustering and IL-2/CD25/STAT5 signaling. Subsequently, IL-4/Gata3-induced selective upregulation of αvβ3 licensed intercellular αvβ3-Thy1 interactions among TH2 cells, enhanced mammalian target of rapamycin (mTOR) signaling, supported differentiation and promoted IL-5/IL-13 production. In mice, αvβ3 was required for efficient, allergen-driven, antigen-specific lung TH2 cell responses. Thus, αvβ3-expressing TH2 cells form multicellular factories to propagate and amplify TH2 cell responses.

Peer reviewed: True, The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4β7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

Interleukin-25 (IL-25) and group 2 innate lymphoid cells (ILC2s) defend the host against intestinal helminth infection and are associated with inappropriate allergic reactions. IL-33–activated ILC2s were previously found to augment protective tissue-specific pancreatic cancer immunity. Here, we showed that intestinal IL-25–activated ILC2s created an innate cancer-permissive microenvironment. Colorectal cancer (CRC) patients with higher tumor IL25 expression had reduced survival and increased IL-25R–expressing tumor-resident ILC2s and myeloid-derived suppressor cells (MDSCs) associated with impaired antitumor responses. Ablation of IL-25 signaling reduced tumors, virtually doubling life expectancy in an Apc mutation–driven model of spontaneous intestinal tumorigenesis. Mechanistically, IL-25 promoted intratumoral ILC2s, which sustained tumor-infiltrating MDSCs to suppress antitumor immunity. Therapeutic antibody-mediated blockade of IL-25 signaling decreased intratumoral ILC2s, MDSCs, and adenoma/adenocarcinoma while increasing antitumor adaptive T cell and interferon-γ (IFN-γ)–mediated immunity. Thus, the roles of innate epithelium-derived cytokines IL-25 and IL-33 as well as ILC2s in cancer cannot be generalized. The protumoral nature of the IL-25–ILC2 axis in CRC highlights this pathway as a potential therapeutic target against CRC.

Naive CD4

Summary Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated “5x polychromILC” transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues., Highlights • Five-color “polychromILC” transcription factor reporter mice define ILC precursors • ILC precursors give rise to ILC1, ILC2, and ILC3 and retain NK potential • A RorcKat allele allows resolution of extremely rare ILC3 progenitors • Detection of divergent trajectories for ILC2 and common ILC1, ILC3, and NK development, Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. Walker et al. demonstrate via five-reporter polychrome mice the distinct divergence of ILC2 development from common ILC1, ILC3, and NK progenitors.

The HumaPen Luxura HD insulin pen (Eli Lilly and Company, Indianapolis, IN) was originally designed to deliver accurate doses in half-unit increments from 1 to 30 units. Laboratory testing examined the accuracy of the initial 0.5-unit dose within a 95/95% tolerance interval with respect to a specification of +/-0.5 unit (+/-0.005 ml).After priming, operators recorded the first 0.5 unit. Data were analyzed using k-value targets.While examining 577 half-unit doses per device lot, test temperature, operator, or test liquid, at least 95% of the doses were accurate with 95% confidence. All data points were within +/-0.5 unit (+/-0.005 ml).Dose accuracy of the initial half-unit is achieved with the HumaPen Luxura HD insulin pen.

This study compared injection force (measured by glide force [GF] and glide force variability [GFV]) and dosing accuracy of the Humalog KwikPen * (prefilled insulin lispro [Humalog dagger] pen, Eli Lilly and Company, Indianapolis, IN) and the Next Generation FlexPen double dagger (prefilled insulin aspart [NovoRapid section sign] pen, Novo Nordisk A/S, Bagsvaerd, Denmark). * Humalog KwikPen is a registered trademark of Eli Lilly and Company, Indianapolis, IN, USA. dagger Humalog is a registered trademark of Eli Lilly and Company, Indianapolis, IN, USA. double dagger FlexPen is a registered trademark of Novo Nordisk A/S, Bagsvaerd, Denmark. section sign NovoRapid is a registered trademark of Novo Nordisk A/S, Bagsvaerd, Denmark.A total of 100 prefilled insulin pens (50 insulin lispro pens, 50 insulin aspart pens) were tested using two dose sizes (30 U and 60 U). In all, 50 devices (25 of each type) were tested at 10 U/s dosing speed and 50 were tested at 6.6 U/s. Devices were used per manufacturer instructions. Dose accuracy (represented as absolute dose error %), maximum and average GF, and GFV data were automatically collected by the test system for all datasets (dose size/dosing speed/device type). The test system controlled for potential dosing errors.The insulin lispro pen demonstrated a significantly lower median maximum GF at both dosing speeds: (2.83 vs. 3.92 lbs [30 U] and 3.00 vs. 4.14 lbs [60 U]) at 10 U/s; (1.85 vs. 2.93 lbs [30 U] and 2.14 vs. 3.02 lbs [60 U]) at 6.6 U/s, all p0.0001. For all datasets, the median GFV was significantly lower for the insulin lispro pen, p0.0001. Median dose error was comparable between device types when tested at 10 U/s dosing speed; however, at 6.6 U/s, the median dose error was significantly lower for insulin lispro pen compared to insulin aspart pen (0.47 vs. 0.67% [30 U] and 0.50 vs. 0.78% [60 U], both p0.05).The insulin lispro pen had significantly lower median GF and GFV compared with insulin aspart pen when tested at two dose sizes and two dosing speeds. Median dose error was similar between the device types at the 10 U/s dosing speed, but median dose error was significantly lower for the insulin lispro pen at the 6.6 U/s dosing speed. A limitation of this study was that it was executed as an open label study.

Objective: Comprehensive analysis of the 9p21 locus including the CDKN2A , ARF , and CDKN2B genes in 53 individuals from melanoma index cases considered to be at heightened risk of melanoma. Methods and Results: Using a combination of DNA sequencing, gene copy number by real time quantitative PCR, linkage analysis, and transcript analysis in haploid somatic cell hybrids, we found no evidence for germline alteration in either coding or non-coding domains of CDKN2A and CDKN2B . However, we identified a p14ARF exon 1β missense germline mutation (G16D) in a melanoma-neural system tumour syndrome (CMM+NST) family and a 8474 bp germline deletion from 196 bp upstream of p14ARF exon 1β initiation codon to 11233 bp upstream of exon 1α of p16INK4A in a family with five melanoma cases. For three out of 10 families with at least three melanoma cases, the disease gene was unlinked to the 9p21 region, while linkage analysis was not fully conclusive for seven families. Conclusions: These data reinforce the hypothesis that ARF is a melanoma susceptibility gene and suggest that germline deletions specifically affecting p14ARF may not be solely responsible for NST susceptibility. Predisposition to CMM+NST could either be due to complete disruption of the CDKN2A locus or be the result of more complex genetic inheritance. In addition, the absence of any genetic alteration in 50 melanoma prone families or patients suggests the presence of additional tumour suppressor genes possibly in the 9p21 region, and on other chromosomes.

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

The alternative product of the human INK4a/ARF locus, p14ARF, has the potential to act as a tumour suppressor by binding to and inhibiting the p53 antagonist MDM2. Current models propose that ARF function depends on its ability to sequester MDM2 in the nucleolus. Here we describe situations in which stabilization of MDM2 and p53 occur without relocalization of endogenous MDM2 from the nucleoplasm. Conversely, forms of ARF that do not accumulate in the nucleolus retain the capacity to stabilize MDM2 and p53. We therefore propose that nucleolar localization is not essential for ARF function but may enhance the availability of ARF to inhibit MDM2.

The development of cluster primary ion sources such as Aun+, Bin+, SF5+, C60+, and Arn+ has been an exciting advancement in SIMS analysis. Relative to atomic primary ion sources, cluster ion sources provide higher secondary ion yields. Furthermore, C60+ and Arn+ impart significantly less chemical damage to the sample thus enabling molecular depth profiling. Molecular depth profiling using cluster primary ion sources is routinely used to characterize a wide range of commercially important materials, including organic light emitting diode, biomaterials and pharmaceuticals, adhesives, and architectural paints and coatings. This paper highlights the application of time of flight secondary ion mass spectrometry (ToF-SIMS) to study contact lenses and acrylic-based paints. In the first application, ToF-SIMS was used to investigate the surface composition of two commercial contact lenses. Lens material I is composed of 2-hydroxy-ethyl methacrylate (HEMA) and glycerol methacrylate while lens material II is compose...

To determine patient ease of use and preference for the Humalog KwikPen* (prefilled insulin lispro [Humalog dagger] pen, Eli Lilly and Company, Indianapolis, IN, USA) (insulin lispro pen) versus the Next Generation FlexPen double dagger (prefilled insulin aspart [NovoRapid section sign ] pen, Novo Nordisk A/S, Bagsvaerd, Denmark) (insulin aspart pen).This was a randomized, open-label, 2-period, 8-sequence crossover study in insulin pen-naïve patients with diabetes. Randomized patients (N = 367) received device training, then simulated low- (15 U) and high- (60 U) dose insulin injections with an appliance. Patients rated pens using an ease of use questionnaire and were asked separately for final pen preferences.The Insulin Device 'Ease of Use' Battery is a 10-item questionnaire with a 7-point scale (higher scores reflect greater ease of use). The primary objective was to determine pen preference for 'easy to press to inject my dose' (by comparing composite scores [low- plus high-dose]). Secondary objectives were to determine pen preference on select questionnaire items (from composite scores), final pen preference, and summary responses for all questionnaire items.On the primary endpoint, 'easy to press to inject my dose,' a statistically significant majority of patients with a preference chose the insulin lispro pen over the insulin aspart pen (68.4%, 95% CI = 62.7-73.6%). Statistically significant majorities of patients with a preference also favored the insulin lispro pen on secondary items: 'easy to hold in my hand when I inject' (64.9%, 95% CI = 58.8-70.7%), 'easy to use when I am in a public place' (67.5%, 95% CI = 61.0-73.6%), and 'overall easy to use' (69.9%, 95% CI = 63.9-75.4%). A statistically significant majority of patients had a final preference for the insulin lispro pen (67.3%, 95% CI = 62.2-72.1%).Among pen-naïve patients with diabetes who had a preference, the majority preferred the insulin lispro pen over the insulin aspart pen with regard to ease of use. Study limitations included open-label design and injection simulation, use of an unvalidated questionnaire, and enrollment of mostly insulin-naïve patients.

The ARF protein, encoded by alternate exon usage within the CDKN2A locus, provides a link between the retinoblastoma (pRb) and p53 tumor suppressor pathways. Agents that disable pRb or otherwise impinge on the E2F family of transcription factors induce expression of ARF, resulting in stabilization of p53 and activation of p53-regulated genes. However, in some cell types ARF is not induced upon cell cycle re-entry, as expected of a conventional E2F target gene, leading to the suggestion that the ARF promoter only responds to supra-physiological or aberrant levels of E2F. These properties have recently been attributed to a variant E2F binding site but attempts to map specific response elements within the ARF promoter have generally yielded confusing answers. Here we show that in IL2-dependent T-lymphocytes, ARF expression is induced as cells progress from G(0) into S phase, in parallel with other bona fide E2F target genes. This is accompanied by increased association of E2F1 with the endogenous ARF promoter. Our findings suggest that the ability of ARF to register normal proliferative cues depends on the levels of E2F generated in different settings and argue against the idea that it reacts exclusively to oncogenic signals.

The small basic protein p14ARF, encoded by one of the alternative transcripts from the human INK4A/ARF locus, interferes with MDM2-mediated ubiquitination of the p53 tumour suppressor protein. The resultant stabilization of p53 leads to increased expression of p53-regulated genes, such as MDM2 itself and the cyclin-dependent kinase inhibitor p21(CIP1). Here we relate physical interactions between p14ARF and MDM2, as determined using synthetic peptides and systematic deletions of p14ARF, with consequential effects on p53 stabilization and transcriptional activity. The data imply that the amino terminal half of p14ARF, encoded by the alternative first exon (exon 1beta) contacts MDM2 through multiple domains that can independently impede MDM2-mediated degradation of p53, provided that they are localized in the cell nucleus. As well as identifying previously unrecognized functional domains, our findings offer an explanation for the relative paucity of missense mutations in exon 1beta in human tumours.

We have studied the gene of the human β2 integrin subunit (CD18) and found it to be organised into 16 exons spanning a region of about 40 kb. All exon/intron boundaries conform to the GT/AG splicing consensus. The exons coding for the cysteine-rich region, which has been postulated to consist of 3 or 4 repeating elements, are not organised correspondingly. Transcription of the gene initiates from multiple sites which may be due to the absence of an upstream TATA box. The polyadenylation site is also heterogeneous. Five different sites were identified over a stretch of 10 bases.

Most human cancers show perturbation of growth regulation mediated by the tumour-suppressor proteins retinoblastoma (RB) and p53 (ref. 1), indicating that loss of both pathways is necessary for tumour development. Loss of RB function leads to abnormal proliferation related to the deregulation of the E2F transcription factors, but also results in the activation of p53, which suppresses cell growth. Here we show that E2F-1 directly activates expression of the human tumour-suppressor protein p14ARF (the mouse homologue is called p19ARF), which binds to the MDM2-p53 complex and prevents p53 degradation2,5. These results complete a pathway linking abnormal proliferative signals, such as loss of RB, with the activation of a p53 response, through E2F-1 and p14ARF. They suggest that E2F-1, a protein inherently activated by cell-cycle progression, is part of a fail-safe mechanism to protect against aberrant cell growth.