Supp tables 1 and 2. Supplementary Table 1 Table of patient clinical characteristics for patient derived samples Supplementary Table 2 PCR Primer sequences

Abstract Background We aimed to predict response to biologics in inflammatory bowel disease (IBD) using computerized image analysis of probe confocal laser endomicroscopy (pCLE) in vivo and assess the binding of fluorescent-labeled biologics ex vivo. Additionally, we investigated genes predictive of anti-tumor necrosis factor (TNF) response. Methods Twenty-nine patients (15 with Crohn’s disease [CD], 14 with ulcerative colitis [UC]) underwent colonoscopy with pCLE before and 12 to 14 weeks after starting anti-TNF or anti-integrin α4β7 therapy. Biopsies were taken for fluorescein isothiocyanate–labeled infliximab and vedolizumab staining and gene expression analysis. Computer-aided quantitative image analysis of pCLE was performed. Differentially expressed genes predictive of response were determined and validated in a public cohort. Results In vivo, vessel tortuosity, crypt morphology, and fluorescein leakage predicted response in UC (area under the receiver-operating characteristic curve [AUROC], 0.93; accuracy 85%, positive predictive value [PPV] 89%; negative predictive value [NPV] 75%) and CD (AUROC, 0.79; accuracy 80%; PPV 75%; NPV 83%) patients. Ex vivo, increased binding of labeled biologic at baseline predicted response in UC (UC) (AUROC, 83%; accuracy 77%; PPV 89%; NPV 50%) but not in Crohn’s disease (AUROC 58%). A total of 325 differentially expressed genes distinguished responders from nonresponders, 86 of which fell within the most enriched pathways. A panel including ACTN1, CXCL6, LAMA4, EMILIN1, CRIP2, CXCL13, and MAPKAPK2 showed good prediction of anti-TNF response (AUROC >0.7). Conclusions Higher mucosal binding of the drug target is associated with response to therapy in UC. In vivo, mucosal and microvascular changes detected by pCLE are associated with response to biologics in inflammatory bowel disease. Anti-TNF–responsive UC patients have a less inflamed and fibrotic state pretreatment. Chemotactic pathways involving CXCL6 or CXCL13 may be novel targets for therapy in nonresponders.

Supplementary Figure Legends. Supplementary Figure Legends for Supp Figs 1-5 Contains Legends for: Supplementary Figure 1: Arginase II is the principal arginase isoform in neuroblastoma Supplementary Figure 2: Arginase II expression by neuroblastoma tumours creates a suppressive microenvironment Supplementary Figure 3: Neuroblastoma alters myeloid cell frequency in a murine model Supplementary Figure 4: Neuroblastoma arginase inactivates CD14+ cells and CAR T cells Supplementary Figure 5: Arginase 2 and CD14 expression are associated with a worse overall survival.

Supplemental figures 1-5. Supplementary Figure 1: Arginase II is the principal arginase isoform in neuroblastoma Supplementary Figure 2: Arginase II expression by neuroblastoma tumours creates a suppressive microenvironment Supplementary Figure 3: Neuroblastoma alters myeloid cell frequency in a murine model Supplementary Figure 4: Neuroblastoma arginase inactivates CD14+ cells and CAR T cells Supplementary Figure 5: Arginase 2 and CD14 expression are associated with a worse overall survival.

Supplementary Materials and Methods. Supplementary Materials and Methods

Supported work internship programmes for young adults with disability are an evidence-based model, leading to greater employment outcomes. This mixed methods pilot study evaluated the experiences of students, supervisors and a project coordinator, who participated in an Integrated Practical Placement (IPP) programme for students with disability in Australia. Intervention students (n = 10) completed 3, 9-week rotations, and accessed personal placement support and employment coaches. Comparison students (n = 38) completed 3, 2-week placements without additional support. Intervention students perceived significantly greater initial changes in work skills ( p < 0.01) and work readiness ( p < 0.05). Intervention students reported development of communication and self-organisational skills and stressed the value of staff support. Post programme 70% of intervention students gained employment, compared with 15.4% of comparison students. The findings suggest an evidence-based supported employment programme emphasising personalised assessment and training, could provide individuals with disability the required skills to enable successful employment.

Risk prediction for psychosis has advanced to the stage at which it could feasibly become a clinical reality. Neuroimaging biomarkers play a central role in many risk prediction models. Using such models to predict the likelihood of transition to psychosis in individuals known to be at high risk has the potential to meaningfully improve outcomes, principally through facilitating early intervention. However, this compelling benefit must be evaluated in light of the broader ethical ramifications of this prospective development in clinical practice. This paper advances ethical discussion in the field in two ways: firstly, through in-depth consideration of the distinctive implications of the clinical application of predictive tools; and, secondly, by evaluating the manner in which newer predictive models incorporating neuroimaging alter the ethical landscape. We outline the current state of the science of predictive testing for psychosis, with a particular focus on emerging neuroimaging biomarkers. We then proceed to ethical analysis employing the four principles of biomedical ethics as a conceptual framework. We conclude with a call for scientific advancement to proceed in tandem with ethical consideration, informed by empirical study of the views of high risk individuals and their families. This collaborative approach will help ensure that predictive testing progresses in an ethically acceptable manner that minimizes potential adverse effects and maximizes meaningful benefits for those at high risk of psychosis.

Aims Several factors are known to increase risk for cerebrovascular disease and dementia, but there is limited evidence on associations between multiple vascular risk factors (VRFs) and detailed aspects of brain macro- and microstructure in large community-dwelling populations across middle- and older age. Methods and Results Associations between VRFs (smoking, hypertension, pulse pressure, diabetes, hypercholersterolaemia, BMI, and waist-hip ratio) and both global and regional brain structural and diffusion MRI markers were examined in UK Biobank ( N = 9722, age range 44-77 years). A larger number of VRFs was associated with greater brain atrophy, lower grey matter volume, and poorer white matter health. Effect sizes were small (brain structural R 2 ≤ 1.8%). Higher aggregate vascular risk was related to multiple regional MRI hallmarks associated with dementia risk: lower frontal and temporal cortical volumes, lower subcortical volumes, higher white matter hyperintensity volumes, and poorer white matter microstructure in association and thalamic pathways. Smoking pack years, hypertension and diabetes showed the most consistent associations across all brain measures. Hypercholesterolaemia was not uniquely associated with any MRI marker. Conclusion Higher levels of VRFs were associated with poorer brain health across grey and white matter macro- and microstructure. Effects are mainly additive, converging upon frontal and temporal cortex, subcortical structures, and specific classes of white matter fibres. Though effect sizes were small, these results emphasise the vulnerability of brain health to vascular factors even in relatively healthy middle and older age, and the potential to partly ameliorate cognitive decline by addressing these malleable risk factors.

There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6+CD4+ and AhR+CD4+ T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1β. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies.

The search for diagnostic and prognostic biomarkers in schizophrenia care and treatment is the focus of many within the research community. Longitudinal cohorts of patients presenting at elevated genetic and clinical risk have provided a wealth of data that has informed our understanding of the development of schizophrenia and related psychotic disorders. Imaging follow-up of high-risk cohorts has demonstrated changes in cerebral grey matter of those that eventually transition to schizophrenia that predate the onset of symptoms and evolve over the course of illness. Longitudinal follow-up studies demonstrate that observed grey matter changes can be employed to differentiate those who will transition to schizophrenia from those who will not prior to the onset of the disorder. In recent years our understanding of the genetic makeup of schizophrenia has advanced significantly. The development of modern analysis techniques offers researchers the ability to objectively quantify genetic risk; these have been successfully applied within a high-risk paradigm to assist in differentiating between high-risk individuals who will subsequently become unwell and those who will not. This chapter will discuss the application of imaging and genetic biomarkers within high-risk groups to predict future transition to schizophrenia and related psychotic disorders. We aim to provide an overview of current approaches focussing on grey matter changes that are predictive of future transition to illness, the developing field of genetic risk scores and other methods being developed to aid clinicians in diagnosis and prognosis.

Sex differences in the human brain are of interest for many reasons: for example, there are sex differences in the observed prevalence of psychiatric disorders and in some psychological traits that brain differences might help to explain. We report the largest single-sample study of structural and functional sex differences in the human brain (2750 female, 2466 male participants; mean age 61.7 years, range 44-77 years). Males had higher raw volumes, raw surface areas, and white matter fractional anisotropy; females had higher raw cortical thickness and higher white matter tract complexity. There was considerable distributional overlap between the sexes. Subregional differences were not fully attributable to differences in total volume, total surface area, mean cortical thickness, or height. There was generally greater male variance across the raw structural measures. Functional connectome organization showed stronger connectivity for males in unimodal sensorimotor cortices, and stronger connectivity for females in the default mode network. This large-scale study provides a foundation for attempts to understand the causes and consequences of sex differences in adult brain structure and function.

Background & Aims:cd T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human cd T cells is primarily based on peripheral blood subsets, while the immunobiology of tissueassociated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of cd T cells in the human liver. Methods:We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating cd T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vd2 cd subset, which is implicated in liver immunopathology. Results: Intrahepatic Vd2 cd T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27 effector lymphocytes, whereas naïve CD27, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA Vd2 cd effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vd2 cd T cell pool also included a phenotypically distinct CD45RA effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vd2 cd cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli.

OPINION article Front. Immunol., 25 September 2018Sec. T Cell Biology Volume 9 - 2018 | https://doi.org/10.3389/fimmu.2018.02106

Background & Aims: gamma delta T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human gamma delta T cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of gamma delta T cells in the human liver. Methods: We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating gamma delta T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated V delta 2(-) gamma delta subset, which is implicated in liver immunopathology. Results: Intrahepatic V delta 2(-) gamma delta T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27(lo/-) effector lymphocytes, whereas naive CD27(hi), TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA(hi) V delta 2(-) gamma delta effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver V delta 2(-) gamma delta T cell pool also included a phenotypically distinct CD45RA(lo) effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating V delta 2(-) gamma delta cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli. Conclusion: These findings suggest that the ability of V delta 2(-) gamma delta T cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues, such as the liver, which results in functionally distinct peripheral and liver-resident memory gamma delta T cell subsets. They also highlight the inherent functional plasticity within the V delta 2(-) gamma delta T cell compartment and provide information that could be used for the design of cellular therapies that suppress liver inflammation or combat liver cancer. Lay summary: gamma delta T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic gamma delta T cells are enriched for clonally expanded effector T cells, whereas naive gamma delta T cells are largely excluded. Moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident gamma delta T cells was also evident. Our findings suggest that factors triggering gamma delta T cell clonal selection and differentiation, such as infection, can drive enrichment of gamma delta T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance. (C) 2018 European Association for the Study of the Liver. Published by Elsevier B.V.

Sex differences in the human brain are of interest, for example because of sex differences in the observed prevalence of psychiatric disorders and in some psychological traits. We report the largest single-sample study of structural and functional sex differences in the human brain (2,750 female, 2,466 male participants; 44-77 years). Males had higher volumes, surface areas, and white matter fractional anisotropy; females had thicker cortices and higher white matter tract complexity. There was considerable distributional overlap between the sexes. Subregional differences were not fully attributable to differences in total volume or height. There was generally greater male variance across structural measures. Functional connectome organization showed stronger connectivity for males in unimodal sensorimotor cortices, and stronger connectivity for females in the default mode network. This large-scale study provides a foundation for attempts to understand the causes and consequences of sex differences in adult brain structure and function.

Vδ2+ T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2+ compartment comprises both innate-like and adaptive subsets. Vγ9+ Vδ2+ T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9- Vδ2+ T-cell subset that typically has a CD27hiCCR7+CD28+IL-7Rα+ naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27loCD45RA+CX3CR1+granzymeA/B+ effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9- Vδ2+ T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2+ T-cell compartment into innate-like (Vγ9+) and adaptive (Vγ9-) subsets, which have distinct functions in microbial immunosurveillance.

γδ T cells are unconventional lymphocytes commonly described as 'innate-like' in function, which can respond in both a T cell receptor (TCR)-independent and also major histocompatibility complex (MHC)-unrestricted TCR-dependent manner. While the relative importance of TCR recognition had remained unclear, recent studies revealed that human Vδ1 T cells display unexpected parallels with adaptive αβ T cells. Vδ1 T cells undergo profound and highly focussed clonal expansion from an initially diverse and private TCR repertoire, most likely in response to specific immune challenges. Concomitantly, they differentiate from a Vδ1 T cell naïve (Tnaïve) to a Vδ1 T cell effector (Teffector) phenotype, marked by the downregulation of lymphoid homing receptors and upregulation of peripheral homing receptors and effector markers. This suggests that an adaptive paradigm applies to Vδ1 T cells, likely involving TCR-dependent but MHC-unrestricted responses to microbial and non-microbial challenges.

γδ T cells are considered to be innate-like lymphocytes that respond rapidly to stress without clonal selection and differentiation. Here we use next-generation sequencing to probe how this paradigm relates to human Vδ2neg T cells, implicated in responses to viral infection and cancer. The prevalent Vδ1 T cell receptor (TCR) repertoire is private and initially unfocused in cord blood, typically becoming strongly focused on a few high-frequency clonotypes by adulthood. Clonal expansions have differentiated from a naive to effector phenotype associated with CD27 downregulation, retaining proliferative capacity and TCR sensitivity, displaying increased cytotoxic markers and altered homing capabilities, and remaining relatively stable over time. Contrastingly, Vδ2+ T cells express semi-invariant TCRs, which are present at birth and shared between individuals. Human Vδ1+ T cells have therefore evolved a distinct biology from the Vδ2+ subset, involving a central, personalized role for the γδ TCR in directing a highly adaptive yet unconventional form of immune surveillance.

A disease-associated variant of an activating ligand engages receptors so strongly that it impairs NK cell–mediated killing.

Introduction Innate lymphoid cells (ILCs) involve in the initiation, regulation and resolution of inflammation. Although the role of ILCs in murine liver fibrosis and biliary proliferation has been reported, the phenotypic characteristics and functional role of these cells in human liver disease remains undefined. We explored the detailed phenotype of human intrahepatic ILCs to gain insight on their function. Methods Liver infiltrating lymphocytes from normal liver tissue, autoimmune liver diseases (AILD), alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH) explants were phenotyped with flow cytometry. Results Total intrahepatic ILC (CD3− lineage− CD45− CD127+ ) comprised 1% (IQR 0.4–1.7%) of the CD3− CD45+ population in normal liver and 0.4% for ALD/NASH and autoimmune livers. The ILC1 subset constituted the majority of intrahepatic ILC and its frequency is higher in normal liver compared to diseased livers (85% vs. 67%) (p < 0.05). In contrast, CD161+CRTH2+ ILC2 subset and ILC3 subset frequency are higher in diseased livers compared to normal livers. Interestingly, frequency of ILC3 is significantly higher in ALD compared to normal liver (p < 0.01). Intrahepatic ILC subsets are in activated and tissue resident state (CD69 75–90%). All subsets expressed the liver tissue homing chemokine receptor, CXCR3. Expression frequency is highest on diseased ILC1 (AILD 50%, ALD/NASH 56%,) compared to ILC2 (AILD 24%; ALD/NASH 15%) and ILC3 (AILD 38%, ALD/NASH 28%). Biliary tropic receptor CCR6 was expressed more highly by ILC subsets in diseased compared to normal livers. The fibronectin receptor, VLA5 and laminin receptor VLA6 were both highly expressed on all intrahepatic ILC subsets (60–90% and 55–80% respectively). In terms of the cytokine receptors expressed, on all ILC subsets, IL-6R was near undetectable (2%) and low expressions of ST2 (3–6%), IL-25R (2–5%) and IL-23R (2–7%) were detected. In contrast there was almost ubiquitous expression of IL18Ralpha. CD25 was also very highly expressed. Of note, CD25 was significantly higher in ILC1 and ILC3 subsets from normal compared to AILD (p < 0.01) livers (40% vs. 80%; 66% vs. 93%) while intrahepatic ILC2 and ILC3 subsets in diseased states highly expressed CD25 (>90%). Of likely functional importance, intrahepatic ILC1 expressed IFN-γ (40%) while ILC2 expressed IL-13 (20–50%) in diseased states. Conclusion We report for the first time the presence of all three ILC subsets within the human liver immune cell infiltrates. CXCR3+ IFN-γ expressing ILC1 subset is enriched in both normal and inflamed diseased livers. Higher frequencies of CCR6+ VLA5+ VLA6+ IL-13 expressing ILC2 are observed in diseased livers suggesting that this subset may play a role in biliary pathology and peri-biliary fibrosis. Disclosure of Interest None Declared