FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses.

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture.

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.

CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity.

CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.

Machining water through laser cutting of nanoparticle-encased water pancakes.

Due to the inherent disorder and fluidity of water, precise machining of water through laser cutting are challenging. Herein we report a strategy that realizes the laser cutting machining of water through constructing hydrophobic silica nanoparticle-encased water pancakes with sub-millimeter depth. Through theoretical analysis, numerical simulation, and experimental studies, the developed process of nanoparticle-encased water pancake laser cutting and the parameters that affect cutting accuracy are verified and elucidated. We demonstrate that laser-fabricated water patterns can form diverse self-supporting chips (SSCs) with openness, transparency, breathability, liquid morphology, and liquid flow control properties. Applications of laser-fabricated SSCs to various fields, including chemical synthesis, biochemical sensing, liquid metal manipulation, patterned hydrogel synthesis, and drug screening, are also conceptually demonstrated. This work provides a strategy for precisely machining water using laser cutting, addressing existing laser machining challenges and holding significance for widespread fields involving fluid patterning and flow control in biological, chemical, materials and biomedical research.

Th1 Cells Alter the Inflammatory Signature of IL-6 by Channeling STAT Transcription Factors to Alu-like Retroelements.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

Active maintenance of CD8+ T cell naïvety through regulation of global genome architecture.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

Helminth Infection-Induced Increase in Virtual Memory CD8 T Cells Is Transient, Driven by IL-15, and Absent in Aged Mice.

CD8 virtual memory T (TVM) cells are Ag-naive CD8 T cells that have undergone partial differentiation in response to common γ-chain cytokines, particularly IL-15 and IL-4. TVM cells from young individuals are highly proliferative in response to TCR and cytokine stimulation but, with age, they lose TCR-mediated proliferative capacity and exhibit hallmarks of senescence. Helminth infection can drive an increase in TVM cells, which is associated with improved pathogen clearance during subsequent infectious challenge in young mice. Given the cytokine-dependent profile of TVM cells and their age-associated dysfunction, we traced proliferative and functional changes in TVM cells, compared with true naive CD8 T cells, after helminth infection of young and aged C57BL/6 mice. We show that IL-15 is essential for the helminth-induced increase in TVM cells, which is driven only by proliferation of existing TVM cells, with negligible contribution from true naive cell differentiation. Additionally, TVM cells showed the greatest proliferation in response to helminth infection and IL-15 compared with other CD8 T cells. Furthermore, TVM cells from aged mice did not undergo expansion after helminth infection due to both TVM cell-intrinsic and -extrinsic changes associated with aging.

Augmenting Adoptive T-cell Immunotherapy by Targeting the PD-1/PD-L1 Axis.

Cancer immunotherapy utilizing checkpoint blockade antibodies or adoptive cellular therapy (ACT) with tumor-specific T cells has led to unprecedented clinical responses in patients with cancer and has been considered one of the most significant breakthroughs in cancer treatment in the past decade. Nevertheless, many cancers remain refractory to these therapies due to the presence of an immunosuppressive tumor microenvironment. This has led to the innovative idea of combining ACT with checkpoint inhibition. A landmark 2004 study by Blank and colleagues published in Cancer Research was one of the original demonstrations that adoptive transfer of T cells lacking the negative T-cell regulator, PD-1, was able to restore functional T-cell antitumor activity, resulting in rapid regression of established tumors in a preclinical model. This work was instrumental in not only driving clinical studies utilizing checkpoint inhibition but also a new wave of recent trials involving checkpoint blockade in the setting of ACT.See related article by Blank and colleagues, Cancer Res 2004;64:1140-5.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.

KDM6B-dependent chromatin remodeling underpins effective virus-specific CD8+ T cell differentiation.

Naive CD8+ T cell activation results in an autonomous program of cellular proliferation and differentiation. However, the mechanisms that underpin this process are unclear. Here, we profile genome-wide changes in chromatin accessibility, gene transcription, and the deposition of a key chromatin modification (H3K27me3) early after naive CD8+ T cell activation. Rapid upregulation of the histone demethylase KDM6B prior to the first cell division is required for initiating H3K27me3 removal at genes essential for subsequent T cell differentiation and proliferation. Inhibition of KDM6B-dependent H3K27me3 demethylation limits the magnitude of an effective primary virus-specific CD8+ T cell response and the formation of memory CD8+ T cell populations. Accordingly, we define the early spatiotemporal events underpinning early lineage-specific chromatin reprogramming that are necessary for autonomous CD8+ T cell proliferation and differentiation.

A Three-Dimensional Paper-Based Isoelectric Focusing Device for Direct Analysis of Proteins in Physiological Samples.

On-site protein analysis is crucial for disease diagnosis in community and family medicine in which microfluidic paper-based analytical devices (µPADs) have attracted growing attention. However, the practical applications of µPADs in protein analysis for physiological samples with high complexity is still limited. Herein, we developed a three-dimensional (3D) paper-based isoelectric focusing (IEF) platform, which is composed of power supply, reservoirs, and separation channel and made by the origami and stacking method, to simultaneously separate and enrich proteins in both low-salt and high-salt samples. Under the optimized experimental conditions, standard proteins (bovine hemoglobin (BHb) and phycocyanin (Phy)) were separated within 18 min under a 36 V power supply and obtained a 10-fold enrichment using the 3D paper-based IEF platform. Then, the capability of the 3D paper-based IEF platform for direct pretreatment of high-salt samples using a 12 V battery as power supply was measured through separating three standard proteins in saline (0.9% NaCl) with separation resolution (SR) > 1.29. Through further coupling with colorimetric and lateral flow strip measurements, the 3D paper-based IEF platform was applied to directly pretreat and quantitatively analyze microalbuminuria and C-reactive proteins in clinical urine and serum samples with analytical results with relative deviations of <8.4% and < 13.1%, respectively, to the clinical test results. This work proposes a new strategy to minimize the difficulty of directly processing high-salt samples with the traditional IEF system and provides a versatile, miniaturized, and low voltage demand analytical platform for on-site analysis of proteins in physiological samples.

Mucosal-Associated Invariant T Cell Effector Function Is an Intrinsic Cell Property That Can Be Augmented by the Metabolic Cofactor α-Ketoglutarate.

Mucosal-associated invariant T (MAIT) cells are an innate-like population of unconventional T cells that respond rapidly to microbial metabolite Ags or cytokine stimulation. Because of this reactivity and surface expression of CD45RO+, CD45RA-, and CD127+, they are described as effector memory cells. Yet, there is heterogeneity in MAIT cell effector response. It is unclear what factors control MAIT cell effector capacity, whether it is fixed or can be modified and if this differs based on whether activation is TCR dependent or independent. To address this, we have taken a systematic approach to examine human MAIT cell effector capacity across healthy individuals in response to ligand and cytokine stimulation. We demonstrate the heterogenous nature of MAIT cell effector capacity and that the ability to produce an effector response is not directly attributable to TCR clonotype or coreceptor expression. Global gene transcription analysis revealed that the MAIT cell effector capacity produced in response to TCR stimulation is associated with increased expression of the epigenetic regulator lysine demethylase 6B (KDM6B). Addition of a KDM6B inhibitor did not alter MAIT cell effector function to Ag or cytokine stimulation. However, addition of the KDM6B cofactor α-ketoglutarate greatly enhanced MAIT cell effector capacity to TCR-dependent stimulation in a partially KDM6B-dependent manner. These results demonstrate that the TCR-dependent effector response of MAIT cells is epigenetically regulated and dependent on the availability of metabolic cofactors.

Dental students' knowledge of and attitudes towards complementary and alternative medicine in Australia - An exploratory study.

OBJECTIVES: While complementary and alternative medicine (CAM) is used by more than half of Australians, its role in dentistry is rarely covered in the curricula of Australian dental schools. This study aimed to investigate dental students' knowledge of and attitudes towards CAM. DESIGN: Australian dental students in the final two years of their study were invited to undertake a survey. These questions consisted of five clinically relevant case vignettes, for which there was only one correct answer amongst four possible options and seven self-reported perspective-based questions. Results were analysed using descriptive statistics. RESULTS: Of the 185 students that participated, the mean correct response rate for the five clinical scenarios was 3.46 ±â€¯0.95 (range: 0-5 out of 5). Of 185 students, 157 (85 %) answered more than half of the questions correctly. All five questions were answered correctly by 19 (10 %) students. Most (74 %) students self-reported to have little to no knowledge. More than two-thirds of dental students reported an interest in and belief in the effectiveness of CAM. A similar proportion expressed a desire for the integration of CAM content into their undergraduate curriculum. CONCLUSIONS: Despite approximately three out of four of dental students in this study having little to no knowledge on CAM, attitudes towards CAM therapies, and the need to integrate them into the dental curriculum, were generally positive. Future research into identifying specific knowledge gaps could help to redesign improved dental curricula.

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria.

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.

Liquid Plasticine Integrated with Isoelectric Focusing for Miniaturized Protein Analysis.

Developing miniaturized and rapid protein analytical platforms is urgently needed for on-site protein analysis, which is important for disease diagnosis and monitoring. Liquid marbles (LMs), a kind of particle-coated droplets, as ideal microreactors have been used in various fields. However, their application as analytical platforms is limited due to the difficulty of pretreating complex samples in simple LMs. Herein, inspired by the microfluidic chip, we propose a strategy through fabricating fluid channels using deformable LM, termed liquid plasticine (LP), to achieve sample pretreatment function. Through combining isoelectric focusing (IEF) with an LP channel, an LP-IEF platform with simultaneous protein separation and concentration functions is realized. The pretreatment capability of the LP-IEF system for proteins in physiological samples is proven using standard proteins and human serum with the results of a clear separation, 10-fold concentration, and a resolution of 0.03 pH. Through cutting the LP after IEF to LMs and transiting isolated LMs containing target proteins for further downstream colorimetric and mass spectrometry measurements, the quantitative analysis of clinical microalbuminuria and identification of α-1-microglobulin/bikunin precursor in clinical diabetic urine samples are achieved. This work proposes a strategy to develop LMs/LPs as a multifunctional integrated analytical platform and the miniaturized LP-IEF device as a rapid protein analytical platform.

Smart Glove Integrated with Tunable MWNTs/PDMS Fibers Made of a One-Step Extrusion Method for Finger Dexterity, Gesture, and Temperature Recognition.

Flexible wearable devices have proven to be emerging tools for motion monitoring, personal healthcare, and rehabilitation training. The development of a multifunctional, flexible sensor and the integration of sensors and a smart chip for signal reading and transmission play a critical role in building a smart wearable device. In this work, a smart glove based on multiwalled carbon nanotubes/poly(dimethylsiloxane) (MWNTs/PDMS) fibers is developed for gesture and temperature recognition. First, the well-tunable, stretchable, and thermal-sensitive MWNTs/PDMS fibers are fabricated via a facile and cost-effective one-step extrusion method. The obtained fibers exhibit an outstanding linear relationship between resistance change and strain in the range of 0-120% and excellent cyclic stability and durability after 20 000 cycles of 50% tension. They also present a linear relationship of resistance change and temperature of 0.55% °C-1 with a correlation coefficient of 0.998 in the range of 0-100 °C. The fibers, as parts of wearable sensors, are then integrated into a smart glove along with a custom-made data acquisition chip to recognize finger dexterity, gestures, and temperature signals and output them through a screen display, an audio system, and Bluetooth transmission. The highly integrated, low-cost, and multifunctional glove holds great potential for various applications, such as sign language recognition, rehabilitation training, and telemedicine in the Internet-of-Things era.

The Impact of MHC Class I Dose on Development and Maintenance of the Polyclonal Naive CD8+ T Cell Repertoire.

Naive CD8+ T cell survival in the periphery is critically dependent on tonic TCR signaling through peptide + MHC class I (MHCI) recognition; however, little is known about how natural variation in MHCI levels impacts the naive CD8+ T cell repertoire. Using mice that are hemizygous or homozygous for a single MHCI allele, we showed that despite a reduction in peripheral CD8+ T cell numbers of ∼50% in MHCI hemizygous mice, MHCI levels had no notable impact on the rate of thymic generation or emigration of CD8 single-positive T cells. Moreover, the peripheral T cell repertoire in hemizygous mice showed selective retention of T cell clonotypes with a greater competitive advantage as evidenced by increased expression of CD5 and IL-7Rα. The qualitative superiority of CD8+ T cells retained in hemizygous mice was also seen during influenza A virus infection, in which epitope-specific CD8+ T cells from hemizygous mice had a higher avidity for pMHCI and increased cytokine polyfunctionality, despite a reduced response magnitude. Collectively, this study suggests that natural variation in MHCI expression levels has a notable and biologically relevant impact on the maintenance, but not generation, of the naive CD8+ T cell repertoire.