Shared and distinct biological circuits in effector, memory and exhausted CD8+ T cells revealed by temporal single-cell transcriptomics and epigenetics.

Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.

Human epigenetic and transcriptional T cell differentiation atlas for identifying functional T cell-specific enhancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

Developmental Relationships of Four Exhausted CD8+ T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms.

CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

Asymmetric distribution of parental H3K9me3 in S phase silences L1 elements.

In eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 trimethylation (H3K9me3). Loss of silencing of the repeat elements leads to genome instability and human diseases, including cancer and ageing1-3. Although the role of H3K9me3 in the establishment and maintenance of heterochromatin silencing has been extensively studied4-6, the pattern and mechanism that underlie the partitioning of parental H3K9me3 at replicating DNA strands are unknown. Here we report that H3K9me3 is preferentially transferred onto the leading strands of replication forks, which occurs predominantly at long interspersed nuclear element (LINE) retrotransposons (also known as LINE-1s or L1s) that are theoretically transcribed in the head-on direction with replication fork movement. Mechanistically, the human silencing hub (HUSH) complex interacts with the leading-strand DNA polymerase Pol ε and contributes to the asymmetric segregation of H3K9me3. Cells deficient in Pol ε subunits (POLE3 and POLE4) or the HUSH complex (MPP8 and TASOR) show compromised H3K9me3 asymmetry and increased LINE expression. Similar results were obtained in cells expressing a MPP8 mutant defective in H3K9me3 binding and in TASOR mutants with reduced interactions with Pol ε. These results reveal an unexpected mechanism whereby the HUSH complex functions with Pol ε to promote asymmetric H3K9me3 distribution at head-on LINEs to suppress their expression in S phase.

Stromal changes in the aged lung induce an emergence from melanoma dormancy.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

Paradoxical Role for Wild-Type p53 in Driving Therapy Resistance in Melanoma.

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

Sugarcane streak mosaic virus P1 protein inhibits unfolded protein response through direct suppression of bZIP60U splicing.

The unfolded protein response (UPR) is a cell-designated strategy that maintains the balance of protein folding in the endoplasmic reticulum (ER). UPR features a network of signal transduction pathways that reprogram the transcription, mRNA translation, and protein post-translational modification to relieve the ER stresses from unfolded/misfolded proteins. Infection with plant viruses can induce the UPR, and activated UPR often promotes plant viral infections in turn. However, the mechanism used by plant viruses to balance UPR and achieve robust infection remain largely unknown. In this study, P1SCSMV was identified as a virus-encoded RNA silencing suppressor (VSR). Heterologous overexpression of P1SCSMV via potato virus X (PVX) was found lead to programmed cell death (PCD) in Nicotiana benthamiana. Furthermore, P1SCSMV was also found to inhibit the PVX infection-triggered UPR by downregulating UPR-related genes and directly induced the distortion and collapse of the ER polygonal meshes on PVX-P1SCSMV infected N. benthamiana. Moreover, self-interaction, VSR activity, UPR inhibition, and cell death phenotype of P1SCSMV were also found to be dependent on its bipartite nuclear localization signal (NLS) (251RKRKLFPRIPLK262). P1SCSMV was found to directly bind to the stem-loop region of NbbZIP60U via its NLS and inhibit the UPR pathways, ultimately resulting in a PCD phenotype in PVX-P1SCSMV infected N. benthamiana leaves. This study also revealed the balancing role of potyviruses encoded P1SCSMV in the UPR pathway to achieve robust viral infection. This may represent a novel virulence strategy for plant viruses.

CD45 Phosphatase Inhibits STAT3 Transcription Factor Activity in Myeloid Cells and Promotes Tumor-Associated Macrophage Differentiation.

Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy.

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.

Author Correction: PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.

TWAS Atlas: a curated knowledgebase of transcriptome-wide association studies.

Transcriptome-wide association studies (TWASs), as a practical and prevalent approach for detecting the associations between genetically regulated genes and traits, are now leading to a better understanding of the complex mechanisms of genetic variants in regulating various diseases and traits. Despite the ever-increasing TWAS outputs, there is still a lack of databases curating massive public TWAS information and knowledge. To fill this gap, here we present TWAS Atlas (https://ngdc.cncb.ac.cn/twas/), an integrated knowledgebase of TWAS findings manually curated from extensive literature. In the current implementation, TWAS Atlas collects 401,266 high-quality human gene-trait associations from 200 publications, covering 22,247 genes and 257 traits across 135 tissue types. In particular, an interactive knowledge graph of the collected gene-trait associations is constructed together with single nucleotide polymorphism (SNP)-gene associations to build up comprehensive regulatory networks at multi-omics levels. In addition, TWAS Atlas, as a user-friendly web interface, efficiently enables users to browse, search and download all association information, relevant research metadata and annotation information of interest. Taken together, TWAS Atlas is of great value for promoting the utility and availability of TWAS results in explaining the complex genetic basis as well as providing new insights for human health and disease research.

Banff 2022 Vascularized Composite Allotransplantation Meeting Report: Diagnostic criteria for vascular changes.

As more data become available, the Banff 2007 working classification of skin-containing vascularized composite allograft (VCA) pathology is expected to evolve and develop. This report represents the Banff VCA Working Group's consensus on the first revision of the 2007 scoring system. Prior to the 2022 Banff-CanXadian Society of Transplantation Joint Meeting, 83 clinicians and/or researchers were invited to a virtual meeting to discuss whether the 2007 Banff VCA system called for a revision. Unanimously, it was determined that the vascular changes were to be included in the first revision. Subsequently, 2 international online surveys, each followed by virtual discussions, were launched. The goals were (1) to identify which changes define severe rejection, (2) to grade their importance in the evaluation of severe rejection, and (3) to identify emerging criteria to diagnose rejection. A final hybrid (in-person and virtual) discussion at the Banff/Canadian Society of Transplantation Joint Meeting finalized the terminology, the definition, a scoring system, and a reporting system of the vascular changes. This proposal represents an international consensus on this topic and establishes the first revision of the Banff 2007 working classification of skin-containing vascularized composite allograft pathology.

Development of a Bioorthogonal Click-to-Release Reaction for Hydrogen Polysulfide (H2Sn) Detection.

In this study, we present an innovative "click-to-release" strategy for the design of highly specific H2Sn bioorthogonal probes that undergo a specific click reaction with H2Sn and release fluorophores by a following rearrangement. A library of cyclooctyne derivatives was established and successfully demonstrated the availability of the release strategy. Then, a model probe CM-CT was synthesized, which can achieve effective fluorophore release (>80%) in the presence of a H2Sn donor. To further validate the application of this class of probes, a new probe QN-RHO-CT based on Rhodamine 110 was developed. This probe showed good water solubility (>160 µM) and fast release kinetics and can achieve selective H2Sn detection in living cells. We used this probe to study the process of H2S-mediated protein S-persulfidation and demonstrated that excess H2S would directly react with protein persulfides to generate H2S2 and reduce the persulfides to thiols. Additionally, we elucidated the click-to-release mechanism in our design through a detailed mechanistic study, confirming the generation of the key intermediate α, ß-unsaturated cyclooctanethione. This bioorthogonal click-to-release reaction provides a useful tool for investigating the function of H2Sn and paves the way for biological studies on H2Sn.

Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections.

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

Artificial intelligence-based evaluation of prognosis in cirrhosis.

Cirrhosis represents a significant global health challenge, characterized by high morbidity and mortality rates that severely impact human health. Timely and precise prognostic assessments of liver cirrhosis are crucial for improving patient outcomes and reducing mortality rates as they enable physicians to identify high-risk patients and implement early interventions. This paper features a thorough literature review on the prognostic assessment of liver cirrhosis, aiming to summarize and delineate the present status and constraints associated with the application of traditional prognostic tools in clinical settings. Among these tools, the Child-Pugh and Model for End-Stage Liver Disease (MELD) scoring systems are predominantly utilized. However, their accuracy varies significantly. These systems are generally suitable for broad assessments but lack condition-specific applicability and fail to capture the risks associated with dynamic changes in patient conditions. Future research in this field is poised for deep exploration into the integration of artificial intelligence (AI) with routine clinical and multi-omics data in patients with cirrhosis. The goal is to transition from static, unimodal assessment models to dynamic, multimodal frameworks. Such advancements will not only improve the precision of prognostic tools but also facilitate personalized medicine approaches, potentially revolutionizing clinical outcomes.

A Fluorination Strategy and Low-Acidity Anchoring Group in Self-Assembled Molecules for Efficient and Stable Inverted Perovskite Solar Cells.

Herein, we synthesized two donor-acceptor (D-A) type small organic molecules with self-assembly properties, namely MPA-BT-BA and MPA-2FBT-BA, both containing a low acidity anchoring group, benzoic acid. After systematically investigation, it is found that, with the fluorination, the MPA-2FBT-BA demonstrates a lower highest occupied molecular orbital (HOMO) energy level, higher hole mobility, higher hydrophobicity and stronger interaction with the perovskite layer than that of MPA-BT-BA. As a result, the device based-on MPA-2FBT-BA displays a better crystallization and morphology of perovskite layer with larger grain size and less non-radiative recombination. Consequently, the device using MPA-2FBT-BA as hole transport material achieved the power conversion efficiency (PCE) of 20.32 % and remarkable stability. After being kept in an N2 glove box for 116 days, the unsealed PSCs' device retained 93 % of its initial PCE. Even exposed to air with a relative humidity range of 30±5 % for 43 days, its PCE remained above 91 % of its initial condition. This study highlights the vital importance of the fluorination strategy combined with a low acidity anchoring group in SAMs, offering a pathway to achieve efficient and stable PSCs.

Associations between Sleep Disorders and Impulsive-Compulsive Behaviors in Parkinson's Disease: A Prospective Cohort Study.

OBJECTIVE: To examine the associations of excessive daytime sleepiness (EDS) and probable rapid eye movement sleep behavior disorder (pRBD), respectively, with impulsive-compulsive behaviors (ICBs) over a 5-year follow-up in patients with early Parkinson's disease (PD). METHODS: The Parkinson's Progression Markers Initiative is a multicenter cohort study based on an ongoing and open-ended registry. Longitudinal associations of sleep disorders with ICB over 5-year follow-up visits were estimated using generalized linear mixed-effects models among PD participants. RESULTS: A total of 825 PD participants were enrolled at baseline. The study sample had a median baseline age of 63.1 (interquartile range: 55.6-69.3) years and comprised 496 (61.5%) men. Among them, 201 (24.9%) had ICB at baseline. In the generalized mixed-effects models, EDS (odds ratio [OR] = 1.09, 95% confidence interval [CI] 1.05, 1.12) and RBD (OR = 1.07, 95% CI 1.03, 1.12) were substantially associated with higher odds of developing ICB over time in PD patients, after multivariate adjustment including age, gender, family history, GDS score, STAI-Y score, MDS-UPDRS part III score, LEDD, and disease duration. Consistent results were observed when stratifying by age at baseline, gender, and PD family history. CONCLUSIONS: The study findings suggest a longitudinal association between EDS and pRBD with an increased risk of developing ICB in patients with PD. The findings emphasize the significance of evaluating and addressing sleep disorders in PD patients as a potential approach to managing ICB.

Low-dose anti-thymocyte globulin plus low-dose posttransplant cyclophosphamide-based regimen for prevention of graft-versus-host disease in haploidentical peripheral blood stem cell transplantation for pediatric patients with hematologic malignancies.

The low-dose anti-thymocyte globulin (ATG) plus low-dose post transplantation cyclophosphamide (PTCy) -based (low-dose ATG/PTCy-based) regimen had a promising activity in preventing of graft-versus-host disease (GVHD) in adult patients. However, its efficacy in pediatric patients remain to be defined. Here, we presented the findings from 35 pediatric patients undergoing haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) with the new regimen for GVHD prophylaxis. The cumulative incidences (CIs) of grades II-III and III-IV acute GVHD (aGVHD) were 34% (95% CI, 17-48%) and 11% (95% CI, 0-21%) within 180 days post-transplantation, respectively. The CIs of chronic GVHD (cGVHD) and moderate-to-severe cGVHD within 2 years were 26% (95% CI, 7-41%) and 12% (95% CI, 0-25%), respectively. The 2-year probabilities of overall survival, relapse-free survival, and graft-versus-host disease and relapse-free survival were 89% (95% CI, 78-100%), 82% (95% CI, 68-98%) and 59% (95% CI, 43-80%), respectively. The CIs of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivation by day 180 were 37% (95% CI, 19-51%) and 20% (95% CI, 6-32%) respectively. These results strongly advocate for the efficacy of the low-dose ATG/PTCy-based regimen as a robust strategy for GVHD prevention in haplo-PBSCT for pediatric patients.

Experimental and Quantum Chemical Calculations on the High-Efficiency Transesterification of Dimethyl Carbonate with Alcohol Catalyzed by Calcium Oxide.

Typical catalysts used in dimethyl carbonate (DMC) transesterification encounter challenges in terms of environmental sustainability and economic viability. Calcium oxide (CaO), being an environmentally friendly and cost-effective catalyst, exhibits favorable compatibility with the criteria above. It has been conclusively demonstrated that CaO performs high efficiency as a catalyst for the transesterification between alcohols and DMC. The optimal conditions for the CaO-catalyzed transesterification of DMC and 1-octanol were determined (90 °C, 17 h, and CaO/1-octanol/DMC molar ratio = 0.3:1.0:40.0), under which the conversion of 1-octanol reaches 98.3%, while the yield and selectivity of methyl octyl carbonate are 98.1 and 99.9%, and CaO has been proven to have the efficient ability to be recycled three times. Meanwhile, the CaO-catalyzed reaction mechanism of the transesterification of DMC with alcohol is illustrated in the quantum chemical method based on the M06-2X functional, and the structures of the corresponding transition states are simultaneously derived. The activation energy barrier is proven to be effectively decreased by the catalytic effect of CaO. In addition, the electrostatic potential diagram verifies the proposed reaction sites. This research constructs the theoretical basis for CaO-based DMC chemistry and expands the green catalysts available for the synthesis of dialkyl carbonates.

Sequencing of Immunotherapy and Outcomes in Operable Clinical Stage III Melanoma: A National Cohort Study.

BACKGROUND AND OBJECTIVES: The impact of neoadjuvant immunotherapy (NIT) on overall survival (OS) in patients with resectable stage III melanoma remains unknown. We sought to identify factors associated with receipt of NIT and survival outcomes in patients with clinical stage III melanoma undergoing surgery. METHODS: The National Cancer Database (2016-2020) was used to identify patients with clinical stage III melanoma who underwent surgery and received either NIT or adjuvant immunotherapy (AIT) only. Multivariable regression, Kaplan-Meier, and Cox proportional hazard methods were used to analyze variables of interest. RESULTS: Patients with clinical N3 disease had 2.5 times the odds of NIT compared to those with N1 disease (95% CI 1.74-3.49). There was no difference in 3-year OS between the two cohorts: 79% (95% CI 73%-85%) for NIT patients and 75% (95% CI 73%-76%) for AIT patients (p = 0.078). Patients with N2/N3 disease had improved 3-year OS of 79% with NIT versus 71% for AIT-only (HR 0.61, 95% CI 0.38-0.97, p = 0.037). CONCLUSIONS: NIT is given more selectively to clinical stage III patients with more advanced N category disease. Despite significant differences in N category between groups, there was no difference in OS observed at 3 years, and NIT was associated with a survival advantage among N2/N3 patients.