CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy.

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Insm1 regulates mTEC development and immune tolerance.

The expression of self-antigens in medullary thymic epithelial cells (mTECs) is essential for the establishment of immune tolerance, but the regulatory network that controls the generation and maintenance of the multitude of cell populations expressing self-antigens is poorly understood. Here, we show that Insm1, a zinc finger protein with known functions in neuroendocrine and neuronal cells, is broadly coexpressed with an autoimmune regulator (Aire) in mTECs. Insm1 expression is undetectable in most mimetic cell populations derived from mTECs but persists in neuroendocrine mimetic cells. Mutation of Insm1 in mice downregulated Aire expression, dysregulated the gene expression program of mTECs, and altered mTEC subpopulations and the expression of tissue-restricted antigens. Consistent with these findings, loss of Insm1 resulted in autoimmune responses in multiple peripheral tissues. We found that Insm1 regulates gene expression in mTECs by binding to chromatin. Interestingly, the majority of the Insm1 binding sites are co-occupied by Aire and enriched in superenhancer regions. Together, our data demonstrate the important role of Insm1 in the regulation of the repertoire of self-antigens needed to establish immune tolerance.

DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance.

BACKGROUND: Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. RESULTS: We report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth. CONCLUSIONS: Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

A pan-cancer blueprint of the heterogeneous tumor microenvironment revealed by single-cell profiling.

The stromal compartment of the tumor microenvironment consists of a heterogeneous set of tissue-resident and tumor-infiltrating cells, which are profoundly moulded by cancer cells. An outstanding question is to what extent this heterogeneity is similar between cancers affecting different organs. Here, we profile 233,591 single cells from patients with lung, colorectal, ovary and breast cancer (n = 36) and construct a pan-cancer blueprint of stromal cell heterogeneity using different single-cell RNA and protein-based technologies. We identify 68 stromal cell populations, of which 46 are shared between cancer types and 22 are unique. We also characterise each population phenotypically by highlighting its marker genes, transcription factors, metabolic activities and tissue-specific expression differences. Resident cell types are characterised by substantial tissue specificity, while tumor-infiltrating cell types are largely shared across cancer types. Finally, by applying the blueprint to melanoma tumors treated with checkpoint immunotherapy and identifying a naïve CD4+ T-cell phenotype predictive of response to checkpoint immunotherapy, we illustrate how it can serve as a guide to interpret scRNA-seq data. In conclusion, by providing a comprehensive blueprint through an interactive web server, we generate the first panoramic view on the shared complexity of stromal cells in different cancers.

Preparation and characterization of highly transparent hydrophobic nanocellulose film using corn husks as main material.

In this paper, nanocellulose was extracted from agricultural waste corn husks. Transparent hydrophobic membranes containing silica were fabricated through two facile methods including surface coating and internal grafting. The results showed that: the nanocellulose prepared by TEMPO-mediated oxidation and high pressure homogenization not only retained the type and crystal structure of the original cellulose, but also increased the crystallinity to 64.5% and improved the thermal stability. Both surface coating and internal grafting methods had successfully loaded silica onto films. The internal grafting film had a silica content of 10.5%, which was mainly present inside the film. The light transmittance of this film was 84.4% and the surface contact angle to water was 152.6°. The content of silica on the surface coating film was 5.7%, and they were mainly distributed on the surface of the film to form a nano-scale rough surface. The light transmittance of the surface coating film was 87.8% and the surface contact angle to water was 165.7°. Compared to the film prepared by internal grafting method, the nanocellulose film prepared by surface coating method contained less nano silica and had better properties including higher transparency, higher surface roughness and excellent hydrophobic anti-fouling properties.

Facile Approach to Develop Hierarchical Roughness fiber@SiO2 Blocks for Superhydrophobic Paper.

Papers with nanoscaled surface roughness and hydrophobically modification have been widely used in daily life. However, the relatively complex preparation process, high costs and harmful compounds have largely limited their applications. This research aims to fabricate superhydrophobic papers with low cost and nontoxic materials. The surface of cellulose fibers was initially coated with a film of SiO2 nanoparticles via sol-gel process. After papermaking and subsequent modification with hexadecyltrimethoxysilane through a simple solution-immersion process, the paper showed excellent superhydrophobic properties, with water contact angles (WCA) larger than 150°. Moreover, the prepared paper also showed superior mechanical durability against 10 times of deformation. The whole preparation process was carried out in a mild environment, with no intricate instruments or toxic chemicals, which has the potential of large-scale industrial production and application.

Epigenetic Co-Deregulation of EZH2/TET1 is a Senescence-Countering, Actionable Vulnerability in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) cells lack the expression of ER, PR and HER2. Thus, TNBC patients cannot benefit from hormone receptor-targeted therapy as non-TNBC patients, but can only receive chemotherapy as the systemic treatment and have a worse overall outcome. More effective therapeutic targets and combination therapy strategies are urgently needed to improve the treatment effectiveness. Methods: We analyzed the expression levels of EZH2 and TET1 in TCGA and our own breast cancer patient cohort, and tested their correlation with patient survival. We used TNBC and non-TNBC cell lines and mouse xenograft tumor model to unveil novel EZH2 targets and investigated the effect of EZH2 inhibition or TET1 overexpression in cell proliferation and viability of TNBC cells. Results: In TNBC cells, EZH2 decreases TET1 expression by H3K27me3 epigenetic regulation and subsequently suppresses anti-tumor p53 signaling pathway. Patients with high EZH2 and low TET1 presented the poorest survival outcome. Experimentally, targeting EZH2 in TNBC cells with specific inhibitor GSK343 or shRNA genetic approach could induce cell cycle arrest and senescence by elevating TET1 expression and p53 pathway activation. Using mouse xenograft model, we have tested a novel therapy strategy to combine GSK343 and chemotherapy drug Adriamycin and could show drastic and robust inhibition of TNBC tumor growth by synergistic induction of senescence and apoptosis. Conclusions: We postulate that the well-controlled dynamic pathway EZH2-H3K27me3-TET1 is a novel epigenetic co-regulator module and provide evidence regarding how to exploit it as a novel therapeutic target via its pivotal role in senescence and apoptosis control. Of clinical and therapeutic significance, the present study opens a new avenue for TNBC treatment by targeting the EZH2-H3K27me3-TET1 pathway that can modulate the epigenetic landscape.

Predominant patterns of splicing evolution on human, chimpanzee and macaque evolutionary lineages.

Although splicing is widespread and evolves rapidly among species, the mechanisms driving this evolution, as well as its functional implications, are not yet fully understood. We analyzed the evolution of splicing patterns based on transcriptome data from five tissues of humans, chimpanzees, rhesus macaques and mice. In total, 1526 exons and exon sets from 1236 genes showed significant splicing differences among primates. More than 60% of these differences represent constitutive-to-alternative exon transitions while an additional 25% represent changes in exon inclusion frequency. These two dominant evolutionary patterns have contrasting conservation, regulation and functional features. The sum of these features indicates that, despite their prevalence, constitutive-to-alternative exon transitions do not substantially contribute to long-term functional transcriptome changes. Conversely, changes in exon inclusion frequency appear to be functionally relevant, especially for changes taking place in the brain on the human evolutionary lineage.

Signatures of personality on dense 3D facial images.

It has long been speculated that cues on the human face exist that allow observers to make reliable judgments of others' personality traits. However, direct evidence of association between facial shapes and personality is missing from the current literature. This study assessed the personality attributes of 834 Han Chinese volunteers (405 males and 429 females), utilising the five-factor personality model ('Big Five'), and collected their neutral 3D facial images. Dense anatomical correspondence was established across the 3D facial images in order to allow high-dimensional quantitative analyses of the facial phenotypes. In this paper, we developed a Partial Least Squares (PLS) -based method. We used composite partial least squares component (CPSLC) to test association between the self-tested personality scores and the dense 3D facial image data, then used principal component analysis (PCA) for further validation. Among the five personality factors, agreeableness and conscientiousness in males and extraversion in females were significantly associated with specific facial patterns. The personality-related facial patterns were extracted and their effects were extrapolated on simulated 3D facial models.

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.

Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling.

Gene expression profiling provides a tool to analyze the internal states of cells or organisms, and their responses to perturbations. While global measurements of mRNA levels have thus been widely used for many years, it is only through the recent development of the ribosome profiling technique that an analogous examination of global mRNA translation programs has become possible. Ribosome profiling reveals which RNAs are being translated to what extent and where the translated open reading frames are located. In addition, different modes of translation regulation can be distinguished and characterized. Here, we present an optimized, step-by-step protocol for ribosome profiling. Although established in Caenorhabditis elegans, our protocol and optimization approaches should be equally usable for other model organisms or cell culture with little adaptation. Next to providing a protocol, we compare two different methods for isolation of single ribosomes and two different library preparations, and describe strategies to optimize the RNase digest and to reduce ribosomal RNA contamination in the libraries. Moreover, we discuss bioinformatic strategies to evaluate the quality of the data and explain how the data can be analyzed for different applications. In sum, this article seeks to facilitate the understanding, execution, and optimization of ribosome profiling experiments.

Organization and evolution of brain lipidome revealed by large-scale analysis of human, chimpanzee, macaque, and mouse tissues.

Lipids are prominent components of the nervous system. Here we performed a large-scale mass spectrometry-based analysis of the lipid composition of three brain regions as well as kidney and skeletal muscle of humans, chimpanzees, rhesus macaques, and mice. The human brain shows the most distinct lipid composition: 76% of 5,713 lipid compounds examined in our study are either enriched or depleted in the human brain. Concentration levels of lipids enriched in the brain evolve approximately four times faster among primates compared with lipids characteristic of non-neural tissues and show further acceleration of change in human neocortical regions but not in the cerebellum. Human-specific concentration changes are supported by human-specific expression changes for corresponding enzymes. These results provide the first insights into the role of lipids in human brain evolution.

Exceptional evolutionary divergence of human muscle and brain metabolomes parallels human cognitive and physical uniqueness.

Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys.

Widespread splicing changes in human brain development and aging.

While splicing differences between tissues, sexes and species are well documented, little is known about the extent and the nature of splicing changes that take place during human or mammalian development and aging. Here, using high-throughput transcriptome sequencing, we have characterized splicing changes that take place during whole human lifespan in two brain regions: prefrontal cortex and cerebellum. Identified changes were confirmed using independent human and rhesus macaque RNA-seq data sets, exon arrays and PCR, and were detected at the protein level using mass spectrometry. Splicing changes across lifespan were abundant in both of the brain regions studied, affecting more than a third of the genes expressed in the human brain. Approximately 15% of these changes differed between the two brain regions. Across lifespan, splicing changes followed discrete patterns that could be linked to neural functions, and associated with the expression profiles of the corresponding splicing factors. More than 60% of all splicing changes represented a single splicing pattern reflecting preferential inclusion of gene segments potentially targeting transcripts for nonsense-mediated decay in infants and elderly.