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Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types 1 – 4 . Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients 5 , 6 . Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control 2 , 4 , 7 – 10 , in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8 + TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein–Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8 + TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8 + TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39 − CD8 + TILs. Furthermore, frequencies of CD39 expression among CD8 + TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells. Human lung and colorectal tumours contain a population of tumour-infiltrating lymphocytes that are specific for tumour-unrelated antigens and, unlike tumour-antigen-specific tumour-infiltrating lymphocytes, do not express CD39.

Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, structure and function of these enzymes have been published in the last half-dozen years, revealing that they carry out key functions in diverse other plants. These recent discoveries about plant apyrases include, among others, novel findings on its crystal structures, its biochemistry, its roles in plant stress responses and its induction of major changes in gene expression when its expression is suppressed or enhanced. This review will describe and discuss these recent advances and the major questions about plant apyrases that remain unanswered.

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies. A synthetic yeast-based therapeutic that secretes an ATP-degrading enzyme in response to pro-inflammatory extracellular ATP in the gut reduces intestinal inflammation, fibrosis and dysbiosis in mouse models of colitis and enteritis.

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA. Adoption of DNA as a data storage medium could be accelerated with specialized synthesis processes and codecs. The authors describe TdT-mediated DNA synthesis in which data is stored in transitions between non-identical nucleotides and the use of synchronization markers to provide error tolerance.

Apyrases (APYs) directly regulate intra- and extra-cellular ATP homeostasis and play a key role in the process of plants adapting to various stresses. In this study, we identified and characterized soybean APY (GmAPY) family members at the genomic level. The results identified a total of 18 APYRASE homologous genes with conserved ACR domains. We conducted a bioinformatics analysis of GmAPYs, including sequence alignment, phylogenetic relationships, and conserved motifs. According to the phylogenetic and structural characteristics, GmAPYs in soybeans are mainly divided into three groups. The characteristics of these GmAPYs were systematically evaluated, including their collinearity, gene structure, protein motifs, cis-regulatory elements, tissue expression patterns, and responses to aluminum stress. A preliminary analysis of the function of GmAPY1-4 was also conducted. The results showed that GmAPY1-4 was localized in the nucleus, presenting relatively high levels in roots and root nodules and demonstrating high sensitivity and positive responses under aluminum stress circumstances. Further functional characterization revealed that the overexpression of GmAPY1-4 in hairy roots not only induced root growth under normal growth conditions but also significantly prevented root growth inhibition under aluminum stress conditions and contributed to maintaining a relatively higher fresh root weight. By contrast, RNAi interference with the expression of GmAPY1-4 in hairy roots inhibited root growth under both normal and aluminum stress conditions, but it exerted no significant influence on the dry or fresh root weight. To sum up, these findings support the significant functional role of GmAPY1-4 in root growth and the aluminum stress response. These findings not only enhance our comprehension of the aluminum stress response mechanism by identifying and characterizing the APY gene family in the soybean genome but also provide a potential candidate gene for improving aluminum tolerance in soybeans in the future.

Microglia, the brain’s resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival 1 . Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A 1 R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease. Microglia, the brain’s immune cells, suppress neuronal activity in response to synaptic ATP release and alter behavioural responses in mice.

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO₂ fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103 + CD39 + tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103 + CD39 + CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103 + CD39 + CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103 + CD39 + CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies. Identifying and enumerating tumor-specific CD8 T cells are important for assessing cancer prognosis and therapy efficacy. Here the authors show that CD39 and CD103 mark a subset of tumor-infiltrating CD8 T cells that are tumor-reactive and exhibit characteristics of exhausted or tissue-resident memory T cells.

Plant cell growth involves coordination of numerous processes and signaling cascades among the different cellular compartments to concomitantly enlarge the protoplast and the surrounding cell wall. The cell wall integrity-sensing process involves the extracellular LRX (LRR-Extensin) proteins that bind RALF (Rapid ALkalinization Factor) peptide hormones and, in vegetative tissues, interact with the transmembrane receptor kinase FERONIA (FER). This LRX/RALF/FER signaling module influences cell wall composition and regulates cell growth. The numerous proteins involved in or influenced by this module are beginning to be characterized. In a genetic screen, mutations in Apyrase 7 ( APY7 ) were identified to suppress growth defects observed in lrx1 and fer mutants. APY7 encodes a Golgi-localized NTP-diphosphohydrolase, but opposed to other apyrases of Arabidopsis, APY7 revealed to be a negative regulator of cell growth. APY7 modulates the growth-inhibiting effect of RALF1, influences the cell wall architecture and -composition, and alters the pH of the extracellular matrix, all of which affect cell growth. Together, this study reveals a function of APY7 in cell wall formation and cell growth that is connected to growth processes influenced by the LRX/RALF/FER signaling module.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson–Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.