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Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers 1 – 10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity 11 – 15 , although these have been less well-studied in ICB treatment 16 . A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling 17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter 18 ) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets. Multiomic profiling of several cohorts of patients treated with immune checkpoint blockade highlights the presence and potential role of B cells and tertiary lymphoid structures in promoting therapy response.

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000–200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans. Analysis of blood from a healthy human show that haematopoietic stem cells increase rapidly in numbers through early life, reaching a stable plateau in adulthood, and contribute to myeloid and B lymphocyte populations throughout life.

The colorectal adenoma–carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer 1 . However, our understanding of the earliest phases of colorectal neoplastic changes—which may occur in morphologically normal tissue—is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer. Genome sequencing of hundreds of normal colonic crypts from 42 individuals sheds light on mutational processes and driver mutations in normal colorectal epithelial cells.

A single-cell sequencing method is developed that uses transcriptomics and CRISPR–Cas9 technology to investigate clonal relationships in cells present in different zebrafish tissues. Tracing single cells from embryo to adult Determining the adult fate of progenitor cells present during embryonic development is a challenging task because it requires simultaneous knowledge about the lineage and identity of the cells at a single-cell level. Alexander van Oudenaarden and colleagues have developed a new method to tackle this challenge. ScarTrace relies on single-cell transcriptome sequencing and barcodes ('scars') introduced by CRISPR–Cas9 in individual progenitor cells. The authors use ScarTrace to investigate lineage relationships in cells present in different zebrafish tissues. In the future, such a method could make it possible to match all embryonic cell types to all adult cell types, and to reconstruct how the body emerges from a single cell. Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development 1 , 2 , monitoring the heritable expression of genetically encoded fluorescent proteins 3 and, more recently, using next-generation sequencing technologies that exploit somatic mutations 4 , microsatellite instability 5 , transposon tagging 6 , viral barcoding 7 , CRISPR–Cas9 genome editing 8 , 9 , 10 , 11 , 12 , 13 and Cre– loxP recombination 14 . Single-cell transcriptomics 15 provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases 1 , 2 . However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation 3 . Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo 4 . Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies. A culture system allows the long-term expansion of human haematopoietic stem cells (HSCs) in vivo without the use of recombinant cytokines or albumin, with potential applications for clinical therapies involving HSCs.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5 + cells in basal organoids revealed a distinct population of ITGA6 + ITGB4 + mitotic cells, whose offspring further segregated into a TNFRSF12A hi subfraction that comprised about ten per cent of KRT5 + basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia. A long-term culture method for organoids derived from single adult human lung cells is used to identify progenitor cells and study SARS-CoV-2 infection.

Transposon tagging to clonally trace progenitors and stem cells provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ . New blood lineage Generating new blood, or haematopoiesis, relies on a pool of stem cells that produces multipotent progenitors and differentiated blood and immune cells. Different models have been proposed to explain the hierarchical organization and fate decision of these cells, mainly using transplantation to assess lineage potential. Fernando Camargo and colleagues use transposon tagging to trace progenitors and stem cells clonally in unperturbed haematopoiesis and apply single-cell RNA sequencing to assess the transcriptome of the cells produced. They find the coexistence of unilineage- and oligolineage-producing clones and suggest that the megakaryocyte lineage arises largely independently of other haematopoietic fates, and can do so from long-term haematopoietic stem cells directly. Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting at the very top 1 , 2 . Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ .

A mouse model of liver damage has identified a population of Lrg5 + liver stem cells that can generate hepatoctyes and bile ducts in vivo. Wake-up call for liver stem cells Hans Clevers and colleagues have identified a quiescent population of adult liver stem cells that can be 'woken up' by damage. In mice subject to liver damage, small cells expressing the Wnt target gene Lgr5 accumulate near the bile ducts. One of these cells was used to grow large numbers of bipotent stem cells in vitro . The stem cells were converted to functional hepatocytes in vitro , and when liver organoids were transplanted into a mouse model of tyrosinemia type I liver disease, islands of apparently normal hepatocytes appeared in the liver. Whether these hepatocytes are fully functional is not yet known, but the results are promising for regenerative approaches in the liver. The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon 1 , stomach 2 and hair follicles 3 . A three-dimensional culture system allows long-term clonal expansion of single Lgr5 + stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture 2 , 4 , 5 . A crucial component of the culture medium is the Wnt agonist RSPO1 6 , the recently discovered ligand of LGR5 7 , 8 . Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ + cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5 + cells generate hepatocytes and bile ducts in vivo . Single Lgr5 + cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah −/− mice. These findings indicate that previous observations concerning Lgr5 + stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function. Single-cell transcriptomic analysis of neurogenic niches in young and old mice reveals that T cells infiltrate the neurogenic niches of old mice and inhibit the proliferation of neural stem cells, in part through expression of interferon-γ.

An artificial recombination locus, Polylox , that can generate hundreds of thousands of individual barcodes is used to trace the fates of haematopoietic stem cells in mice. Barcode tracking blood stem cells Transplantation-based assays of haematopoietic stem cells (HSCs) and progenitors isolated on the basis of the expression of their surface markers have inferred that the haematopoietic lineage follows a tree-like structure that starts from a long-term multipotent HSC at its base and splits into a few major branches. However, recent data question the existence of this structure, instead supporting the idea that the blood lineage is sustained by several fate-restricted progenitors. Hans-Reimer Rodewald and colleagues have developed a DNA recombination locus based on the Cre– loxP system that can tag single cells using several hundred thousand barcodes. They introduce the labelling in mouse embryos and track HSCs during their life. Surprisingly, the adult HSC compartment is a mosaic of HSC clones derived from embryos and contributes with different proportion to blood lineage, some multilineage and others of restricted fates, according to a pattern that is consistent within clones. However, they define an early split of fate between myeloid erythroid and lymphocyte development which agrees with the tree-like structure. Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping 1 has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites 2 , viral barcodes 3 , and strategies based on transposons 4 and CRISPR–Cas9 genome editing 5 ; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox ) that enables broadly applicable endogenous barcoding based on the Cre– loxP recombination system 6 , 7 . Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo . Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs 8 , 9 , 10 . We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid–erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.