Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Genome-wide maps of nuclear lamina interactions in single human cells.

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.

Haemopedia RNA-seq: a database of gene expression during haematopoiesis in mice and humans.

During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.

Massive reshaping of genome-nuclear lamina interactions during oncogene-induced senescence.

Cellular senescence is a mechanism that virtually irreversibly suppresses the proliferative capacity of cells in response to various stress signals. This includes the expression of activated oncogenes, which causes Oncogene-Induced Senescence (OIS). A body of evidence points to the involvement in OIS of chromatin reorganization, including the formation of senescence-associated heterochromatic foci (SAHF). The nuclear lamina (NL) is an important contributor to genome organization and has been implicated in cellular senescence and organismal aging. It interacts with multiple regions of the genome called lamina-associated domains (LADs). Some LADs are cell-type specific, whereas others are conserved between cell types and are referred to as constitutive LADs (cLADs). Here, we used DamID to investigate the changes in genome-NL interactions in a model of OIS triggered by the expression of the common BRAFV600E oncogene. We found that OIS cells lose most of their cLADS, suggesting the loss of a specific mechanism that targets cLADs to the NL. In addition, multiple genes relocated to the NL. Unexpectedly, they were not repressed, implying the abrogation of the repressive activity of the NL during OIS. Finally, OIS cells displayed an increased association of telomeres with the NL. Our study reveals that senescent cells acquire a new type of LAD organization and suggests the existence of as yet unknown mechanisms that tether cLADs to the NL and repress gene expression at the NL.

The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells.

Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewing's sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.

Plasmacytoid dendritic cell heterogeneity is defined by CXCL10 expression following TLR7 stimulation.

Plasmacytoid dendritic cells (pDCs) play a critical role in bridging the innate and adaptive immune systems. pDCs are specialized type I interferon (IFN) producers, which has implicated them as initiators of autoimmune pathogenesis. However, little is known about the downstream effectors of type I IFN signaling that amplify autoimmune responses. Here, we have used a chemokine reporter mouse to determine the CXCR3 ligand responses in DCs subsets. Following TLR7 stimulation, conventional type 1 and type 2 DCs (cDC1 and cDC2, respectively) uniformly upregulate CXCL10. By contrast, the proportion of chemokine positive pDCs was significantly less, and stable CXCL10+ and CXCL10- populations could be distinguished. CXCL9 expression was induced in all cDC1s, in half of the cDC2 but not by pDCs. The requirement for IFNAR signaling for chemokine reporter expression was interrogated by receptor blocking and deficiency and shown to be critical for CXCR3 ligand expression in Flt3-ligand-derived DCs. Chemokine-producing potential was not concordant with the previously identified markers of pDC heterogeneity. Finally, we show that CXCL10+ and CXCL10- populations are transcriptionally distinct, expressing unique transcriptional regulators, IFN signaling molecules, chemokines, cytokines, and cell surface markers. This work highlights CXCL10 as a downstream effector of type I IFN signaling and suggests a division of labor in pDCs subtypes that likely impacts their function as effectors of viral responses and as drivers of inflammation.

Reduced lymphocyte longevity and homeostatic proliferation in lamin B receptor-deficient mice results in profound and progressive lymphopenia.

The lamin B receptor (LBR) is a highly unusual inner nuclear membrane protein with multiple functions. Reduced levels are associated with decreased neutrophil lobularity, whereas complete absence of LBR results in severe skeletal dysplasia and in utero/perinatal lethality. We describe a mouse pedigree, Lym3, with normal bone marrow and thymic development but profound and progressive lymphopenia particularly within the T cell compartment. This defect arises from a point mutation within the Lbr gene with only trace mutant protein detectable in homozygotes, albeit sufficient for normal development. Reduced T cell homeostatic proliferative potential and life span in vivo were found to contribute to lymphopenia. To investigate the role of LBR in gene silencing in hematopoietic cells, we examined gene expression in wild-type and mutant lymph node CD8 T cells and bone marrow neutrophils. Although LBR deficiency had a very mild impact on gene expression overall, for common genes differentially expressed in both LBR-deficient CD8 T cells and neutrophils, gene upregulation prevailed, supporting a role for LBR in their suppression. In summary, this study demonstrates that LBR deficiency affects not only nuclear architecture but also proliferation, cell viability, and gene expression of hematopoietic cells.

Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease.

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice.

Hematopoietic stem cells (HSCs) are rare residents of the bone marrow responsible for the lifelong production of blood cells. Regulation of the balance between HSC self-renewal and differentiation is central to hematopoiesis, allowing precisely regulated generation of mature blood cells at steady state and expanded production at times of rapid need, as well as maintaining ongoing stem cell capacity. Erg, a member of the Ets family of transcription factors, is deregulated in cancers; and although Erg is known to be required for regulation of adult HSCs, its precise role has not been defined. We show here that, although heterozygosity for functional Erg is sufficient for adequate steady-state HSC maintenance, Erg(+/Mld2) mutant mice exhibit impaired HSC self-renewal after bone marrow transplantation or during recovery from myelotoxic stress. Moreover, although mice functionally compromised for either Erg or Mpl, the receptor for thrombopoietin, a key regulator of HSC quiescence, maintained sufficient HSC activity to sustain hematopoiesis, Mpl(-/-) Erg(+/Mld2) compound mutant mice displayed exacerbated stem cell deficiencies and bone marrow failure. Thus, Erg is a critical regulator of adult HSCs, essential for maintaining self-renewal at times of high HSC cycling.

Regulation of hematopoietic stem cells by their mature progeny.

Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalization and degradation. Here, we demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem-cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. HSCs from Myb(Plt4/Plt4) mice show altered expression of TPO-responsive genes and, like HSCs from Tpo and Mpl mutant mice, exhibit increased cycling and a decline in the number of HSCs with age. These studies suggest that disorders of platelet number can have profound effects on the HSC compartment via effects on the feedback regulation of circulating TPO concentration.

Critical roles for c-Myb in lymphoid priming and early B-cell development.

c-Myb is a transcription factor with functions in many hematopoietic lineages. c-Myb-deficient mice display reduced numbers of B cells; however, it is unknown what role c-Myb plays in B lymphopoiesis because no critical target genes have been identified in the B-cell lineage. We demonstrate that conditional deletion of c-Myb in B-cell progenitors completely abolishes B-cell development. c-Myb is required for lymphoid progenitors to respond to the cytokines interleukin-7 and thymic stromal lymphopoietin; in the absence of sufficient c-Myb activity, mice display a B lymphopenia that closely resembles that observed in interleukin-7 receptor alpha-deficient animals. Analysis of the multipotent progenitor compartment indicates that c-Myb is also required for up-regulation of multiple lymphoid-associated genes, including Il7r, and for the subsequent development of the common lymphoid progenitor population. These data show that c-Myb plays a critical role in the regulatory pathways governing lymphoid specification and early B-cell differentiation.

Estimating the proportion of microarray probes expressed in an RNA sample.

A fundamental question in microarray analysis is the estimation of the number of expressed probes in different RNA samples. Negative control probes available in the latest microarray platforms, such as Illumina whole genome expression BeadChips, provide a unique opportunity to estimate the number of expressed probes without setting a threshold. A novel algorithm was proposed in this study to estimate the number of expressed probes in an RNA sample by utilizing these negative controls to measure background noise. The performance of the algorithm was demonstrated by comparing different generations of Illumina BeadChips, comparing the set of probes targeting well-characterized RefSeq NM transcripts with other probes on the array and comparing pure samples with heterogenous samples. Furthermore, hematopoietic stem cells were found to have a larger transcriptome than progenitor cells. Aire knockout medullary thymic epithelial cells were shown to have significantly less expressed probes than matched wild-type cells.

What can we learn from mice lacking pro-survival BCL-2 proteins to advance BH3 mimetic drugs for cancer therapy?

In many human cancers the control of apoptosis is dysregulated, for instance as a result of the overexpression of pro-survival BCL-2 proteins. This promotes tumorigenesis by protecting nascent neoplastic cells from stress and renders malignant cells resistant to anti-cancer agents. Therefore, several BH3 mimetic drugs targeting distinct pro-survival proteins have been developed. The BCL-2 inhibitor Venetoclax/ABT-199, has been approved for treatment of certain blood cancers and tens of thousands of patients have already been treated effectively with this drug. To advance the clinical development of MCL-1 and BCL-XL inhibitors, a more detailed understanding of their distinct and overlapping roles in the survival of malignant as well as non-transformed cells in healthy tissues is required. Here, we discuss similarities and differences in pro-survival BCL-2 protein structure, subcellular localisation and binding affinities to the pro-apoptotic BCL-2 family members. We summarise the findings from gene-targeting studies in mice to discuss the specific roles of distinct pro-survival BCL-2 family members during embryogenesis and the survival of non-transformed cells in healthy tissues in adults. Finally, we elaborate how these findings align with or differ from the observations from the clinical development and use of BH3 mimetic drugs targeting different pro-survival BCL-2 proteins.

Polycomb repressive complex 2 (PRC2) restricts hematopoietic stem cell activity.

Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Using mice with an N-ethyl-N-nitrosourea (ENU)-induced mutation in Suppressor of Zeste 12 (Suz12), a core component of Polycomb Repressive Complex 2 (PRC2), we show here that loss of Suz12 function enhances hematopoietic stem cell (HSC) activity. In addition to these effects on a wild-type genetic background, mutations in Suz12 are sufficient to ameliorate the stem cell defect and thrombocytopenia present in mice that lack the thrombopoietin receptor (c-Mpl). To investigate the molecular targets of the PRC2 complex in the HSC compartment, we examined changes in global patterns of gene expression in cells deficient in Suz12. We identified a distinct set of genes that are regulated by Suz12 in hematopoietic cells, including eight genes that appear to be highly responsive to PRC2 function within this compartment. These data suggest that PRC2 is required to maintain a specific gene expression pattern in hematopoiesis that is indispensable to normal stem cell function.

A mouse model of harlequin ichthyosis delineates a key role for Abca12 in lipid homeostasis.

Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis.

Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes.

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes.

Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl-/- mice.

In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl(-/-) mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl(-/-) p300(Plt6/+) mice displayed elevated platelet counts relative to Mpl(-/-) p300(+/+) controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300(Plt6) mutation, or recipients of Mpl(+/+) p300(Plt6/Plt6) bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl(-/-) thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial thrombopoietin (TPO)-independent platelet production.

miR17~92 restrains pro-apoptotic BIM to ensure survival of haematopoietic stem and progenitor cells.

The miR17~92 cluster plays important roles in haematopoiesis. However, it is not clear at what stage of differentiation and through which targets miR17~92 exerts this function. Therefore, we generated miR17~92fl/fl; RosaCreERT2 mice for inducible deletion of miR17~92 in haematopoietic cells. Bone marrow reconstitution experiments revealed that miR17~92-deleted cells were not capable to contribute to mature haematopoietic lineages, which was due to defects in haematopoietic stem/progenitor cells (HSPCs). To identify the critical factor targeted by miR17~92 we performed gene expression analysis in HSPCs, demonstrating that mRNA levels of pro-apoptotic Bim inversely correlated with the expression of the miR17~92 cluster. Strikingly, loss of pro-apoptotic BIM completely prevented the loss of HSPCs caused by deletion of miR17~92. The BIM/miR17~92 interaction is conserved in human CD34+ HSPCs, as miR17~92 inhibition or blockade of its binding to the BIM 3'UTR reduced the survival and growth of these cells. Despite the prediction that miR17~92 functions by impacting a plethora of different targets, the absence of BIM alone is sufficient to prevent all defects caused by deletion of miR17~92 in haematopoietic cells.

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).