Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors.

Single-cell RNA sequencing is a powerful technology for assessing heterogeneity within defined cell populations. Here, we describe the heterogeneity of a B220+CD117intCD19-NK1.1- uncommitted hematopoietic progenitor having combined lymphoid and myeloid potential. Phenotypic and functional assays revealed four subpopulations within the progenitor with distinct lineage developmental potentials. Among them, the Ly6D+SiglecH-CD11c- fraction was lymphoid-restricted exhibiting strong B-cell potential, whereas the Ly6D-SiglecH-CD11c- fraction showed mixed lympho-myeloid potential. Single-cell RNA sequencing of these subsets revealed that the latter population comprised a mixture of cells with distinct lymphoid and myeloid transcriptional signatures and identified a subgroup as the potential precursor of Ly6D+SiglecH-CD11c- Subsequent functional assays confirmed that B220+CD117intCD19-NK1.1- single cells are, with rare exceptions, not bipotent for lymphoid and myeloid lineages. A B-cell priming gradient was observed within the Ly6D+SiglecH-CD11c- subset and we propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Therefore, the apparent multipotency of B220+CD117intCD19-NK1.1- progenitors results from underlying heterogeneity at the single-cell level and highlights the validity of single-cell transcriptomics for resolving cellular heterogeneity and developmental relationships among hematopoietic progenitors.

George Gabriel Stokes as a biologist.

It is universally acknowledged that George Gabriel Stokes (1819-1903) was a polymath who made major contributions to the fields of mathematics, chemistry, physics, fluidics and optics. However, his contributions to biology have received far less attention and this brief communication examines two of Stokes' major biological contributions, namely his description of the phenomenon of fluorescence and his studies on the changes in the colour of blood following oxidation and reduction. The paper on fluorescence is discussed because in it, Stokes demonstrates his wide-ranging biological knowledge and because the use of fluorescence is an invaluable experimental tool in biology. It was by developing the experimental approaches and equipment used to investigate fluorescence that Stokes then applied these to other investigations, including that of blood. From what we now know, what Stokes was describing in his paper on blood were the changes in the configuration of the haemoglobin molecule upon the acquisition and release of oxygen. This article is part of the theme issue 'Stokes at 200 (part 2)'.

A Database of Human Immune Receptor Alleles Recovered from Population Sequencing Data.

High-throughput sequencing data from TCRs and Igs can provide valuable insights into the adaptive immune response, but bioinformatics pipelines for analysis of these data are constrained by the availability of accurate and comprehensive repositories of TCR and Ig alleles. We have created an analytical pipeline to recover immune receptor alleles from genome sequencing data. Applying this pipeline to data from the 1000 Genomes Project we have created Lym1K, a collection of immune receptor alleles that combines known, well-supported alleles with novel alleles found in the 1000 Genomes Project data. We show that Lym1K leads to a significant improvement in the alignment of short read sequences from immune receptors and that the addition of novel alleles discovered from genome sequence data are likely to be particularly significant for comprehensive analysis of populations that are not currently well represented in existing repositories of immune alleles.

Permissive roles of cytokines interleukin-7 and Flt3 ligand in mouse B-cell lineage commitment.

Hematopoietic cells are continuously generated throughout life from hematopoietic stem cells, thus making hematopoiesis a favorable system to study developmental cell lineage commitment. The main factors incorporating environmental signals to developing hematopoietic cells are cytokines, which regulate commitment of hematopoietic progenitors to the different blood lineages by acting either in an instructive or a permissive manner. Fms-like tyrosine kinase-3 (Flt3) ligand (FL) and Interleukin-7 (IL-7) are cytokines pivotal for B-cell development, as manifested by the severely compromised B-cell development in their absence. However, their precise role in regulating B-cell commitment has been the subject of debate. In the present study we assessed the rescue of B-cell commitment in mice lacking IL-7 but simultaneously overexpressing FL. Results obtained demonstrate that FL overexpression in IL-7-deficient mice rescues B-cell commitment, resulting in significant Ebf1 and Pax5 expression in Ly6D+CD135+CD127+CD19- precursors and subsequent generation of normal numbers of CD19+ B-cell progenitors, therefore indicating that IL-7 can be dispensable for commitment to the B-cell lineage. Further analysis of Ly6D+CD135+CD127+CD19- progenitors in IL-7- or FL-deficient mice overexpressing Bcl2, as well as in IL-7 transgenic mice suggests that both FL and IL-7 regulate B-cell commitment in a permissive manner: FL by inducing proliferation of Ly6D+CD135+CD127+CD19- progenitors and IL-7 by providing survival signals to these progenitors.

Pro-B cells propagated in stromal cell-free cultures reconstitute functional B-cell compartments in immunodeficient mice.

Up to now long-term in vitro growth of pro-B cells was thought to require stromal cells. However, here we show that fetal liver (FL) and bone marrow (BM) derived pro-B cells can be propagated long-term in stromal cell-free cultures supplemented with IL-7, stem cell factor and FLT3 ligand. Within a week, most cells expressed surface CD19, CD79A, λ5, and VpreB antigens and had rearranged immunoglobulin D-J heavy chain genes. Both FL and BM pro-B cells reconstituted the B-cell compartments of immuno-incompetent Rag2-deficient mice, with FL pro-B cells generating follicular, marginal zone (MZB) and B1a B cells, and BM pro-B cells giving rise mainly to MZB cells. Reconstituted Rag2-deficient mice generated significant levels of IgM and IgG antibodies to a type II T-independent antigen; mice reconstituted with FL pro-B cells generated surprisingly high IgG1 titers. Finally, we show for the first time that mice reconstituted with mixtures of pro-B and pro-T cells propagated in stromal cell-free in vitro cultures mounted a T-cell-dependent antibody response. This novel stromal cell-free culture system facilitates our understanding of B-cell development and might be applied clinically.

The changing face of hematopoiesis: a spectrum of options is available to stem cells.

For more than 30 years, the scheme whereby bone marrow hematopoietic stem cells give rise to the many different types of blood and immune cells has been represented as a lineage tree diagram. In this model, hematopoietic stem cells follow a preferred route to each of the end-cell types and gradually restrict their other lineage options via a series of intermediate oligo-potent progenitors. Recent findings of lineage biases or affiliations within hematopoietic stem and progenitor cells that are either pluripotent or uni-potent show that a continuum of fate options is open to hematopoietic stem cells. These results support the view that in order to close down developmental options, hematopoietic stem cells can make an immediate lineage choice rather than become gradually committed as they progress step-wise through a series of intermediate progenitors. In this scenario, there is inherent versatility in that developing cells are still able to move sideways to adopt an alternative lineage fate. Here, we examine the information that is leading toward this very different viewpoint of blood cell development.

Anti-donor antibody induction following intramuscular injections of allogeneic mesenchymal stromal cells.

Allogeneic mesenchymal stromal cells (allo-MSC) are a promising "off-the-shelf" therapy with anti-inflammatory and pro-repair properties. This study investigated humoral immune responses to intramuscular (IM) injections of allo-MSC. Total and isotype-specific anti-donor IgG and donor-specific complement-mediated lysis were determined in sera from healthy mice 2 weeks after single or repeated IM injections of fully mismatched-MHC allo-MSC with comparison to mice receiving syngeneic MSC, allogeneic splenocytes or saline. In mice subjected to hind limb ischemia (HLI), anti-donor IgG was analyzed following IM allo-MSC injection with and without administration of the T-cell immunosuppressant tacrolimus. Recipients of single and repeated IM allo-MSC developed readily-detectable anti-donor IgG. Serum anti-donor IgG levels were similar to those of allo-splenocyte recipients but had higher IgG1/IgG2a ratio and variable capacity for complement-mediated lysis of donor cells. The induced anti-donor IgG bound readily to allo-MSC and this binding was increased following allo-MSC pretreatment with interferon gamma. In mice with HLI, IM injection of allo-MSC into the ischemic limb was also associated with induction of anti-donor IgG but this was abrogated by tacrolimus (FK-506). The results indicate that allo-MSC are inherently immunogenic when delivered intramuscularly to healthy and ischemic mouse hind limb, but induce an IgG1-skewed humoral response that is suppressed by tacrolimus.

Phenotypic and functional heterogeneity of human intermediate monocytes based on HLA-DR expression.

Human blood monocytes are subclassified as classical, intermediate and nonclassical. In this study, it was shown that conventionally defined human intermediate monocytes can be divided into two distinct subpopulations with mid- and high-level surface expression of HLA-DR (referred to as DRmid and DRhi intermediate monocytes). These IM subpopulations were phenotypically and functionally characterized in healthy adult blood by flow cytometry, migration assays and lipoprotein uptake assays. Their absolute numbers and proportions were then compared in blood samples from obese and nonobese adults. DRmid and DRhi intermediate monocytes differentially expressed several proteins including CD62L, CD11a, CX3CR1 and CCR2. Overall, the DRmid intermediate monocytes surface profile more closely resembled that of classical monocytes while DRhi intermediate monocytes were more similar to nonclassical. However, in contrast to classical monocytes, DRmid intermediate monocytes migrated weakly to CCL2, had reduced intracellular calcium flux following CCR2 ligation and favored adherence to TNFα-activated endothelium over transmigration. In lipid uptake assays, DRmid intermediate monocytes demonstrated greater internalization of oxidized and acetylated low-density lipoprotein than DRhi intermediate monocytes. In obese compared to nonobese adults, proportions and absolute numbers of DRmid , but not DRhi intermediate monocytes, were increased in blood. The results are consistent with phenotypic and functional heterogeneity within the intermediate monocytes subset that may be of specific relevance to lipoprotein scavenging and metabolic health.

LymAnalyzer: a tool for comprehensive analysis of next generation sequencing data of T cell receptors and immunoglobulins.

The adaptive immune system includes populations of B and T cells capable of binding foreign epitopes via antigen specific receptors, called immunoglobulin (IG) for B cells and the T cell receptor (TCR) for T cells. In order to provide protection from a wide range of pathogens, these cells display highly diverse repertoires of IGs and TCRs. This is achieved through combinatorial rearrangement of multiple gene segments in addition, for B cells, to somatic hypermutation. Deep sequencing technologies have revolutionized analysis of the diversity of these repertoires; however, accurate TCR/IG diversity profiling requires specialist bioinformatics tools. Here we present LymAnalzyer, a software package that significantly improves the completeness and accuracy of TCR/IG profiling from deep sequence data and includes procedures to identify novel alleles of gene segments. On real and simulated data sets LymAnalyzer produces highly accurate and complete results. Although, to date we have applied it to TCR/IG data from human and mouse, it can be applied to data from any species for which an appropriate database of reference genes is available. Implemented in Java, it includes both a command line version and a graphical user interface and is freely available at https://sourceforge.net/projects/lymanalyzer/.

The Making of Hematopoiesis: Developmental Ancestry and Environmental Nurture.

Evidence from studies of the behaviour of stem and progenitor cells and of the influence of cytokines on their fate determination, has recently led to a revised view of the process by which hematopoietic stem cells and their progeny give rise to the many different types of blood and immune cells. The new scenario abandons the classical view of a rigidly demarcated lineage tree and replaces it with a much more continuum-like view of the spectrum of fate options open to hematopoietic stem cells and their progeny. This is in contrast to previous lineage diagrams, which envisaged stem cells progressing stepwise through a series of fairly-precisely described intermediate progenitors in order to close down alternative developmental options. Instead, stem and progenitor cells retain some capacity to step sideways and adopt alternative, closely related, fates, even after they have "made a lineage choice." The stem and progenitor cells are more inherently versatile than previously thought and perhaps sensitive to lineage guidance by environmental cues. Here we examine the evidence that supports these views and reconsider the meaning of cell lineages in the context of a continuum model of stem cell fate determination and environmental modulation.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRß locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.

Promiscuous mRNA splicing under the control of AIRE in medullary thymic epithelial cells.

MOTIVATION: The expression of tissue-restricted antigens (TRAs) in the thymus is required to ensure efficient negative selection of potentially auto-reactive T lymphocytes and avoid autoimmune disease. This promiscuous expression is under the control of the autoimmune regulator (AIRE), a transcription factor expressed in medullary thymic epithelial cells (mTECs). Tissue-specific alternative splicing may also produce TRAs but the extent to which splice isoforms that are restricted to specific tissues are expressed in mTECs is yet to be investigated. RESULTS: We reanalyzed microarray and RNA-Seq datasets from mouse mTECs and other epithelial and non-epithelial cell types and found that the diversity of splice isoforms in mTECs was greater than in any of the other cell types or tissues studied. We identified tissue-specific isoforms from a panel of mouse tissues and found several examples of such isoforms that are expressed in mTECs. The number of isoforms with restricted expression found in mTECs was significantly higher than for comparable cell types. Furthermore, we found evidence that AIRE influences the increased splicing diversity observed in mTECs as the genes for which tissue restricted isoforms are produced in mTECs were significantly more likely than other genes to be differentially spliced between AIRE knock-out and wild-type samples. Our results suggest that developing T lymphocytes are exposed to diverse tissue-restricted splice isoforms in the thymus and that AIRE has a direct or indirect role in this process, representing a novel aspect of its role in the maintenance of immune self-tolerance. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The key role of IL-7 in lymphopoiesis.

In mice, interleukin 7, although initially thought to be predominantly a cytokine acting on B cells, appears to have potent survival and growth activity during both B and T lymphopoiesis. Although acting on both lineages at the very immature stages, T cell differentiation becomes independent of IL-7 at the intermediate stages before regaining dependence on IL-7 for survival and proliferation at the mature T cell stage. In contrast, although essential for B lymphopoiesis, mature B cell survival is independent of IL-7. In this review, we focus on and discuss the similarities and differences between the role of IL-7 in these two processes.

Versatility of stem and progenitor cells and the instructive actions of cytokines on hematopoiesis.

For many years, developing hematopoietic cells have been strictly compartmentalized into a rare population of multi-potent self-renewing hematopoietic stem cells (HSC), multi-potent hematopoietic progenitor cells (MPP) that are undergoing commitment to particular lineage fates, and recognizable precursor cells that mature towards functional blood and immune cells. A single route to each end-cell type is prescribed in the "classical" model for the architecture of hematopoiesis. Recent findings have led to the viewpoint that HSCs and MPPs are more versatile than previously thought. Underlying this are multiple routes to a particular fate and cells having clandestine fate options even when they have progressed some way along a pathway. The primary role of cytokines during hematopoiesis has long been seen to be regulation of the survival and proliferation of developing hematopoietic cells. Some cytokines now clearly have instructive actions on cell-fate decisions. All this leads to a new way of viewing hematopoiesis whereby versatile HSC and MPP are directed towards lineage outcomes via cytokine regulated cell-fate decisions. This means greater flexibility to the shaping of hematopoiesis.

Hypoxia enhances the radioresistance of mouse mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are radioresistant bone marrow progenitors that support hematopoiesis and its reconstitution following total body irradiation. MSCs reside in hypoxic niches within the bone marrow and tumor microenvironments. The DNA damage response (DDR) represents a network of signaling pathways that enable cells to activate biological responses to DNA damaging agents. Hypoxia-mediated alterations in the DDR contribute to the increased radioresistance of hypoxic cancer cells, limiting therapeutic efficacy. The DDR is important in mediating mouse MSC radioresistance. However, the effects of hypoxia on MSC radioresistance are currently unknown. In this report, hypoxia was found to (a) increase MSC proliferation rate and colony size; (b) increase long-term survival post-irradiation (IR), and (c) improve MSC recovery from IR-induced cell cycle arrest. DNA double-strand break (DSB) repair in MSCs was upregulated in hypoxia, accelerating the resolution of highly genotoxic IR-induced DNA DSBs. In addition, HIF-1α was found to contribute to this enhanced DSB repair by regulating (a) the expression of DNA ligase IV and DNA-PKcs and (b) Rad51 foci formation in response to DNA DSBs in hypoxic MSCs. We have demonstrated, for the first time, that hypoxia enhances mouse MSC radioresistance in vitro. These findings have important implications for our understanding of MSC functions in supporting allogeneic bone marrow transplantation and in tumorigenesis.

The influence of hypoxia on the differentiation capacities and immunosuppressive properties of clonal mouse mesenchymal stromal cell lines.

Multipotent mesenchymal stromal cells are multipotent cells capable of differentiating into different mesodermal cell types. Enigmatically, mesenchymal stromal cells present in the bone marrow support early lymphopoiesis yet can inhibit mature lymphocyte growth. Critical features of the bone marrow microenvironment, such as the level of oxygen, play an important role in mesenchymal stromal cell biology. Herein, we show that a panel of continuously growing mouse mesenchymal stromal cell lines, namely OP9, MS5, PA6, ST2 and B16-14, exhibit mesenchymal stromal cell characteristic phenotypes and respond physiologically to oxygen deprivation. Culturing freshly isolated bone marrow-derived mesenchymal stromal cells or cell lines at 5% O2 resulted in a dramatic increase in expression of hypoxia-inducible factor family members and of key genes involved in their differentiation. Phenotypically, their osteogenic and adipogenic differentiation capacity was generally improved in hypoxia, whereas their inhibitory effects on in vitro T-cell proliferation were preserved. Taken together, we conclude that these continuously growing mouse cell lines behave as canonical mesenchymal stromal cells and respond physiologically to hypoxia, thereby providing a potent tool for the study of different aspects of mesenchymal stromal cell biology.

Multiple facets of the DNA damage response contribute to the radioresistance of mouse mesenchymal stromal cell lines.

The regeneration of the hematopoietic system following total body irradiation is supported by host-derived mesenchymal stromal cells (MSCs) within the bone marrow. The mechanisms used by MSCs to survive radiation doses that are lethal to the hematopoietic system are poorly understood. The DNA damage response (DDR) represents a cohort of signaling pathways that enable cells to execute biological responses to genotoxic stress. Here, we examine the role of the DDR in mediating the resistance of MSCs to ionizing radiation (IR) treatment using two authentic clonal mouse MSC lines, MS5 and ST2, and primary bulk mouse MSCs. We show that multiple DDR mechanisms contribute to the radio-resistance of MSCs: robust DDR activation via rapid γ-H2AX formation, activation of effective S and G(2)/M DNA damage checkpoints, and efficient repair of IR-induced DNA double-strand breaks. We show that MSCs are intrinsically programmed to maximize survival following IR treatment by expressing high levels of key DDR proteins including ATM, Chk2, and DNA Ligase IV; high levels of the anti-apoptotic, Bcl-2 and Bcl-(XL); and low levels of the pro-apoptotic, Bim and Puma. As a result, we demonstrate that irradiated mouse MSCs withstand IR-induced genotoxic stress, continue to proliferate, and retain their capacity to differentiate long-term along mesenchymal-derived lineages. We have shown, for the first time, that the DDR plays key roles in mediating the radioresistance of mouse MSCs which may have important implications for the study and application of MSCs in allogeneic bone marrow transplantation, graft-versus-host disease, and cancer treatment.

Concise review: adult mesenchymal stromal cell therapy for inflammatory diseases: how well are we joining the dots?

Mesenchymal stromal (stem) cells (MSCs) continue to be a strong area of focus for academic- and industry-based researchers who share the goal of expanding their therapeutic use for diverse inflammatory and immune-mediated diseases. Recently, there has been an accelerated rate of scientific publication, clinical trial activity, and commercialisation in the field. This has included the reporting of exciting new developments in four areas that will be of key importance to future successful use of MSC-based therapies in large numbers of patients: (a) fundamental biology of the primary cells in bone marrow and other tissues that give rise to MSCs in culture. (b) Mechanisms by which MSCs modulate immune and inflammatory responses in vivo. (c) Insights into MSC kinetics, safety, and efficacy in relevant animal disease models. (d) Isolation, definition, and clinical trial-based testing of human MSCs by biomedical companies and academic medical centers. Despite this progress, it remains unclear whether MSCs will enter mainstream therapeutic practice as a frequently used alternative to pharmacotherapy or surgical/radiological procedures in the foreseeable future. In this review, we summarize some of the most significant new developments for each of the four areas that contribute to the process of translating MSC research to the clinical arena. In the context of this recent progress, we discuss key challenges and specific knowledge gaps which, if not addressed in a coordinated fashion, may hinder the creation of robust "translational pipelines" for consolidating the status of MSC-based therapies.