A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

FlowAtlas: an interactive tool for high-dimensional immunophenotyping analysis bridging FlowJo with computational tools in Julia.

As the dimensionality, throughput and complexity of cytometry data increases, so does the demand for user-friendly, interactive analysis tools that leverage high-performance machine learning frameworks. Here we introduce FlowAtlas: an interactive web application that enables dimensionality reduction of cytometry data without down-sampling and that is compatible with datasets stained with non-identical panels. FlowAtlas bridges the user-friendly environment of FlowJo and computational tools in Julia developed by the scientific machine learning community, eliminating the need for coding and bioinformatics expertise. New population discovery and detection of rare populations in FlowAtlas is intuitive and rapid. We demonstrate the capabilities of FlowAtlas using a human multi-tissue, multi-donor immune cell dataset, highlighting key immunological findings. FlowAtlas is available at https://github.com/gszep/FlowAtlas.jl.git.

Multimodal profiling reveals tissue-directed signatures of human immune cells altered with age.

The immune system comprises multiple cell lineages and heterogeneous subsets found in blood and tissues throughout the body. While human immune responses differ between sites and over age, the underlying sources of variation remain unclear as most studies are limited to peripheral blood. Here, we took a systems approach to comprehensively profile RNA and surface protein expression of over 1.25 million immune cells isolated from blood, lymphoid organs, and mucosal tissues of 24 organ donors aged 20-75 years. We applied a multimodal classifier to annotate the major immune cell lineages (T cells, B cells, innate lymphoid cells, and myeloid cells) and their corresponding subsets across the body, leveraging probabilistic modeling to define bases for immune variations across donors, tissue, and age. We identified dominant tissue-specific effects on immune cell composition and function across lineages for lymphoid sites, intestines, and blood-rich tissues. Age-associated effects were intrinsic to both lineage and site as manifested by macrophages in mucosal sites, B cells in lymphoid organs, and T and NK cells in blood-rich sites. Our results reveal tissue-specific signatures of immune homeostasis throughout the body and across different ages. This information provides a basis for defining the transcriptional underpinnings of immune variation and potential associations with disease-associated immune pathologies across the human lifespan.

Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice.

We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.

Derivation of pluripotent epiblast stem cells from mammalian embryos.

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.

The immunogenicity of midbrain dopaminergic neurons and the implications for neural grafting trials in Parkinson's disease.

Dopaminergic (DA) cell replacement therapies are a promising experimental treatment for Parkinson's disease (PD) and a number of different types of DA cell-based therapies have already been trialled in patients. To date, the most successful have been allotransplants of foetal ventral midbrain but even then, the results have been inconsistent. This coupled to the ethical and logistical problems with using this tissue has meant that an alternative cell source has been sought of which human pluripotent stem cells (hPSCs) sources have proven very attractive. Robust protocols for making mesencephalic DA (mesDA) progenitor cells from hPSCs now exist and the first in-human clinical trials have or are about to start. However, while their safety and efficacy are well understood, relatively little is known about their immunogenicity and in this review, we briefly summarise this with reference mainly to the limited literature on human foetal DA cells.

Therapeutically expanded human regulatory T-cells are super-suppressive due to HIF1A induced expression of CD73.

The adoptive transfer of regulatory T-cells (Tregs) is a promising therapeutic approach in transplantation and autoimmunity. However, because large cell numbers are needed to achieve a therapeutic effect, in vitro expansion is required. By comparing their function, phenotype and transcriptomic profile against ex vivo Tregs, we demonstrate that expanded human Tregs switch their metabolism to aerobic glycolysis and show enhanced suppressive function through hypoxia-inducible factor 1-alpha (HIF1A) driven acquisition of CD73 expression. In conjunction with CD39, CD73 expression enables expanded Tregs to convert ATP to immunosuppressive adenosine. We conclude that for maximum therapeutic benefit, Treg expansion protocols should be optimised for CD39/CD73 co-expression.

NKG2D-RAE-1 receptor-ligand variation does not account for the NK cell defect in nonobese diabetic mice.

NK cells from NOD mice induced with poly(I:C) in vivo exhibit low cytotoxicity against a range of target cells, but the genetic mechanisms controlling this defect are yet to be elucidated. Defects in the expression of NKG2D and its ligands, the RAE-1 molecules, have been hypothesized to contribute to the reduced NK function present in NOD mice. In this study, we show that segregation of the NK-mediated killing phenotype did not correlate with the NOD Raet1 haplotype and that the large alterations in NKG2D expression previously reported on NK cells expanded in vitro were not observed in primary, poly(I:C)-elicited NK cells in vivo. Additional studies indicate a complex genetic control of defective NOD NK cells including genes linked to the MHC and possibly those that are associated with an altered cytokine response to the TLR3-agonist poly(I:C).

The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome.

It would be extremely advantageous to the analysis of disease mechanisms in the spontaneous mouse model of type 1 diabetes, the nonobese diabetic (NOD) strain, if genes in this strain could be modified in vivo using embryonic stem (ES) cells and homologous recombination. However, a NOD ES cell line with adequate germline transmission has not yet been reported. We report the development of highly germline-competent ES cell lines from the F1 hybrid of NOD and 129 for use in NOD gene targeting. Consequently, we developed ES cell lines derived from (NOD x 129)F1 x 129 backcross 1 mice, which were intercrossed to select for homozygosity of particular regions of NOD genome known to contain disease loci.

Commonality in the genetic control of Type 1 diabetes in humans and NOD mice: variants of genes in the IL-2 pathway are associated with autoimmune diabetes in both species.

Variants within the IL-2 (interleukin 2) and CD25 genes are associated with T1DM (Type 1 diabetes mellitus) in mice and humans respectively. Both gene products are essential for optimal immune tolerance and a partial failure to tolerize is linked to the autoimmune responses to insulin and other beta-cell proteins that precede T1DM onset. Gene variants that contribute to common disease susceptibility often alter gene expression only modestly. Small expression changes can be technically challenging to measure robustly, especially since biological variation usually contributes negatively to this goal. The present review focuses on allele-specific expression assays that can be used to quantify genotype-determined expression differences such as those observed for IL-2, where the susceptibility allele is transcribed 2-fold less than the resistance allele.

The effect of inhibiting DNA replication in the one-cell mouse embryo.

Inhibition of DNA replication in the 1-cell mouse embryo causes arrest in interphase. In such arrested cells the pattern of protein synthesis progresses as normal through the first cell cycle but does not show the characteristic changes that are associated with mitosis. Synthesis does occur of the 68/70 kd heat shock-like proteins, on the earliest detectable embryonic transcripts synthesised shortly after first cleavage in control embryos. However, the quantitative change in the pattern of protein synthesis, indicative of the major activation of the embryonic genome that normally occurs during G2 of the second cell cycle, is prevented.