Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-ß superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Interleukin-17-Producing γδ T Cells Originate from SOX13+ Progenitors that Are Independent of γδTCR Signaling.

Lineage-committed αß and γδ T cells are thought to originate from common intrathymic multipotent progenitors following instructive T cell receptor (TCR) signals. A subset of lymph node and mucosal Vγ2+ γδ T cells is programmed intrathymically to produce IL-17 (Tγδ17 cells), however the role of the γδTCR in development of these cells remains controversial. Here we generated reporter mice for the Tγδ17 lineage-defining transcription factor SOX13 and identified fetal-origin, intrathymic Sox13+ progenitors. In organ culture developmental assays, Tγδ17 cells derived primarily from Sox13+ progenitors, and not from other known lymphoid progenitors. Single cell transcriptome assays of the progenitors found in TCR-deficient mice demonstrated that Tγδ17 lineage programming was independent of γδTCR. Instead, generation of the lineage committed progenitors and Tγδ17 cells was controlled by TCF1 and SOX13. Thus, T lymphocyte lineage fate can be prewired cell-intrinsically and is not necessarily specified by clonal antigen receptor signals.

The Autoimmunity-Associated Gene CLEC16A Modulates Thymic Epithelial Cell Autophagy and Alters T Cell Selection.

CLEC16A variation has been associated with multiple immune-mediated diseases, including type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, celiac disease, Crohn's disease, Addison's disease, primary biliary cirrhosis, rheumatoid arthritis, juvenile idiopathic arthritis, and alopecia areata. Despite strong genetic evidence implicating CLEC16A in autoimmunity, this gene's broad association with disease remains unexplained. We generated Clec16a knock-down (KD) mice in the nonobese diabetic (NOD) model for type 1 diabetes and found that Clec16a silencing protected against autoimmunity. Disease protection was attributable to T cell hyporeactivity, which was secondary to changes in thymic epithelial cell (TEC) stimuli that drive thymocyte selection. Our data indicate that T cell selection and reactivity were impacted by Clec16a variation in thymic epithelium owing to Clec16a's role in TEC autophagy. These findings provide a functional link between human CLEC16A variation and the immune dysregulation that underlies the risk of autoimmunity.

A biomimetic five-module chimeric antigen receptor (5MCAR) designed to target and eliminate antigen-specific T cells.

T cells express clonotypic T cell receptors (TCRs) that recognize peptide antigens in the context of class I or II MHC molecules (pMHCI/II). These receptor modules associate with three signaling modules (CD3γε, δε, and ζζ) and work in concert with a coreceptor module (either CD8 or CD4) to drive T cell activation in response to pMHCI/II. Here, we describe a first-generation biomimetic five-module chimeric antigen receptor (5MCAR). We show that 1) chimeric receptor modules built with the ectodomains of pMHCII assemble with CD3 signaling modules into complexes that redirect cytotoxic T lymphocyte (CTL) specificity and function in response to the clonotypic TCRs of pMHCII-specific CD4+ T cells, and 2) surrogate coreceptor modules enhance the function of these complexes. Furthermore, we demonstrate that adoptively transferred 5MCAR-CTLs can mitigate type I diabetes by targeting autoimmune CD4+ T cells in NOD mice. This work provides a framework for the construction of biomimetic 5MCARs that can be used as tools to study the impact of particular antigen-specific T cells in immune responses, and may hold potential for ameliorating diseases mediated by pathogenic T cells.

Young, proliferative thymic epithelial cells engraft and function in aging thymuses.

The thymus reaches its maximum size early in life and then begins to shrink, producing fewer T cells with increasing age. This thymic decline is thought to contribute to age-related T cell lymphopenias and hinder T cell recovery after bone marrow transplantation. Although several cellular and molecular processes have been implicated in age-related thymic involution, their relative contributions are not known. Using heterochronic parabiosis, we observe that young circulating factors are not sufficient to drive regeneration of the aged thymus. In contrast, we find that resupplying young, engraftable thymic epithelial cells (TECs) to a middle-aged or defective thymus leads to thymic growth and increased T cell production. Intrathymic transplantation and in vitro colony-forming assays reveal that the engraftment and proliferative capacities of TECs diminish early in life, whereas the receptivity of the thymus to TEC engraftment remains relatively constant with age. These results support a model in which thymic growth and subsequent involution are driven by cell-intrinsic changes in the proliferative capacity of TECs, and further show that young TECs can engraft and directly drive the growth of involuted thymuses.

Ly6d marks the earliest stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development.

Common lymphoid progenitors (CLPs) clonally produce both B- and T-cell lineages, but have little myeloid potential in vivo. However, some studies claim that the upstream lymphoid-primed multipotent progenitor (LMPP) is the thymic seeding population, and suggest that CLPs are primarily B-cell-restricted. To identify surface proteins that distinguish functional CLPs from B-cell progenitors, we used a new computational method of Mining Developmentally Regulated Genes (MiDReG). We identified Ly6d, which divides CLPs into two distinct populations: one that retains full in vivo lymphoid potential and produces more thymocytes at early timepoints than LMPP, and another that behaves essentially as a B-cell progenitor.

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

Expression of TCR-Vß peptides by murine bone marrow cells does not identify T-cell progenitors.

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vß8.2 in absence of anti-Cß antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vß8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vß8.2(+) Cß(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-)) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vß8.2(+) Cß(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vß8.2(+) Cß(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors.

The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials.

T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cell.

The conversion of mature somatic cells into pluripotent stem cells, both by nuclear transfer and transduction with specific "reprogramming" genes, represents a major advance in regenerative medicine. Pluripotent stem cell lines can now be generated from an individual's own cells, facilitating the generation of immunologically acceptable stem cell-based therapeutics. Many cell types can undergo nuclear reprogramming, leading to the question of whether the identity of the reprogrammed cell of origin has a biological consequence. Peripheral blood, containing a mixture of T, B, NK, and myeloid cell types, represents one potential source of reprogrammable cells. In this study, we describe the unique case of mice derived from a reprogrammed T cell. These mice have prerearranged T-cell receptor (TCR) genes in all cells. Surprisingly, ≈50% of mice with prerearranged TCR genes develop spontaneous T cell lymphomas, which originate in the thymus. The lymphomas arise from developing T cells, and contain activated Notch1, similar to most human and mouse T-cell acute lymphoblastic lymphomas. Furthermore, lymphomagenesis requires the expression of both prerearranged TCRα and TCRß genes, indicating a critical role for TCR signaling. Furthermore, inhibitors of multiple branches of TCR signaling suppress lymphoma growth, implicating TCR signaling as an essential component in lymphoma proliferation. The lymphomagenesis in mice derived from a reprogrammed T cell demonstrates the deleterious consequences of misregulation of the TCR rearrangement and signaling pathways and illustrates one case of cellular reprogramming where the identity of the cell of origin has profound consequences.

Position-dependent silencing of germline Vß segments on TCRß alleles containing preassembled VßDJßCß1 genes.

The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

Brown adipose tissue-derived MaR2 contributes to cold-induced resolution of inflammation.

Obesity induces chronic inflammation resulting in insulin resistance and metabolic disorders. Cold exposure can improve insulin sensitivity in humans and rodents, but the mechanisms have not been fully elucidated. Here, we find that cold resolves obesity-induced inflammation and insulin resistance and improves glucose tolerance in diet-induced obese mice. The beneficial effects of cold exposure on improving obesity-induced inflammation and insulin resistance depend on brown adipose tissue (BAT) and liver. Using targeted liquid chromatography with tandem mass spectrometry, we discovered that cold and ß3-adrenergic stimulation promote BAT to produce maresin 2 (MaR2), a member of the specialized pro-resolving mediators of bioactive lipids that play a role in the resolution of inflammation. Notably, MaR2 reduces inflammation in obesity in part by targeting macrophages in the liver. Thus, BAT-derived MaR2 could contribute to the beneficial effects of BAT activation in resolving obesity-induced inflammation and may inform therapeutic approaches to combat obesity and its complications.

Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis.

Ongoing thymopoiesis requires continual seeding from progenitors that reside within the bone marrow (BM), but the identity of the most proximate prethymocytes has remained controversial. Here we take a comprehensive approach to prospectively identify the major source of thymocyte progenitors that reside within the BM and blood, and find that all thymocyte progenitor activity resides within a rare Flk2(+)CD27(+) population. The BM Flk2(+)CD27(+) subset is predominantly composed of common lymphoid progenitors (CLPs) and multipotent progenitors. Of these 2 populations, only CLPs reconstitute thymopoiesis rapidly after intravenous injection. In contrast, multipotent progenitor-derived cells reconstitute the thymus with delayed kinetics only after they have reseeded the BM, self-renewed, and generated CLPs. These results identify CLPs as the major source of thymocyte progenitors within the BM.

Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay.

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called "pre-HSCs." Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed "11a- eKLS" (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs.

Flk2+ common lymphoid progenitors possess equivalent differentiation potential for the B and T lineages.

Mature blood cells develop from multipotent hematopoietic stem cells through a series of sequential intermediates in which the developmental potential for particular blood lineages is progressively extinguished. We previously reported the identification of one of these developmental intermediates, the common lymphoid progenitor (CLP), which can give rise to T cells, B cells, dendritic cells (DCs), and natural killer cells (NKs), but lacks myeloid and erythroid potential. Recently, several studies have suggested that the T-cell and DC potential of CLP is limited or absent, and/or that CLP contains significant myeloid potential. Here, we show that the originally identified CLP population can be divided into functionally distinct subsets based on the expression of the tyrosine kinase receptor, Flk2. The Flk2(+) subset contains robust in vivo and in vitro T-cell, B-cell, DC, and NK potential, but lacks myeloid potential and, therefore, represents an oligopotent, lymphoid-restricted progenitor. This population of cells does not appear to be B cell-biased and robustly reconstitutes both B and T lineages in vivo, consistent with its being a physiologic progenitor of both of these subsets. Thus, Flk2 expression defines a homogeneous, readily obtainable subset of bone marrow CLP that is completely lymphoid-committed and can differentiate equivalently well into both B and T lineages.

Isolation of Highly Viable Thymic Epithelial Cells for Use in In Vitro and In Vivo Experiments.

Thymic epithelial cells (TECs) play multiple essential roles in T-cell development and the establishment of immune tolerance, but their isolation can be challenging, and their low viability upon isolation complicates downstream experiments. A method that allows TECs to be isolated easily and to survive afterward will be useful for elucidating key questions in TEC biology. Here, we demonstrate a simple method to isolate highly viable TECs. Primary TECs isolated using papain together with collagenase IV and DNase I survive and proliferate in vitro. Moreover, these primary TECs functionally engraft after intrathymic transplantation into recipient mice. Thus, the methods described herein will be useful for elucidating the roles of TECs and TEC subsets in T-cell development and immune tolerance.

Increased ß-cell proliferation before immune cell invasion prevents progression of type 1 diabetes.

Type 1 diabetes (T1D) is characterized by pancreatic islet infiltration by autoreactive immune cells and a near-total loss of ß-cells1. Restoration of insulin-producing ß-cells coupled with immunomodulation to suppress the autoimmune attack has emerged as a potential approach to counter T1D2-4. Here we report that enhancing ß-cell mass early in life, in two models of female NOD mice, results in immunomodulation of T-cells, reduced islet infiltration and lower ß-cell apoptosis, that together protect them from developing T1D. The animals displayed altered ß-cell antigens, and islet transplantation studies showed prolonged graft survival in the NOD-LIRKO model. Adoptive transfer of splenocytes from the NOD-LIRKOs prevented development of diabetes in pre-diabetic NOD mice. A significant increase in the splenic CD4+CD25+FoxP3+ regulatory T-cell (Treg) population was observed to underlie the protected phenotype since Treg depletion rendered NOD-LIRKO mice diabetic. The increase in Tregs coupled with activation of TGF-ß/SMAD3 signaling pathway in pathogenic T-cells favored reduced ability to kill ß-cells. These data support a previously unidentified observation that initiating ß-cell proliferation, alone, prior to islet infiltration by immune cells alters the identity of ß-cells, decreases pathologic self-reactivity of effector cells and increases Tregs to prevent progression of T1D.

The CD11a and Endothelial Protein C Receptor Marker Combination Simplifies and Improves the Purification of Mouse Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. Stem Cells Translational Medicine 2018;7:468-476.

In Vivo Enrichment of Diabetogenic T Cells.

Dysfunctional T cells can mediate autoimmunity, but the inaccessibility of autoimmune tissues and the rarity of autoimmune T cells in the blood hinder their study. We describe a method to enrich and harvest autoimmune T cells in vivo by using a biomaterial scaffold loaded with protein antigens. In model antigen systems, we found that antigen-specific T cells become enriched within scaffolds containing their cognate antigens. When scaffolds containing lysates from an insulin-producing ß-cell line were implanted subcutaneously in autoimmune diabetes-prone NOD mice, ß-cell-reactive T cells homed to these scaffolds and became enriched. These T cells induced diabetes after adoptive transfer, indicating their pathogenicity. Furthermore, T-cell receptor (TCR) sequencing identified many expanded TCRs within the ß-cell scaffolds that were also expanded within the pancreata of NOD mice. These data demonstrate the utility of biomaterial scaffolds loaded with disease-specific antigens to identify and study rare, therapeutically important T cells.

CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1.

Identification and isolation of hematopoietic stem cells (HSCs) in mice is most commonly based on the expression of surface molecules Kit and Sca-1 and the absence of markers of mature lineages. However, Sca-1 is absent or weakly expressed in hematopoietic progenitors in many strains, including nonobese diabetic (NOD), BALB/c, C3H, and CBA mice. In addition, both Kit and Sca-1 levels are modulated following bone marrow injury. In these cases, other markers and dye exclusion methods have been employed to identify HSCs, yet there is no antibody-based stain that enables identification of HSCs and early progenitors when Kit and Sca-1 are inadequate. CD201 is a marker that is highly restricted to HSCs and progenitors, and CD27 is expressed at moderate-to-high levels on HSCs. We show here that combining CD201 and CD27 enables highly efficient isolation of long-term HSCs in NOD mice as well as in other strains, including SJL, FVB, AKR, BALB/c, C3H, and CBA. We also find that HSCs appear to maintain expression of CD201 and CD27 after hematopoietic injury when Kit expression is downregulated. These results suggest a widely applicable yet simple alternative for HSC isolation in settings where Kit and Sca-1 expression are insufficient.