Summary Somatic cell nuclear transfer and transcription factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. These two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, by different mechanisms and kinetics, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low passage induced pluripotent stem cells (iPSC) derived by factor-based reprogramming harbor residual DNA methylation signatures characteristic of their somatic tissue of origin, which favors their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an “epigenetic memory” of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSC with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSC, consistent with more effective reprogramming. Our data demonstrate that factor-based reprogramming can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modeling or treatment.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Hematopoiesis 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. While DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs1, a comprehensive DNA methylation map of hematopoietic 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). Dramatic 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, suggesting a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome appears to be important in hematopoietic 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.

NKG2D is an activating receptor on CD8(+) T cells and NK cells that has been implicated in immunity against tumors and microbial pathogens. Here we show that RAE-1 is present in prediabetic pancreas islets of NOD mice and that autoreactive CD8(+) T cells infiltrating the pancreas express NKG2D. Treatment with a nondepleting anti-NKG2D monoclonal antibody (mAb) during the prediabetic stage completely prevented disease by impairing the expansion and function of autoreactive CD8(+) T cells. These findings demonstrate that NKG2D is essential for disease progression and suggest a new therapeutic target for autoimmune type I diabetes.

Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named “Gene Expression Commons” (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

Therapeutics, BioNTx, and Polaris. L.L. serves on advisory boards for Servier. S.C.W. and J.P.A. are inventors of a patent application submitted by The University of Texas MD Anderson Cancer Center related to a genetic mouse model of immune checkpoint blockade induced immune-related adverse events. S.C.W. is currently an employee of Spotlight Therapeutics. E.M.W has ownership interest in Pathogenesis, LLC. W.C.M. was supported by funding from the Niels Stensen Fellowship and the Netherlands Heart Institute. D.B.J serves on advisory boards for Array Biopharma, BMS, Merck, Novartis; research funding from BMS and Incyte. J.E.S serves on advisory boards for BMS. J.E.S, D.B.J and J.J.M are inventors of a patent application submitted by The Assistance Publique-Hopitaux de Paris related to abatacept for the treatment of immune-related adverse events associated with immune checkpoint inhibitors. Research.

SUMMARY T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used 2-photon microscopy to visualize the migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4−8−) and double-positive (CD4+8+; DP) thymocytes were strictly confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were excluded from the medulla by an inability to migrate on medullary substrates. In contrast, CD4 single-positive (SP) thymocytes migrated directionally towards the medulla, where they accumulated and moved very rapidly. Our results reveal a requisite two-step process governing CD4 SP medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards the medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus as they mature.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8 lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

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. IntroductionT cells develop in the thymus from immature thymocytes throughout life. Thymocytes themselves are not a self-sustaining population but, instead, are supplied from a constant stream of bone marrow (BM)-derived progenitors. However, the identity of the exact progenitors that leave the BM and home to the thymus has remained controversial. [1][2][3][4] The original identification of the lymphoid-restricted common lymphoid progenitors (CLPs) demonstrated T progenitor activity from this population in vivo, suggesting that it might be the immediate precursor to thymocytes. 5 This model has been steadily challenged by an alternative theory, in which subpopulations of multipotent progenitors (MPPs) are the major source of thymocytes. [6][7][8][9][10][11][12][13] MPPs were first implicated as the major source of thymic seeding cells on the basis of cell surface phenotype similarity between MPP and early thymic progenitors (DN1), the higher proliferative potential of DN1 and MPPs compared with CLPs, and the phenotype of ikaros mutant mice, which lacked phenotypic CLPs but still had some thymopoiesis. 14 Although this study was suggestive, it has since been shown that CLPs rapidly adopt a DN1 phenotype on thymic entry, 15 and the time course of intrathymic differentiation of DN1 and CLP is much more similar than that of MPP. 14,16,17 Nonetheless, consistent with the idea that MPPs are the major source of thymopoiesis, papers from many laboratories [17][18][19][20][21][22][23] have shown that subsets of MPPs, when injected intravenously, give rise to more T cells than do CLPs over several weeks. Indeed, a subset of MPPs has been found to home to the thymus 24 hours after intravenous injection, but the ability of those cells to give rise to T-lineage progeny remains unclear. 22 Two recent publications supported the MPP progenitor model of thymopoiesis by showing, clonally in vitro, that the majority of DN1 cells have myeloid potential. 24,25 Although these results implied that few, if any, DN1 come from the lymphoid restricted CLPs, they contradicted the very low myeloid readout from DN1 observed in other in vitro assays, as well ...