Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1–/– phenotype. Here we demonstrate that cells derived from Bmi1–/– mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1–/– mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

SUMMARY Long-term survival and anti-tumor immunity of adoptively-transferred CD8+ T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically-robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8+ T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8+, CD4+ T-cell subsets and long-term hematopoietic stem cells. This metabolism-based approach to cell-selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Regulatory T cells (T reg s) that constitutively express FOXP3 are instrumental to the maintenance of tolerance and may suppress graft-versus-host disease (GVHD) in humans. To determine whether regulatory T cells in allogeneic stem cell transplants (SCTs) ameliorate GVHD after transplantation, we quantitated the coexpression of FOXP3 on CD4 ؉ T cells in 32 donor SCTs infused into HLA-matched siblings and examined GVHD incidence in respective recipients. High CD4 ؉ FOXP3 ؉ Tcell count in the donor was associated with a reduced risk of GVHD. We monitored T reg s during immune reconstitution in 21 patients with leukemia undergoing a T-cell-depleted allogeneic SCT. Early after SCT, there was a significant expansion in the CD4 ؉ FOXP3 ؉ T-cell compartment. A low CD4 ؉ FOXP3 ؉ Tcell count early after SCT (day 30) was associated with an increased risk of GVHD, and the ratio of CD4 ؉ FOXP3 ؉ T cells to CD4 ؉ CD25 ؉ FOXP3 ؊ T cells was significantly reduced in patients with GVHD, suggesting diminished control of effector T cells. Our findings suggest that graft T reg content may predict for risk of GVHD after SCT. Determining the T reg levels in the donor and manipulating T reg s early after transplantation may provide a new approach to

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IntroductionThe ability to transfer genes into repopulating hematopoietic stem cells ex vivo and to achieve regulated expression in specific lineages following hematopoietic reconstitution would create many therapeutic opportunities. 1 Although the initial use of murine oncoretroviral vectors to transfer genes into primitive murine hematopoietic cells was reported 20 years ago, 2 translation of this approach to clinical application has been slow and has required considerable effort. Despite progress being achieved in the murine system with correction of single gene defects in murine models of human immunodeficiencies [3][4][5][6] and chronic granulomatous disease, 7,8 the much lower efficiency of gene transfer into human stem cells 1 has hampered success. The necessity for high-level oncoretroviral vector gene transfer to achieve therapeutic benefit, however, was circumvented in 2 recent clinical trials designed to cure severe combined immunodeficiency due to a deficiency of the common ␥-chain of the lymphoid cytokine receptor 9 or adenosine deaminase. 10 In these trials, a potent selective repopulating advantage of the gene-corrected lymphoid cells resulted in therapeutically relevant numbers of functional lymphocytes.Despite this success, 2 barriers appear to limit the ability of murine oncoretroviral vectors to achieve adequate transduction of primitive hematopoietic stem cells for treatment of other disorders in which the gene-corrected cells do not have a selective advantage. Because the human homolog of the receptor for murine ecotropic vector particles does not interact with the ecotropic envelope protein, amphotropic particles have been used in both human studies and in large animal models. The amphotropic receptor, however, is expressed at low levels on human stem cells. 11 Alternative envelopes have been tested, such as those derived from the gibbon ape leukemia virus, 12 feline endogenous virus (RD114), 13 or feline leukemia virus type C, 14 the receptors for which are expressed at higher levels on primitive hematopoietic cell populations. However, large animal studies have failed to clearly identify a superior pseudotype that consistently yields high-level stem cell gene transfer. These data suggest that a second barrier-namely, the requirement for mitosis to allow integration of the oncoretroviral vector genome 15 -along with the relative instability of the preintegration complex 16 may be the main limitations of oncoretroviral-mediated stem cell gene transfer. In an effort to induce stem cell cycling, cytokines such as stem cell factor (SCF), Flt-3 ligand (Flt3-L), and megakaryocyte growth and development factor (MGDF) 17,18 are added to culture medium and a fragment of fibronectin is used to colocalize vector particles and target cells, 19 leading to improved stem cell transduction efficiency in large animal models. 12,20,21 Nonetheless, there remains significant variability among animals, with many animals having low marking and only occasional animals having proportions of genetically modifi...

IntroductionA subpopulation of T cells, termed regulatory T cells (Tregs), have been described in humans and animal models; these cells are important because they suppress autoreactive T cells by direct cell contact. [1][2][3][4] Phenotypically, Treg are characterized by cell surface expression of the proteins CD4 and CD25 and by intracellular expression of the transcription factor FOXP3. Only CD4 ϩ CD25hi ϩ FOXP3 ϩ T cells express suppressor functions. FOXP3 is a member of the forkhead/winged-helix family of transcription regulators (FOXP1-4). In humans, mutations in FOXP3 result in an autoimmune syndrome termed immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. 5 The transcription factor NFAT1 induces FOXP3 expression by binding to its promoter. 6 In addition, FOXP3 expresses its repressive effects on cytokine expression and its activating effects on CD25 by cooperation with NFAT1. 7,8 Acquired aplastic anemia is characterized by destruction of hematopoietic stem cells by cytotoxic T lymphocytes. 9 Hematopoietic response and hematologic recovery after successful immunosuppressive treatment represent the most powerful evidence that this rare and complex disease is immune-mediated. 10 Increased T-bet protein levels in T cells probably are responsible for the increased interferon-␥ levels and the Th1 polarization in patients with aplastic anemia. 11 We show that Tregs were decreased in acquired aplastic anemia, as in other autoimmune diseases. [12][13][14][15][16] All patients examined had low levels of FOXP3. Mechanistically, FOXP3 down-regulation appeared to be mediated by the transcription factor NFAT1. Patients, materials, and methods Patients and control subjectsInformed consent was acquired according to protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (NHLBI) and was obtained in accordance with the Declaration of Helsinki for all patients (n ϭ 20; age range, 13-52 years) with acquired aplastic anemia examined (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) and healthy volunteers (n ϭ 14; age range, 18-55 years). Lymphocyte isolation, flow cytometry, and immunoblotsCD4, CD25, and FOXP3 expression was examined in peripheral blood mononuclear cells (PBMC) by 3-color flow cytometry as previously described 11 using an APC-antihuman FOXP3 staining kit (eBioscience, San Diego, CA). 15 Immunoblot experiments were performed as previously described 11 (Document S1). Quantitative real-time polymerase chain reactionFOXP3 gene expression was measured in CD4 ϩ CD25 ϩ T cells as previously described. 15 All polymerase chain reaction assays were performed in duplicate and reported as the mean. Confocal microscopy and T-cell transfectionsNFAT1 and FOXP3 expression was examined by confocal microscopy as previously described 17 (Document S1). Transfections were performed 18,19 using a GFP-wild-type NFAT1 plasmid 20 (a gift from Dr. A. Rao, Harvard University, Cambridge, MA) and examined ...

T helper type 17 (Th17) cells have been characterized based on production of interleukin-17 (IL-17) and association with autoimmune diseases. We studied the role of Th17 cells in aplastic anemia (AA) by isolating Th17 cells from patients blood (n ‫؍‬ 41) and bone marrow (BM) mononuclear cells (n ‫؍‬ 7). The frequency and total number of CD3 ؉ CD4 ؉ IL-17-producing T cells were increased in AA patients at presentation compared with healthy controls (P ‫؍‬ .0007 and .02, respectively) and IntroductionTh17 cells have been characterized recently in mice as a novel subset of CD4 ϩ T cells that produce interleukin-17A (IL-17A), IL-17-F, and IL-22, 1,2 and serve as immune effectors in various settings, including inflammation, infection, and autoimmunity. 3,4 Th17 cells produce a large amount of IL-17A, a cytokine that coordinates tissue inflammation by inducing the expression of proinflammatory cytokines (such as IL-6 and tumor necrosis factor [TNF]), chemokines (such as KC, MCP-1, and MIP-2), and matrix metalloproteases that mediate tissue infiltration and tissue destruction. 5 In mice, the differentiation program of Th17 cells from naive CD4 ϩ T cells requires the activation of the transcription factor, orphan nuclear receptor ROR␥t, 6 and the presence of IL-6 and transforming growth factor-␤ (TGF-␤). 7,8 In humans, Th17 differentiation is under the control of IL-1␤, 10 Several studies have reported the association of IL-17 with inflammatory disorders, such as rheumatoid arthritis, asthma, multiple sclerosis, and lupus, 11 as well as hematologic disorders, such as myelodysplastic syndrome 12,13 and acute myeloid leukemia. 14 Aplastic anemia (AA), a disease characterized by peripheral blood pancytopenia and bone marrow (BM) hypoplasia, 15 is an immunemediated disorder in most cases with active destruction of hematopoietic cells by effector T lymphocytes. 16 Recovery of autologous hematopoiesis in patients who failed to engraft after conditioning and stem cell transplantation, 17 and responsiveness of patients to immunosuppressive therapies, 18 provided powerful evidence for the pivotal role of the immune system in the disease pathophysiology. Immoderate production of interferon-␥ (IFN-␥), TNF-␣, and IL-2 from patients'T cells suggests that the hematopoietic cells are destroyed through a Th1 response, [19][20][21] as illustrated by the up-regulation of the transcription factor T-bet in patient T cells. 22 The description of nonrandom skewing of the V␤ chain families of the T-cell receptor in patient peripheral blood (PB) revealed that expanded oligoclonal or monoclonal specific V␤ subfamilies selectively induced apoptosis of hematopoietic progenitor cells. 23 Regulatory T cells (Tregs), which control and suppress autoreactive T cells, are decreased at disease presentation in almost all patients. 24 We have developed murine models for immune-mediated BM failure by the infusion of allogeneic lymph node (LN) cells into sublethally irradiated recipients for which treatment with limited number of Treg cells, 25 or anti-IFN...

To determine whether the leukemia-associated Wilms tumor antigen (WT1) contributes to a graft-versus-leukemia (GVL) effect after allogeneic stem-cell transplantation (SCT) for acute lymphoblastic leukemia (ALL), we studied CD8(+) T-cell responses to WT1 in 10 human lymphocyte antigen (HLA)-A*0201-positive ALL patients during the early phase of immune recovery after SCT (days 30-120). Seven of 10 patients had detectable WT1 expression in their peripheral blood (PB) before SCT by quantitative reverse-transcription polymerase chain reaction. Using WT1/HLA-A*0201 tetramers and intracellular interferon-gamma (IFN-gamma) staining, WT1(+) CD8(+) T-cell responses after SCT were found only in patients with detectable WT1 expression before SCT (5 of 7 vs. 0 of 3; P < .05). To monitor the kinetics of WT1(+) CD8(+) T-cell responses and disease regression after SCT, absolute WT1(+) CD8(+) T-cell numbers and WT1 expression were studied for each time point. The emergence of WT1(+) CD8(+) T cells was associated with a decrease in WT1 expression, suggesting a WT1-driven GVL effect. Loss of WT1(+) CD8(+) T-cell responses was associated with reappearance of WT1 transcripts, consistent with a molecular relapse (P < .001). WT1(+) CD8(+) T cells had a predominantly effector-memory phenotype (CD45RO(+) CD27(-)CD57(+)) and produced IFN-gamma. Our results support the immunogenicity of WT1 after SCT for ALL and highlight the potential for WT1 vaccines to boost GVL after SCT for ALL.