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Table S1. Differential expression analysis for CD8 T cells.

Table S5. List of Genes Used to Generate NK Cell Activation Scores.

Table S2. Differential expression analysis for Treg CD4 T cells.

Table S3. Differential expression analysis for non-Treg CD4 T cells.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC.Significance:This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Table S4. Differential expression analysis for NK cells.

Supplementary Materials and Methods, Figures, and Figure and Table legends

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. Significance: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Oncogenic FLT3 kinase is a clinically validated target in acute myeloid leukemia (AML), and both multi-targeted and selective FLT3 inhibitors have been developed. Spleen tyrosine kinase (SYK) has been shown to be activated and increased in FLT3-ITD-positive AML patients, and has further been shown to be critical for transformation and maintenance of the leukemic clone in these patients. Further, over-expression of constitutively activated SYK causes resistance to highly selective FLT3 tyrosine kinase inhibitors (TKI). Up to now, the activity of the multi-targeted FLT3 inhibitor, midostaurin, against cells expressing activated SYK has not been explored in the context of leukemia, although SYK has been identified as a target of midostaurin in systemic mastocytosis. We compared the ability of midostaurin to inhibit activated SYK in mutant FLT3-positive AML cells with that of inhibitors displaying dual SYK/FLT3 inhibition, targeted SYK inhibition, and targeted FLT3 inhibition. Our findings suggest that dual FLT3/SYK inhibitors and FLT3-targeted drugs potently kill oncogenic FLT3-transformed cells, while SYK-targeted small molecule inhibition displays minimal activity. However, midostaurin and other dual FLT3/SYK inhibitors display superior anti-proliferative activity when compared to targeted FLT3 inhibitors, such as crenolanib and quizartinib, against cells co-expressing FLT3-ITD and constitutively activated SYK-TEL. Interestingly, additional SYK suppression potentiated the effects of dual FLT3/SYK inhibitors and targeted FLT3 inhibitors against FLT3-ITD-driven leukemia, both in the absence and presence of activated SYK. Taken together, our findings have important implications for the design of drug combination studies in mutant FLT3-positive patients and for the design of future generations of FLT3 inhibitors.

The study of CD8 positive cells in peripheral blood has become an essential part of research in the field of cancer immunotherapies, vaccine development, inflammation, autoimmune disease, etc. In this study, an 8-color flow cytometry panel, containing lineage and functional markers, was developed for the identification of CD8+ cytotoxic T cells in previously cryopreserved peripheral blood mononuclear cells from healthy human donors. By studying functional markers in naïve and CD3/CD28 activated T cells we demonstrate that the panel is capable of detecting protein markers corresponding to different T cell activation statuses. Data generated by flow cytometry were corroborated by different antibody based assay technologies to detect soluble cytokines. Our findings suggest that there is an inter donor variability in both baseline and activation responses. We have also successfully developed an antibody panel for flow cytometry that could be used to study cytotoxic function of CD8 T cells in clinical immunology research areas.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

We have previously shown that immune cells, including regulatory T cells, Th17 cells, myeloid-derived suppressor cells and NK cells are abnormal in multiple myeloma (MM). It is well demonstrated that immune-modulatory drugs like pomalidomide (POM) have shown impact on various immune sub-populations in pre-clinical studies. Relapsed/refractory (RR) MM (N=540) patients were randomized to be bortezomib and dexamethasone (Vd) with or without pomalidomide POM (Vd) in a large Phase III CC4047-MM007 (OPTIMISMM, NCT01734928) study, allowing for critical investigation into the impact of POM on immune cell-subset. We have analyzed 197 RRMM patients utilizing 366 peripheral blood samples collected at screening, day 8 of cycle 1 and day 8 of cycle 3 using 98 immune biomarker-panel with multi-color flow to identify changes with POM exposure in various sub-populations of B, T, & NK cells. The primary objective was to investigate association of PFS with the modulation of predictive and prognostic immune bio-markers by POM. The analyzed patient-treatment cohorts had identical characteristics as the total patient population and was balanced for the 2-treatment arms, overall patient characteristics and response. Among B-Cell-subpopulations, we observed significant up-regulation of B1b cells (CD19+CD43+) and down-regulation of regulatory B cells (Bregs, CD19+CD5+CD43-) by the PVd arm as compared with the Vd arm. A significant increase in MZB cells were observed in both arms. Patients with a higher proportion of either CD19+ B cells or Bregs at screening showed significantly favorable PFS (logrank p value < 0.05) in the PVd arm as compared with the VD arm, which indicates that these markers are predictive of PFS for PVd treatment. Moreover, patients with a higher proportion of either [naïve B cells (CD19+CD27-IgD+)] or [CD95+ B cells that display germinal center differentiation features (CD19+ CD185+) at cycle 1 day 8] showed significantly favorable PFS in PVd arm (logrank p value < 0.05); however, they were not prognostic with the Vd arm alone. This indicates that the early impact of Pom on these cell types may provide a prognostic bio-marker outcome following PVd therapy in B cell-compartment at cycle 1. Among T-Cell subpopulations, we observed that patients with higher proportion of CD4+ T cells at screening showed significantly favorable PFS in PVd arm, indicating a predictive marker. We observed significantly favorable PFS in PVd arm in both patients with a higher number of CD8 T cells expressing OX-40 at cycle 3 day 8 and patients with lower number of CD4 T cells or Tfh cells (CD4+CD185+) expressing PD-1 or regulatory T cells expressing (low CD127+ and high CD25+) HLA-DR at cycle 3 day 8, indicating prognostic markers. The studied markers were neither predictive nor prognostic for the Vd arm alone. The total proportion of NK cells were significantly elevated following the Pom-containing regimen as early as day 8 of cycle 1 and persisted at cycle 3 as compared to the Vd arm. Among NK cell-subpopulations, we observed that the patients with either a higher proportion of NK cells without expressing KIR molecules or a lower proportion of NKT cells expressing CD158b KIR molecule at screening had significantly superior PFS with in the PVd, which indicates that each is a predictive marker. Finally, we showed that patients with either a higher proportion of NK cells expressing activation marker NKG2D, or a lower number of CD158b expressing NK cells at cycle 3 had significantly improved PFS with the PVd arm but not with the Vd arm, which indicates the prognostic nature of these markers. In summary, this large randomized study identified specific immunophenotypes at screening and after exposure to pomalidomide combinations, allowing for the identification of predictive and prognostic markers for favorable PFS, respectively. Disclosures Biyukov: Celgene: Employment, Equity Ownership. Oriol:Celgene, Amgen, Takeda, Jansse: Consultancy, Speakers Bureau. Pierceall:Celgene Corporation: Employment, Equity Ownership. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Anderson:Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder . Thakurta:Celgene: Employment, Equity Ownership. Munshi:Oncopep: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

INTRODUCTION Dendritic cells (DCs) and monocytes consist of multiple specialized subtypes that play a central role in pathogen sensing, phagocytosis, and antigen presentation. However, their identities and interrelationships are not fully understood, as these populations have historically been defined by a combination of morphology, physical properties, localization, functions, developmental origins, and expression of a restricted set of surface markers. RATIONALE To overcome this inherently biased strategy for cell identification, we performed single-cell RNA sequencing of ~2400 cells isolated from healthy blood donors and enriched for HLA-DR + lineage − cells. This single-cell profiling strategy and unbiased genomic classification, together with follow-up profiling and functional and phenotypic characterization of prospectively isolated subsets, led us to identify and validate six DC subtypes and four monocyte subtypes, and thus revise the taxonomy of these cells. RESULTS Our study reveals: 1) A new DC subset, representing 2 to 3% of the DC populations across all 10 donors tested, characterized by the expression of AXL , SIGLEC1 , and SIGLEC6 antigens, named AS DCs. The AS DC population further divides into two populations captured in the traditionally defined plasmacytoid DC (pDC) and CD1C + conventional DC (cDC) gates. This split is further reflected through AS DC gene expression signatures spanning a spectrum between cDC-like and pDC-like gene sets. Although AS DCs share properties with pDCs, they more potently activate T cells. This discovery led us to reclassify pDCs as the originally described “natural interferon-producing cells (IPCs)” with weaker T cell proliferation induction ability. 2) A new subdivision within the CD1C + DC subset: one defined by a major histocompatibility complex class II–like gene set and one by a CD14 + monocyte–like prominent gene set. These CD1C + DC subsets, which can be enriched by combining CD1C with CD32B, CD36, and CD163 antigens, can both potently induce T cell proliferation. 3) The existence of a circulating and dividing cDC progenitor giving rise to CD1C + and CLEC9A + DCs through in vitro differentiation assays. This blood precursor is defined by the expression of CD100 + CD34 int and observed at a frequency of ~0.02% of the LIN – HLA-DR + fraction. 4) Two additional monocyte populations: one expressing classical monocyte genes and cytotoxic genes, and the other with unknown functions. 5) Evidence for a relationship between blastic plasmacytoid DC neoplasia (BPDCN) cells and healthy DCs. CONCLUSION Our revised taxonomy will enable more accurate functional and developmental analyses as well as immune monitoring in health and disease. The discovery of AS DCs within the traditionally defined pDC population explains many of the cDC properties previously assigned to pDCs, highlighting the need to revisit the definition of pDCs. Furthermore, the discovery of blood cDC progenitors represents a new therapeutic target readily accessible in the bloodstream for manipulation, as well as a new source for better in vitro DC generation. Although the current results focus on DCs and monocytes, a similar strategy can be applied to build a comprehensive human immune cell atlas.

Although the main focus of immuno-oncology has been manipulating the adaptive immune system, harnessing both the innate and adaptive arms of the immune system might produce superior tumour reduction and elimination. Tumour-associated macrophages often have net pro-tumour effects, but their embedded location and their untapped potential provide impetus to discover strategies to turn them against tumours. Strategies that deplete (anti-CSF-1 antibodies and CSF-1R inhibition) or stimulate (agonistic anti-CD40 or inhibitory anti-CD47 antibodies) tumour-associated macrophages have had some success. We hypothesized that pharmacologic modulation of macrophage phenotype could produce an anti-tumour effect. We previously reported that a first-in-class selective class IIa histone deacetylase (HDAC) inhibitor, TMP195, influenced human monocyte responses to the colony-stimulating factors CSF-1 and CSF-2 in vitro. Here, we utilize a macrophage-dependent autochthonous mouse model of breast cancer to demonstrate that in vivo TMP195 treatment alters the tumour microenvironment and reduces tumour burden and pulmonary metastases by modulating macrophage phenotypes. TMP195 induces the recruitment and differentiation of highly phagocytic and stimulatory macrophages within tumours. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade in this model significantly enhances the durability of tumour reduction. These data introduce class IIa HDAC inhibition as a means to harness the anti-tumour potential of macrophages to enhance cancer therapy.

SUMMARY PARP inhibitors have shown promising clinical activities for patients with BRCA mutations and are changing the landscape of ovarian cancer treatment. However, the therapeutic mechanisms of action for PARP inhibition in the interaction of tumors with the tumor microenvironment and the host immune system remain unclear. We find that PARP inhibition by olaparib triggers robust local and systemic antitumor immunity involving both adaptive and innate immune responses through a STING-dependent antitumor immune response in mice bearing Brca1-deficient ovarian tumors. This effect is further augmented when olaparib is combined with PD-1 blockade. Our findings thus provide a molecular mechanism underlying antitumor activity by PARP inhibition and lay a foundation to improve therapeutic outcome for cancer patients., In Brief Ding et al. show that PARP inhibition in Brca1-deficient tumors elicits strong antitumor immunity involving activation of both innate and adaptive immune responses, a process that is dependent on STING pathway activation. In addition, they show that addition of PD-1 blockade augments the therapeutic efficacy of PARP inhibitor treatment., Graphical Abstract

An attractive target for therapeutic intervention is constitutively activated, mutant FLT3, which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing, and which is currently in late-stage clinical trials. However, the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel, structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here, we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487, which selectively targets mutant FLT3 protein kinase activity, is also shown to override PKC412 resistance in vitro, and has significant antileukemic activity in an in vivo model of FLT3-ITD+ leukemia. Finally, the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus, we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target, and which could potentially be used to override drug resistance in AML.

Summary To understand the role of FoxO family members in hematopoiesis, we conditionally deleted FoxO1 , FoxO3 , and FoxO4 in the adult hematopoietic system. FoxO- deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1 + , c-Kit + (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO -deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO -deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO -deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential.

Hematopoietic stem cell (HSC) self-renewal is a complicated process, and its regulatory mechanisms are poorly understood. Previous studies have identified tumor necrosis factor (TNF)-α as a pleiotropic cytokine, which, among other actions, prevents various hematopoietic progenitor cells from proliferating and differentiating in vitro. However, its role in regulating long-term repopulating HSCs in vivo has not been investigated. In this study, mice deficient for the p55 or the p75 subunit of the TNF receptor were analyzed in a variety of hematopoietic progenitor and stem cell assays. In older p55−/− mice (>6 mo), we identified significant differences in their hematopoietic system compared with age-matched p75−/− or wild-type counterparts. Increased marrow cellularity and increased numbers of myeloid and erythroid colony-forming progenitor cells (CFCs), paralleled by elevated peripheral blood cell counts, were found in p55-deficient mice. In contrast to the increased myeloid compartment, pre-B CFCs were deficient in older p55−/− mice. In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay. Secondary transplantations of marrow cells from primary recipients of p55−/− marrow revealed impaired self-renewal ability of p55-deficient HSCs. These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.

While tumor-associated macrophages (TAMs) often have net pro-tumor effects, their embedded location and their untapped potential provide impetus to the discovery of strategies to turn them against tumors. We recently reported that a first in class selective class IIa HDAC inhibitor (TMP195) influenced human monocyte responses to colony stimulating factors CSF-1 and CSF-2 in vitro. Here, we utilize a macrophage-dependent autochthonous mouse model of breast cancer to demonstrate that in vivo TMP195 treatment alters the tumor microenvironment and reduces tumor burden and pulmonary metastases through macrophage modulation. TMP195 induces recruitment and differentiation of highly phagocytic and stimulatory macrophages within tumors. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade in this model significantly enhances the durability of tumor reduction. These data introduce class IIa HDAC inhibition as a novel means to harness the antitumor potential of macrophages to enhance cancer therapy. Citation Format: Jennifer L. Guerriero, Alaba Sotayo, Holly Ponichtera, Alexandra Pourzia, Sara Schad, Ruben Carrasco, Suzan Lazo, Roderick Bronson, Anthony Letai. Class IIa HDAC inhibition promotes an antitumor macrophage phenotype that induces breast tumor regression and inhibits metastasis [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B038.

Drug resistance is a growing concern with clinical use of tyrosine kinase inhibitors. Utilizing in vitro models of intrinsic drug resistance and stromal-mediated chemoresistance, as well as functional mouse models of progressive and residual disease, we attempted to develop a potential therapeutic approach designed to suppress leukemia recurrence following treatment with selective kinase inhibitors. The novel IAP inhibitor, LCL161, [corrected] was observed to potentiate the effects of tyrosine kinase inhibition against leukemic disease both in the absence and presence of a stromal-protected [corrected] environment. LCL161 enhanced the proapoptotic effects of nilotinib and PKC412, against leukemic disease in vitro and potentiated the activity of both kinase inhibitors against leukemic disease in vivo. In addition, LCL161 synergized in vivo with nilotinib to reduce leukemia burden significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib. Finally, LCL161 displayed antiproliferative effects against cells characterized by intrinsic resistance to tyrosine kinase inhibitors as a result of expression of point mutations in the protein targets of drug inhibition. These results support the idea of using IAP inhibitors in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease.

Acute graft-versus-host disease (aGVHD) is a major complication after hematopoietic stem cell transplantation (HSCT). The pathophysiology of aGVHD involves priming of naïve donor T cells in host secondary lymphoid tissue, followed by migration of effector T cells to target organs. Mediators of lymphocyte trafficking are believed to play a significant role in this migration. In this retrospective case-controlled study, we analyzed the expression of alpha4beta7 integrin and CCR9, 2 surface T cell molecules specific for intestinal trafficking, from blood samples collected previously from 59 patients after HSCT (20 without aGVHD, 20 with skin aGVHD, and 19 with intestinal aGVHD). All samples had been obtained before the onset of aGVHD symptoms (with 1 sample collected on the day of symptom onset). Analysis by flow cytometry demonstrated that alpha4beta7 integrin was significantly increased on both naïve and memory T cells in patients who subsequently developed intestinal aGVHD, with the most significant differences observed in memory subsets. Immunohistochemical staining on rectal biopsy specimens from patients with intestinal aGVHD showed that expression of alpha4beta7 integrin was concentrated on mononuclear cells in blood vessels within the intestinal mucosa. These results suggest that alpha4beta7 integrin likely is involved in lymphocyte trafficking in intestinal aGVHD and may have potential clinical use as a correlative biomarker or as a target for the treatment and prophylaxis of intestinal aGVHD after HSCT.

Waldenström macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase Cβ (PKCβ) regulates cell survival and growth in many B-cell malignancies. In this study, we demonstrate up-regulation of PKCβ protein in WM using protein array techniques and immunohistochemistry. Enzastaurin, a PKCβ inhibitor, blocked PKCβ activity and induced a significant decrease of proliferation at 48 hours in WM cell lines (IC50, 2.5-10 μM). Similar effects were demonstrated in primary CD19+ WM cells, without cytotoxicity on peripheral blood mononuclear cells. In addition, enzastaurin overcame tumor cell growth induced by coculture of WM cells with bone marrow stromal cells. Enzastaurin induced dose-dependent apoptosis at 48 hours mediated via induction of caspase-3, caspase-8, caspase-9, and PARP cleavage. Enzastaurin inhibited Akt phosphorylation and Akt kinase activity, as well as downstream p-MARCKS and ribosomal p-S6. Furthermore, enzastaurin demonstrated additive cytotoxicity in combination with bortezomib, and synergistic cytotoxicity in combination with fludarabine. Finally, in an in vivo xenograft model of human WM, significant inhibition of tumor growth was observed in the enzastaurin-treated mice (P = .028). Our studies therefore show that enzastaurin has significant antitumor activity in WM both in vitro and in vivo, providing the framework for clinical trials to improve patient outcome in WM.

Reconstitution of T cell function after allogeneic HSCT is dependent on the balanced recovery of CD4+Foxp3+ regulatory T cells (Treg) and CD4+Foxp3- conventional T cells (Tcon). While Tcon are required for effector T cell function, Treg play an essential role in the maintenance of immune tolerance after allogeneic HSCT and prevention of graft versus host disease (GVHD). To examine the reconstitution of Treg and Tcon after HSCT and identify mechanisms that contribute to homeostatic imbalance of these T cell subsets, we undertook a prospective analysis of 188 adult patients (median age 54y) with hematologic malignances who underwent T-cell replete allogeneic HSCT. Patients received either myeloablative (MAC; n=80) or reduced-intensity conditioning (RIC; n=108). GVHD prophylaxis differed in these two cohorts as RIC patients received tacrolimus/methotrexate/sirolimus-based regimens and MAC patients received tacrolimus/methotrexate-based regimens. Serial blood samples (total n=739) obtained at 1, 2, 3, 6, 9 and 12 months after transplant were characterized by flow cytometry with a panel of intracellular and surface markers designed to quantify phenotypically and functionally distinct subsets of Treg, Tcon and CD8 T cells in each sample. Likely reflecting the prophylactic administration of sirolimus for the first 6 months post-HSCT after RIC conditioning, recovery of absolute CD3+, CD4+ and CD8+ T cell counts was significantly greater after MAC-HSCT throughout the first year after HSCT. Total CD4+ Tcon recovery was significantly decreased in RIC patients at all time points but Treg recovery was significantly lower only in the first 2 months after HSCT. Central memory cells (CM; 45RA-62L+) comprise the majority of Treg and Tcon throughout the first year after HSCT. The percentage of Treg-CM is greater than Tcon-CM and the fraction of CM cells within Treg and Tcon is increased in the RIC cohort (Figure 1A). Effector memory cells (EM; 45RA-62L-) also represent a large fraction of Treg and Tcon. Reconstitution of Treg-EM and Tcon-EM is similar but recovery of this subset appears to be strongly affected by sirolimus (Figure 1B). In RIC patients who receive sirolimus, the percentage of EM cells within Treg and Tcon subsets is significantly lower in the first 6 months after HSCT. Naïve Treg and Tcon (CD45RA+CD62L+) represent a relatively small fraction of recovering CD4 T cells during this period. Reconstitution of naïve CD4 T cells identified as recent thymic emigrants (RTE; CD45RA+CD31+) is markedly different in Treg and Tcon (Figure 1C). Within Tcon, the RTE fraction rapidly recovers to levels observed in healthy donors. In contrast, the fraction of RTE-Treg remains very low, with no evidence of improvement for at least 1 year. For both Treg-RTE and Tcon-RTE, recovery is significantly greater after RIC suggesting that either the reduced-intensity of conditioning or sirolimus is thymus protective. In vivo proliferation of each T cell subset was monitored by expression of Ki67. As previously observed in healthy donors, proliferation of Treg was significantly greater than Tcon at all time points. This primarily reflects homeostatic proliferation of Treg memory cells since very few naïve Treg are present. Both Treg and Tcon proliferation was higher in the MAC cohort in the first 1-3 months after HSCT. In vivo susceptibility to apoptosis was monitored by expression of pro-survival Bcl-2 and pro-apoptotic CD95 (FAS) expression. All Treg subsets expressed lower levels of Bcl-2 and higher levels of CD95 compared to Tcon. RIC/sirolimus was associated with higher levels of Bcl-2 and lower levels of CD95 predominately in the first 3 months after transplant. This effect was evident in all Treg and Tcon subsets. These results demonstrate distinctly different patterns of reconstitution of Treg and Tcon after allogeneic HSCT. Reconstitution of Tcon is characterized by rapid recovery of thymic generation, moderate homeostatic proliferation of memory subsets and relative resistance to apoptosis. Reconstitution of Treg is characterized by prolonged impaired thymic generation, high levels of homeostatic proliferation of memory subsets and increased susceptibility to both intrinsic and extrinsic pathways of apoptosis. RIC followed by administration of sirolimus for GVHD prophylaxis appears to selectively delay recovery of Treg and Tcon EM cells while sparing naïve, RTE and CM subsets. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

IL-12 has significant antitumor activity in mice that may be mediated by CD8(+) T cells. We show in this report that repeated subcutaneous injections of IL-12 in patients with cancer resulted in the selective expansion of a subset of peripheral blood CD8(+) T cells. This T cell subset expressed high levels of CD18 and upregulated IL-12 receptor expression after IL-12 treatment in vivo. In normal subjects, these CD3(+)CD8(+)CD18(bright) T cells expressed IL-12 and IL-2 receptors and adhesion/costimulatory molecules to a greater degree than other CD8(+) and CD4(+) T cells. They appeared morphologically as large granular lymphocytes, although they did not express NK cell markers such as CD56. In addition, CD8(+)CD18(bright) T cells were almost exclusively T cell receptor (TCR) alphabeta+, and exhibited a TCR Vbeta repertoire that was strikingly oligoclonal, whereas the Vbeta repertoire of CD18(dim) T cells was polyclonal. Although CD8+CD18(bright) T cells demonstrated little functional responsiveness to IL-12 or IL-2 alone in vitro, they responded to the combination of IL-12+IL-2 with strong IFN-gamma production and proliferation and enhanced non-MHC-restricted cytolytic activity. In contrast, CD18(dim) T cells were not activated by IL-12 or IL-2, alone or in combination. These findings demonstrate that CD8+CD18(bright) T cells are a unique population of peripheral blood lymphocytes with features of both memory and effector cells that are capable of TCR-independent activation through combined stimulation with IL-12+IL-2. As this activation results in IFN-gamma production and enhanced cytolytic activity, these T cells may play a role in innate as well as acquired immunity to tumors and infectious pathogens. Additional studies will be necessary to determine whether CD8+CD18(bright) T cells mediate the antitumor effect of IL-12 or IL-2 administered to cancer patients, and if so, whether maximal activation of these T cells with the combination of IL-12+IL-2 in vivo can augment the clinical effectiveness of these cytokines.

We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures: No relevant conflicts of interest to declare.

CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the maintenance of self-tolerance and immune homeostasis. Interleukin-2 (IL-2) is critical for Treg development, expansion, activity and survival. Lack of either IL-2 or IL-2Rα (CD25) results in Treg deficiency and Treg impairment is associated with loss of tolerance, autoimmunity, and chronic GVHD. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo and clinical improvement of chronic GVHD (Koreth et al. NEJM 2011). We also reported that low-dose IL-2 enhanced Treg resistance to FAS-mediated apoptosis through the extrinsic pathway (Matsuoka et al. Sci Transl Med 2013). However susceptibility of Treg to apoptosis through the intrinsic pathway has not been examined. To examine the mechanisms whereby IL-2 affects susceptibility of Treg to apoptosis through the intrinsic pathway, we used a functional flow cytometry-based assay (BH3 profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). Mitochondria that are more sensitive to the BH3 peptides are more 'primed' for apoptosis, and more sensitive to pro-apoptotic signaling. This assay allowed us to compare “priming” which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization in Treg and Tcon. We also examined cytoplasmic expression of Bcl-2, BclxL, Mcl1, Bim, XIAP, Ki67 and Staurosporine (STS) induced apoptosis as additional measures of susceptibility to apoptosis and proliferation in each subset. In resting blood obtained from healthy donors (n=25), Treg were more “primed” than Tcon when exposed to several BH3 peptides (Bim, Bad, PUMA and BMF). This BH3 peptide pattern suggests that the anti-apoptotic protein, Bcl2 plays a primary role in the priming of these cells. Analysis of pro- and anti-apoptotic proteins revealed that Treg expressed higher levels Bim and BclxL and lower levels of Bcl2 and XIAP than Tcon, but there was no significant difference in Mcl1. Treg expressed higher levels of Ki67 and were more susceptible to both STS- and FAS-induced apoptosis than Tcon. Thus, Treg in healthy individuals have higher proliferative activity and are more susceptible to apoptosis than Tcon through both mitochondrial and death receptor pathways. To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured with different concentrations of IL-2 (10, 100, 1000 IU/ml) with and without TCR stimulation (1ug/ml anti-CD3 + 1ug/ml anti-CD28). BH3 priming was measured after 3 days and results were compared to cells cultured in media alone. Low-concentration IL-2 (10 U/ml) lowered BH3 priming and increased Bcl2 expression in Treg. Similar effects were observed in Tcon, but higher concentrations of IL-2 (>100 U/ml) were required to increase Bcl2 expression and decrease BH3 priming in Tcon. TCR stimulation alone induced higher BH3 priming in Tcon but had little effect in Treg. The combination of TCR stimulation plus IL-2 increased BH3 priming in both Tcon and Treg. TCR stimulation increased proliferation only in Tcon, but the TCR + IL-2 combination increased proliferation in both Tcon and Treg. Thus, IL-2 alone reduced BH3 priming in unstimulated cells but the TCR + IL-2 combination induced higher proliferation and higher BH3 priming. STS-induced apoptosis assays revealed that low-dose IL-2 reduced susceptibility to apoptosis only in Treg. IL-2 did not change the expression of BclxL, Mcl1, and Bim but XIAP expression was increased in both Treg and Tcon. To confirm the effects of low-dose IL-2 on Treg priming were mediated by Bcl2, we examined the effect of ABT-199, a selective Bcl2 inhibitor on BH3 priming in Treg and Tcon. As shown in Figure 1, ABT-199 enhanced priming and spontaneous apoptosis of both Treg and Tcon. IL-2 had no effect on ABT-199-induced priming or apoptosis in Tcon. In contrast, IL-2 reversed the effects of ABT-199 on Treg priming and apoptosis providing further evidence that the inhibition of intrinsic pathway apoptosis mediated by IL-2 in Treg is dependent on Bcl2. Disclosures: Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.

Abstract 652 Long-term survival of patients with acute myeloid leukemia (AML) is poor, and new forms of therapy are needed. Many genetic lesions have been identified and studied, and most patients have chromosome translocations or other mutations that promote self-renewal of leukemic stem cells, block differentiation, enhance growth, and block apoptosis. Only a few of these mutations result in druggable targets (e.g., PML-RARa, Kit, PDGFR, FLT3 for instance). In addition to genetic lesions, epigenetic abnormalities have been shown to be very common in AML, and provide opportunities for novel treatments. Using genome-wide approaches to identify alternative splicing, we have recently shown that AML cells have a high level of aberrantly regulated genome-wide alternative splicing (AS) as a frequent epigenetic event. By comparing samples from 62 AML patients with 10 normal donors (NDs) we identified 428 genes differentially spliced in AML. A list of differentially spliced genes includes 50 oncogenes and 52 tumor suppressor genes, as well as genes encoding proteins involved in cell proliferation and differentiation, and apoptosis. We evaluated splicing event frequency in AML compared to NDs and we observed that on average 527 (range 137–1657) genes were identified as differentially spliced in any given patient, out of 62 analyzed. Also, we found that any given differentially spliced gene, of the 3,108 detected, were spliced on average in 26 (range 1–54) AML patients. Thus, splicing aberrations are highly recurrent in AML patients. To identify the causes of aberrant splicing in AML, we evaluated transcript levels of the 24 major splicing factors (SFs) that are involved in the first and second splicing transesterification reactions. These splicing factors are important proteins involved in spliceosomalassembly. Expression levels of these SFs were evaluated in 20 AML patients exhibiting high levels of AS. Quantitative RT-PCR analysis showed significant (up to 30 fold) upregulation of U2AF2 (P We have obtained a minigene cassette of the p53 inducible PIG3 gene based on previous splicing studies. The minigene cassette was cloned between RFP (red fluorescent prtoein) and GFP (green fluorescent prtoein) in such a way that translation of the normally spliced transcript results in expression of RFP and GFP, while aberrant splicing results in the expression of RFP only. Production of a similar minigene cassette that includes exons/introns of a gene that is subjected to aberrant splicing in AML (NOTCH2, FLT3 and CD13) is in progress. In studies, completed so far, with the PIG3 minigene cassette construct transiently transfected into the HEK293 cells lines, overexpression of PTBP increased aberrant splicing of the PIG3 minigene. Similar studies testing the effects of elevated levels of U2AF2 and PTBP on NOTCH2 and other genes misspliced in AML (such as FLT3 and CD13) will be presented. Our results indicate that aberrant splicing could be an important event in AML, and development of an in vitro, synthetic splicing assay will enable us to better understand the underlying causes of this process in AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3280 CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the maintenance of self-tolerance and immune homeostasis and Treg deficiency contributes to the development of autoimmune diseases. CD4Treg, conventional CD4 T cells (Tcon) and CD8 T cells are derived from lymphocyte progenitor cells that differentiate into distinct functional subsets in the thymus before export to the peripheral circulation. As T cells differentiate and expand in the periphery, each T cell subset is differentially regulated and subjected to distinct homeostatic signals. For example, interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in selective Treg deficiency. In regulating Treg homeostasis, IL-2 has multiple and distinct effects on Treg differentiation, proliferation and susceptibility to apoptosis. To determine the mechanism whereby IL-2 affects susceptibility of Treg to apoptosis, we used a new flow cytometry-based assay (BH3 profiling) to measure the mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assay allowed us to compare “priming” which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization in different T cell subsets, including CD4 Treg, CD4 Tcon and CD8 T cells. We also examined cell surface expression of CD95 death receptor (Fas) and cytoplasmic expression of Bcl-2 and Ki67 as additional measures of susceptibility to apoptosis and proliferation in each subset. In resting blood obtained from healthy donors (n=10), CD4 Treg were more “primed” than either CD4 Tcon or CD8 T cells when exposed to several BH3 peptides (PUMA, BMF and the combination of BAD+NOXA). CD4 Treg were also found to have decreased expression of Bcl-2 and increased expression of CD95 and Ki67 compared to CD4 Tcon or CD8 T cells. Thus, Treg in healthy individuals have higher proliferative activity and are more susceptible to apoptosis than other major T cell subsets through both mitochondrial and death receptor pathways. To establish the functional effects of TCR stimulation and IL-2, CD4 Treg, CD4 Tcon and CD8 T cells were purified by cell sorting and cultured for 5–6 days with or without TCR stimulation (1μg/ml anti-CD3 + 1μg/ml anti-CD28) and IL-2 (100 IU/ml). Results were compared to cells cultured in media alone. Results are summarized in the table below. CD4 Tcon and CD8 T cells responded in a similar fashion to either TCR stimulation alone or TCR plus IL-2. This response included increased BH3 priming, reduced expression of Bcl-2, increased expression of CD95 and increased proliferation (Ki-67). IL-2 alone had no effect on CD4 Tcon or CD8 T cells. In contrast, TCR stimulation alone had no effect on CD4 Treg but IL-2 alone reduced BH3 priming and increased expression of Bcl-2. Combined TCR stimulation plus IL-2 in Treg increased BH3 priming, reduced expression of Bcl-2, increased expression of CD95 and increased proliferation. Thus, TCR stimulation reversed the anti-apoptotic effects of IL-2 alone and markedly increased susceptibility of Treg to apoptosis. When compared with CD4 Tcon and CD8 T cells, these studies demonstrate distinct effects of TCR stimulation and IL-2 on both mitochondrial and death receptor pathways of apoptosis in CD4 Treg and define mechanisms whereby TCR stimulation and IL-2 interact to regulate Treg homeostasis. Table 1. Effects of in vitro TCR stimulation and IL-2 on apoptotic pathways of T cell subsets TCR Stimulation IL-2 TCR + IL2 BH3 priming Bcl-2 CD95 Ki67 BH3 priming Bcl-2 CD95 Ki67 BH3 priming Bcl-2 CD95 Ki67 CD4 Treg – – – – ↓ ↑ – – ↑ ↓ ↑ ↑ CD4 Tcon ↑ ↓ ↑ ↑ – – – – ↑ ↓ ↑ ↑ CD8 ↑ ↓ ↑ ↑ – – – – ↑ ↓ ↑ ↑ Disclosures: No relevant conflicts of interest to declare.

Abstract 225 CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to play a central role in the maintenance of self-tolerance and immune homeostasis. After allogeneic stem cell transplantation, impaired recovery of Treg is associated with the development of cGVHD. Interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in Treg deficiency. In patients with cGVHD, we previously demonstrated that Treg proliferate at high levels but this subset is also highly susceptible to apoptosis leading to inadequate Treg numbers (Matsuoka et al. JCI 2010). We also reported that low-dose IL-2 administration resulted in selective expansion of Treg in vivo and clinical improvement of cGVHD (Koreth et al. NEJM 2011). To identify mechanisms responsible for increased Treg susceptibility to apoptosis in cGVHD we used a new flow cytometry-based assay to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assessment allowed us to compare BH3 peptide-induced mitochondrial membrane depolarization (“priming”) in different T cell subsets, including CD4 Treg, conventional CD4 T cells (CD4 Tcon), and CD8 T cells. Expression of Bcl-2, CD95 and Ki67 were also studied in each T cell subset. We studied peripheral blood samples from 36 patients with hematologic malignancies (median age 59 yr) who are > 2 years post HSCT (27 patients with cGVHD and 9 patients without cGVHD) and 15 patients who received daily subcutaneous IL-2 for 8 weeks for treatment of steroid-refractory cGvHD. Severity of cGVHD was classified according to NIH criteria. In patients without cGVHD, BH3 priming was similar in all 3 T cell subsets (CD4 Treg, CD4 Tcon and CD8). In patients with cGVHD, CD4 Treg were more primed than CD4 Tcon when challenged with BIM, BAD, PUMA, BMF and the combination of BAD + NOXA peptides (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1257 CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to play an important role in the maintenance of peripheral tolerance and control of cGVHD. Recent studies have shown that Treg undergo extensive homeostatic proliferation after allogeneic HSCT. However, highly proliferating Treg are also susceptible to apoptosis and the inability to maintain adequate Treg survival may contribute to the development of cGVHD. Thus, mechanisms of Treg survival and persistence are of great importance. Telomeres are repetitive DNA sequences, located at the ends of each chromosome. Telomeres gradually shorten with each cell division and eventually lead to cell senescence and death. In rapidly dividing cells, telomere length is maintained by telomerase, a reverse transcriptase that prevents premature senescence. Previous studies have shown that abnormal telomerase activity is found in T cells from patients with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, suggesting that telomerase may play a role in the maintenance of immune tolerance. In the present study, we examined whether telomerase activity and telomere length were associated with Treg homeostasis after allogeneic HSCT, particularly in the context of cGVHD. We examined Treg and conventional CD4 T cells (Tcon) in 35 patients with hematologic malignancies (median age 54 yr) who survived without relapse for more than 2 years after HSCT. Patient samples were obtained at 43 months (median, range: 25–99 months) after HSCT. The severity of cGVHD was classified according to NIH criteria. At the time of analysis, 2 patients had no cGVHD, 19 had mild cGVHD, 9 had moderate cGVHD, and 5 had severe cGVHD. CD4+CD25-CD127+ Tcon and CD4+CD25+CD127- Treg were isolated by flow cytometric cell sorting. Relative telomere length was measured by real time PCR. Telomerase activity was measured by PCR-ELISA. Telomere length was significantly shorter in Treg compared to Tcon in all patients (0.05 vs 0.28; p Disclosures: No relevant conflicts of interest to declare.

Hematopoietic development and long-term homeostasis are tightly regulated by a dynamic balance of stem cell self-renewal and differentiation. To understand the role of the FoxO family of transcription factors in these processes, we studied the impact of somatic deletion of all FoxO genes in the adult hematopoietic system of mice engineered with the interferon-inducible Mx-Cre transgene and various conditional FoxO alleles: FoxO1L/L; FoxO3 L/L; and/or FoxO4 L/L. Cre-mediated excision of FoxO1, FoxO3 and FoxO4 (FoxO1/O3/O4L/L), resulted in myeloid lineage expansion, lymphoid developmental abnormalities and a marked decrease of the lineage-negative, Sca-1+, c-Kit+ (LSK) compartment, containing the hematopoietic stem cell (HSC) population, but normal numbers of myeloid progenitors. Furthermore, FoxO1/O3/O4L/L null bone marrow cells were defective in competitive and noncompetitive repopulation assays. This FoxO deficient defect correlated with enhanced cell cycling and increased apoptosis of HSC cells. Notably, there was an HSC-restricted elevation in the level of reactive oxygen species (ROS) that was not observed in committed progenitors, and was associated with decreased catalase and MnSOD expression. Furthermore, in vivo treatment with the anti-oxidative agent N-acetyl-cysteine (NAC) resulted in complete reversion of the FoxO deficient HSC phenotype. Taken together, these results demonstrate that, in the HSC compartment, FoxO proteins are mediators of quiescence and enhanced survival and play essential regulatory roles in the response to physiologic oxidative stress, a function that may contribute to the long-term regenerative potential of the hematopoietic stem cell compartment.