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Figure S4. MIR300 anti-proliferative activity accounts for BMM-induced LSC entry into quiescence. A, Structure of 14q32 DLK1-DIO3 genomic imprinted locus hosting the MEG3-regulated human MIR300. B, MIR300 levels in 5-Aza- or DMSO-treated (24h) Ph+ cells. C, Effect of hypoxia on proliferation of CFSE+CD34+ CML-BC cells. D, left: Effect of MSC (HS-5)-derived CM on LAMA-84 proliferation expressed as fold changes of CFSE mean of fluorescence intensity (MFI)+/-SEM; middle: pro-apoptotic effect of PAD (FTY720; 2.5uM) and DMSO (control) on HS-5-cultured LAMA-84 cells; right: Effect of MSC (HS-5)-derived CM BCR-ABL1 expression (anti-ABL1) and activity anti-PY), phospho-BCR-ABL1, JAK2 expression and activity JAK2 Y1007/1008, PP2A activity (pPP2AY307 inactive form) and GRB2 used as a control (blots are representative of three independent experiments). E, Levels of C/EBPbeta and GRB2 mRNA and protein in HS-5 cells exposed to hypoxia (48h; 1% O2). F, Effect of neutralizing TGFbeta antibody (anti-TGFb Ab; 48h, 1.25 μg/ml) on MIR300 levels in CD34+ CML-BC cells. G, Effect of ectopic C/EBPalpha (MigR1-deltauORF-C/EBPalpha-HA) and C/EBPbeta (MigR1-C/EBPB-ERTAM) on MIR300 levels in K562 cells. Immunoblot shows levels of C/EBPbeta and GRB2 in normoxic and hypoxic K562 cells.

Range values of controls for the indicated experiments

Figure S3. MIR300 acts as master PP2A activator and inhibitor of G1/S transition. A, (left) Additional effects of MIR300 on SET, CCND2 and CDK6 expression; (right) KEGG/GO analysis of MIR300 effects on signal transduction pathways. B, CSmiRTar (filters: bone marrow normal and myeloid leukemia cells) and miRDIP-ComiR integrated analyses show functional clustering of predicted/validated MIR300 targets.

Figure S6. Selective suppression of MIR300 pro-apoptotic but not anti-proliferative activity by TUG1 lncRNA in quiescent LSCs. A, BloodSpot array-based TUG1 expression levels during normal myelopoiesis and in myeloid neoplams. B, GEO Profiles show TUG1 levels in in lineage-negative (Lin-) and -positive (Lin+) CD34- and CD34human stem/progenitor cells from healthy individuals. C, Experimental data- and current literature-based graphic representation of signaling network controlling CML LSC quiescence and survival through the BMM-C/EBPbeta-MIR300 and BMM-TGFbeta-FoxM1 pathways. Dotted lines indicate inactive pathways, line thickness indicates relevance of the signal for LSC quiescence. Red lines indicate signals increasing MIR300 levels. Black lines signals increasing TUG1 levels. (bottom) effects of different TUG1 levels on CML leukemic stem (LSC) and progenitor (LPC) cell fate.

Figure S2. KEGG/GO analysis of MIR300 on PP2A-regulated signal transduction pathways. Cartoon shows the SET-dependent PP2A Inhibitory pathway in CML and the pleiotropic inhibitory effect of PP2A activation on validated and predicted MIR300 targets regulating G1/S cell cycle transition, Wnt-beta-catenin, TGFbeta, JAK-STAT, PI-3K-Akt, RAS-MAPK and Notch signaling pathways.

Figure S5. BMM-induced MIR300 anti-proliferative and PP2A-activating functions impair NK cell immune-response. A, PP2A-dependent regulation of MIR300 and miR-155 validated pathways predicted to occur also in the bone marrow endosteal niche. B, Effect of hypoxia (1% O2, 7 days) on CFSE+NK cell proliferation (% CFSE mean of fluorescence). C, HS-5 exosomal miRNAs reported as negative regulators of NK cell proliferation/activity and their experimentally validated targets. miRNA RNAseq was performed on an Illumina platform using libraries derived from 100 ng RNA/sample from HS-5 exosome purifications (n=3). D, Precursor (pre-miR-155) miR-155 (BIC) levels in resting and IL-12/IL-18 (18h)-stimulated NK cells exposed (48h) to HS-5 exosomes. E, Effect of hypoxia (1% O2) and HS-5 CM (48h) on TUG1 expression in CD56+CD3- primary NK and NK-92 cells. F, Effect of CpG-TUG1-shRNA and CpG-scramble (200-500 nM, 5 days) on IL-2-induced NK cell proliferation (% cell number). Data are represented as mean+/-SEM.

Figure S1. MIR300 activity in quiescent leukemic stem and progenitor cells. CFC-replating assays shows effects of lentiviral-mediated ectopic MIR300 expression, 250 nM and 500 nM CpG-miR-300 on serial replating activity (2nd replating) of leukemic chronic and acute CML and normal UCB CD34+CD38- HSC-enriched cell fractions. Infection with lentiviral empty vector and treatment with CpG-anti-MIR300 and CpG-scramble served as controls.

Acute graft versus host (aGVHD) is the second cause of death after allogeneic-hematopoietic stem cell transplant (allo-HSCT) underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft versus leukemia effect. Histopathology analysis revealed higher amount of leukocyte infiltration in both large intestine and liver of PBS group compared to rEGFL7-treated mice. Furthermore, damage to the gut was reduced in EGFL7 treated mice. Finally, we showed that rEGFL7 treatment results in higher thymocytes, T, B and dendritic cells in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4+ T cell HP during GVHD. However, it is not clear whether these alterations to the peripheral CD4+ T cell niche also contribute to impair CD8+ T cell regeneration during GVHD. We found that IL-7 therapy was sufficient for restoring CD8+ T cell HP in GVHD hosts while forcing DC regeneration with Flt3-L had only a modest effect on CD8+ T cell HP in IL-7 treated mice. Using bone marrow chimeras, we showed that HP of naive CD8+ T cells is primarily regulated by MHC class I on radio-resistant stromal cells, yet optimal recovery of CD8+ T cell counts still requires expression of MHC class I on both radio-resistant and radio-sensitive hematopoietic cells. Thus, IL-7 level is the primary limiting factor that constrains naive CD8+ T cell HP during GVHD, and accessibility of MHC class I on stromal cells explains how IL-7 therapy, as a single agent, can induce robust CD8 + T cell HP in the absence of DCs.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that

For several years, it was believed that the thymus was entirely responsible for maintaining T cell homeostasis. Today, it is well-known that homeostatic peripheral mechanisms are essential in order to maintain T cell numbers and diversity constant in the periphery. Naïve and memory T cells require continual access to self-peptide MHC class I and II molecules and/or cytokines to survive in the periphery. Under normal conditions, homeostatic resources are low, and lymphocytes undergo very slow proliferation and survive. Following T cell depletion, the bioavailability of homeostatic resources is significantly increased, and T cell proliferation is dramatically augmented. The development of lymphopenic mouse models has helped our current understanding of factors involved in the regulation of peripheral T cell homeostasis. In this minireview, we will give a brief overview about basic techniques used to study peripheral T cell homeostasis in mice.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy. Significance: Tumor-naïve microenvironment–induced MIR300 is the only tumor suppressor miRNA that induces CML LSC quiescence while inhibiting NK cell antitumor immune response, and CML LSC/progenitor cell apoptosis through its anti-proliferative and PP2A-activating functions, respectively. Thus, the importance of MIR300 and PP2A-activating drugs for formation/survival and eradication of drug-resistant CML LSCs, respectively. See related commentary by Broxmeyer, p. 13. This article is highlighted in the In This Issue feature, p. 5

For several years, it was believed that the thymus was entirely responsible for maintaining T cell homeostasis. Today, it is well-known that homeostatic peripheral mechanisms are essential in order to maintain T cell numbers and diversity constant in the periphery. Naive and memory T cells require continual access to self-peptide MHC class I and II molecules and/or cytokines to survive in the periphery. Under normal conditions, homeostatic resources are low, and lymphocytes undergo very slow proliferation and survive. Following T cell depletion, the bioavailability of homeostatic resources is significantly increased, and T cell proliferation is dramatically augmented. The development of lymphopenic mouse models has helped our current understanding of factors involved in the regulation of peripheral T cell homeostasis. In this minireview, we will give a brief overview about basic techniques used to study peripheral T cell homeostasis in mice.

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4

Imatinib mesylate (IM) therapy has been shown to induce lower T cell counts in chronic myelogenous leukemia (CML) patients and an interference of IM with T cell receptor (TCR) signaling has been invoked to explain this observation. However, IL-7 and TCR signaling are both essential for lymphocyte survival. This study was undertaken to determine whether IM interferes with IL-7 or TCR signaling to explain lower T cell counts in patients. At diagnosis, CML patients have typically lower CD4+ counts in their blood, yet CD8+ counts are normal or even increased in some. Following the initiation of IM treatment, CD4+ counts were further diminished and CD8+ T lymphocytes were dramatically decreased. In vitro studies confirmed IM interference with TCR signaling through the inhibition of ERK phosphorylation and we showed a similar effect on IL-7 signaling and STAT5 phosphorylation (STAT5-p). Importantly however, using an in vivo mouse model, we demonstrated that IM impaired T cell survival through the inhibition of IL-7 and STAT5-p but not TCR signaling which remained unaffected during IM therapy. Thus, off-target inhibitory effects of IM on IL-7 and STAT5-p explain how T cell lymphopenia occurs in patients treated with IM.

For numerous patients, allogeneic stem cell transplantation (SCT) is the only therapeutic option that could potentially cure their disease. Despite significant progress made in clinical management of allogeneic SCT, acute graft-versus-host disease (aGVHD) remains the second cause of death after disease recurrence. aGVHD is highly immunosuppressive and the adverse effect of allogeneic SCT on T cell regeneration is typically more important than the levels of immunosuppression normally seen after autologous SCT. In these patients, immune reconstitution often takes several years to occur and restoring immunocompetence after allogeneic SCT represents an important challenge, principally because clinical options are limited and current methods used to accelerate immune reconstitution are associated with increased GVHD. Interleukin-7 and IL-15 are both under clinical investigation and demonstrate the greatest potential on peripheral T cells regeneration in mice and humans. However, awareness has been raised about the use of IL-7 and IL-15 after allogeneic SCT with regards to potential adverse effects on aGVHD. In this review, we will discuss about recent progress made in lymphocyte regeneration, the critical role played by IL-7 and IL-15 in T cell homeostasis and how these cytokines could be used to improve immune reconstitution after allogeneic SCT.

PURPOSE: Chronic myelogenous leukemia (CML) is a disorder affecting early hematopoietic stem cells (HSC) and is characterized by excessive proliferation and accumulation of myeloid progenitors and progeny in the periphery. During the chronic phase of the disease, CML patients are normally at low risk of developing infections but such complications tend to rise during the progression of the disease. Gleevec (imatinib mesylate) is currently administered as first line therapy for patients with Philadelphia chromosome-positive CML. Despite the relative high specificity of tyrosine kinase inhibitor (TKI) treatment towards the BCR-ABL fusion protein, off-target multikinase inhibitory effects occur and can interfere with normal hematopoiesis. This study was conducted to evaluate how myeloid and lymphoid immune homeostasis are affected by Imatinib mesylate. METHODS: Healthy volunteer donors (n=25) and CML patients were recruited during their first visit at our CML clinic. Seven CML patients were treated with Imatinib (400mg). The median time of Gleevec treatment was 2.9 years (range: 0.5-10.9). The median time of remission post TKI was 1.1 years (range: 0.3-3). Phenotypic analysis of dendritic cell (DCs) subsets: plasmacytoid (pDCs) and myeloid type 1, 2 and 3 (mDC1, mDC2, mDC3) were evaluated by flow cytometry. Percentage and absolute numbers of naive and memory CD4+ and CD8+ T cells, NK cells and B cells were also evaluated. DCs were differentiated from purified CD34+ cells culturedwith GM-CSF (800 U/ml) or Flt3-L (50ng/ml), IL-4 (10 U/ml) and TNFa (50 U/ml), in the presence of varying concentrations of Imatinib mesylate (0 to 5µM/mL). TCR and IL-7 signaling were evaluated based on ERK-phosphorylation (-p) and STAT5-p after incubation with 3µM of Imatinib. RESULTS AND CONCLUSION: At diagnosis, several CML patients have a deficit in DCs resulting from a severe skewing affecting BM progenitor cells. After initiating Gleevec therapy, normalization of stem cell progenitors occurs but DC counts remain well below normal levels in all CML patients. We demonstrated a direct and dose dependent interference of Imatinib on GM-CSF and Flt3-L pathways for DC differentiation from CD34+ stem cells. For T lymphocytes, Imatinib interfered with TCR and IL-7 signaling through the inhibition of ERK and STAT5 phosphorylation respectively. The failure to maintain adequate numbers of DCs combined to diminished homeostatic response to cytokines and TCR stimuli explains T cell lymphopenia in these patients. Such immune dysfunction is at least in part responsible for infectious complications that are often increased in patients treated with Imatinib. Disclosures No relevant conflicts of interest to declare.