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Acute myeloid leukemia (AML) patients refractory to induction therapy or relapsed within 1 year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in preclinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single-cell RNA sequencing using prospectively acquired patient specimens identified IFN response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well tolerated, with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies. Significance: This study demonstrates a favorable safety profile and promising clinical activity of ficlatuzumab and cytarabine in high-risk AML, thus supporting further investigation of this combination in a randomized trial. It also shows the utility of a novel application using multiplexed single-cell analyses to detect on-target activity and identify biomarkers of response. This article is highlighted in the In This Issue feature, p. 403

INTRODUCTION AND OBJECTIVE:Recently single-cell RNA sequencing (scRNA-Seq) has increased our depth of understanding of genomic features of distinct cellular populations. To reveal the dynamics of b...

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

Background Haplo-HCT emerged as a reliable option for patients with high risk hematological malignancies with no HLA-matched related or unrelated donors available. Retrospective registry data suggest comparable outcomes to HLA-matched donors; however, relatively little is known regarding predictors of disease relapse following haplo-HCT. We hypothesize that attainment of full-donor CD3 and CD33 chimerism at day 30 (D30) and 100 (D100) is associated with lower incidence of relapse after haplo-HCT. Methods We undertook a retrospective analysis of 78 patients who underwent haplo-HCT at the Ohio State University James Cancer Hospital between January 2013 and December 2017. The associations between patient characteristics including CD3/CD33 donor chimerisms and the cumulative incidence rate (CIR) of relapse were evaluated using proportional sub-distribution hazards model, treating death without relapse as the competing risk. The models were built adopting a landmark analysis approach at D30 and D100. Progression-free survival (PFS) was estimated using the method of Kaplan-Meier. Results Median age at haplo-HCT was 56 (20-74) years and 68% were males. 50% had acute myeloid leukemia/myelodysplastic syndrome, and 54% had intermediate disease risk index (DRI). Most patients (85%) received reduced-intensity conditioning and 50% received bone marrow (BM) grafts. Complete (100%) CD3 donor chimerism was observed in 63 (84%) patients at D30, and subsequently 59 (95%) patients at D100. Complete CD33 donor chimerism was observed in 67 (89%) patients at D30, and subsequently 56 (88%) patients at D100. In univariable D30 landmark analysis, the CIR of relapse was significantly lower in patients who achieved complete CD3 (and CD33) chimerism compared with patients with less than complete chimerism (Figure 1A; p Conclusions Complete CD3 and CD33 donor chimerisms at D30 and D100 predict a patient's risk for relapse following haplo-HCT. We observed a strong association between CD3 chimeric status at D30 with DRI, as well suggestive that high risk disease may be associated with impaired immune reconstitution after transplant and perhaps a less robust graft vs disease effect.

Blaser et al. use live imaging of the zebrafish hematopoietic niche to show that cxcl8/cxcr1 signaling positively regulates HSPC engraftment by increasing HSPC-niche interactions, HSPC mitotic rate, niche size, and expression of cxcl12a in a niche-autonomous manner., The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.

The sinusoidal endothelial cell niche is a component of the hematopoietic microenvironment with an important role in regulating hematopoietic stem cell (HSC) fate. Mechanisms by which the niche control the long-term functional and phylogenetic diversity of the hematopoietic system are poorly understood. We performed gene expression profiling on FACS-purified endothelial cells from the zebrafish caudal hematopoietic territory (CHT), the sinusoidal endothelial niche equivalent to the mammalian fetal liver. Kdrl:GFP+lyve1b:DsRed+ CHT endothelial cells were compared to kdrl:GFP+lyve1b:DsRed- non-CHT endothelial cells by bulk RNA-seq and 106 genes were downregulated in the CHT with q Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Cellarity: Consultancy; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder.

Single cell RNA sequencing (scRNA-seq) is a powerful method to understand gene expression changes in specific subsets of heterogeneous cell populations and to provide novel insight into mechanisms of drug resistance. However, substantial cost and interpatient variability have limited its application as a correlate for prospective clinical trials. To overcome these challenges, we employed a genetic polymorphism-based multiplexing approach to assay peripheral blood samples prospectively collected from patients with relapsed/refractory AML treated on a Phase 1b study using the anti-hepatocyte growth factor monoclonal antibody, Ficlatuzumab (NCT02109627). Ficlatuzumab was administered on study days 1, 15, 29 and 42-44 in combination with high-dose cytarabine. Peripheral blood was collected from 11 subjects on study days 0, 1, 2, 3 and 42-44. N = 7 subjects achieved a complete response to therapy and N = 4 did not respond to therapy. A peripheral blood sample from a healthy volunteer was collected and processed in parallel. 57 samples were multiplexed into 5 pools of 11 to 12 samples using a Latin square design to maximize genetic diversity within each pool and minimize batch effects. Each pool was assayed in duplicate on a 10X Chromium single cell isolation system using 3'-capture technology, targeting 40,000 cells per replicate pool. After initial bioinformatic processing, 175,667 unique cell barcodes were recovered. Genotype-free demultiplexing was performed using Freemuxlet (https://github.com/yelabucsf/popscle). Single nucleotide variants were annotated within the aligned reads from each pool using the 1000 Genomes variant database. A Bayesian clustering algorithm was applied to assign each cell to a group of cells with like genotype. Reference sample genotypes were determined using the Infinium OmniExpressExome-8 BeadChip (Illumina) and these were used to map each cluster to known subject identity. This approach unambiguously assigned 105,363 cells (60.0%) to single subjects and 60,787 (34.6%) to inter-sample doublets. 103,415 cells were retained after removal of intra-sample doublets and low-quality cells. Dimensionality reduction and clustering identified 3 groups of cells that expressed AML blast markers including CD33, CD34, KIT and HLA-DR. We hypothesized that induction of HGF expression by AML blasts might be a mechanism of resistance to anti-HGF therapy. AML blasts were stratified by treatment day and multivariable regression was performed using cluster assignment as a potential confounder. This showed that non-response to study therapy was independently correlated with higher HGF levels on study D0 (q = 0.008), D1 (q = 0.02), and D42-44 (q = 5x10-7). To identify additional biomarkers of resistance to anti-HGF therapy, we performed unsupervised gene module analysis and found 20 groups of genes that were co-regulated within the dataset. One module was strongly induced in non-responders compared to responders at D0 (q = 0), D1 (q = 1x10-278), and D42-44 (q = 0). This module was highly enriched for genes related to type-1 interferon signaling and antiviral response including IL6, IRF7, IRF2, STAT1, STAT2 and APOBEC3A. Unsupervised hierarchical clustering based on gene module scores segregated non-responders from complete responders and showed that the normal control cells were most similar to complete responders at D42-44. These data suggest that multiplexing using genetic polymorphisms increases the number of scRNA-seq samples it is feasible to analyze in the context of a prospective clinical trial. Gene module analysis is able to identify pathways as potential biomarkers of drug resistance in an unsupervised way. These approaches will aid studies in which interpatient variability is a challenge to interpretation of scRNA-seq data. Disclosures Andreadis: Merck: Research Funding; Gilead/Kite: Consultancy; Karyopharm: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Research Funding; Genentech: Consultancy, Current equity holder in publicly-traded company; BMS/Celgene/Juno: Honoraria, Research Funding; Incyte: Consultancy.

BackgroundA significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC.MethodsImmunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein.ResultsWe found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/− MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (pConclusionsThese results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.

Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro–derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro–derived HSCs as a therapeutic modality.

We currently have little understanding of the mechanisms by which hematopoietic stem and progenitor cells (HSPCs) gain a selective advantage in patients with clonal hematopoiesis and other myeloid neoplasms. The chemokine CXCL8 is elevated in a subset of patients with myeloid neoplasms. Our previous work in zebrafish has discovered a novel role for cxcl8 and its receptor, cxcr1, in supporting colonization of HSPCs within the sinusoidal endothelial cell niche of the embryonic zebrafish known as the caudal hematopoietic tissue (CHT). We hypothesized that mosaic overexpression of cxcl8 in a population of HSPCs during development would alter HSPC-niche interactions, selectively favor HSPCs expressing cxcl8 and lead to their expansion in adults. To test this hypothesis, we microinjected DNA constructs encoding cxcl8-2A-GFP or GFP alone under the control of the HSPC-specific Runx1+23 enhancer into zebrafish embryos at the single-cell stage. Time lapse fluorescence video microscopy and single-cell tracking was performed on HSPCs within the CHT. Overexpression of cxcl8 nearly doubled the amount of time HSPCs resided within the CHT when compared to expression of GFP alone as a control (cxcl8: 4.94 ± 0.86 h vs GFP: 2.54 ± 0.18 h, p=0.01, N=142 tracked cells). Substitution of WT cxcl8 with a mutant cxcl8 construct lacking the ELRCXC motif required for receptor binding reduced these effects (WT cxcl8: 6.6 ± 0.48 h vs ELRCXC-cxcl8: 5.3 ± 0.33 h, p=0.02, N=355 tracked cells). To observe HSPC-niche interactions, kdrl:mCherry endothelial cell reporter zebrafish were microinjected with Runx1+23:cxcl8-2A-GFP or Runx1+23:GFP DNA constructs. The percent of time individual HSPCs spent closely interacting with a single group of CHT endothelial cells (endothelial cell cuddling) was quantified over the period from 52 to 72 hours post-fertilization. Overexpression of cxcl8 by HSPCs increased HSPC-endothelial cell cuddling time by 30% (cxcl8: 87% vs GFP: 57%, p=0.001). To directly test competition between wild type and cxcl8 overexpressing HSPCs, zebrafish embryos were microinjected with a 1:1 molar ratio of Runx1+23:cxcl8-2A-mCherry and Runx1+23:clover DNA. Single cxcl8-2A-mCherry+ and clover+ competitor cells were tracked by time-lapse fluorescence confocal microscopy. HSPCs expressing cxcl8 resided longer within the CHT than competitor HSPCs when quantified over the period from 72 to 96 hours post-fertilization (cxcl8: 4.0 ± 0.20 h vs competitor: 2.5 ± 0.25 h, p=2.0 x 10-6, n=426 tracked cells). Single cell RNA-sequencing (scRNA-seq) of zebrafish embryos with mosaic expression of cxcl8 in HSPCs showed upregulation of cxcl12a in endothelial cells compared to endothelial cells from control embryos (p=5.19 x 10-3), suggesting a possible mechanism to explain the increased CHT residency time. Zebrafish with mosaic expression of Runx1+23:cxcl8 were raised to adulthood and the kidney marrow cells were analyzed by flow cytometry. Compared to clutchmate controls, Runx1+23:cxcl8 mosaic transgenics had a higher hematopoietic progenitor/precursor to lymphocyte ratio, suggesting a mild differentiation block and possible lineage skewing (cxcl8: 2.0 ± 0.15 vs control: 1.6 ± 0.10, p=0.048, N=25 animals). Taken together, these data support a model in which pre-malignant HSPC clones aberrantly express cxcl8 and acquire a selective advantage over normal clones through enhanced interactions with the endothelial cell niche. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

Background: Haploidentical hematopoietic cell transplantation (haplo-HCT) is an advancing and potentially curative therapy for patients with hematologic malignancies when the availability of HLA-matched donors are limited. The common source for haplo-HCT is donor bone marrow, but peripheral blood (PB) haplo-HCT is an emerging and less invasive option with similar survival outcomes. However, multiple series showed an increased incidence of culture negative fever early after haplo-HCT, which has been attributed to cytokine release syndrome (CRS) and more commonly observed with PB haplo-HCT. In this study, we investigated the incidence and impact of CRS on clinical outcomes and financial toxicity in PB haplo-HCT patients treated at a single center. Methods: A total of 40 patients who underwent PB haplo-HCT in the Ohio State University between Jan 2015 - Dec 2017 were included. CRS was defined and graded based on the criteria proposed by Lee, et al (Blood, 2014). Symptoms occurring before day +14 were included, patients with a documented infection (e.g. positive culture data) and clinical suspicion of sepsis were excluded. Overall survival (OS) was defined as the time from transplantation until death. Transplant-related mortality (TRM) was defined as death before day +28 from any cause and patients who died after day +28 with no evidence of disease. Results:. The median age at HCT was 53 years (range, 19 to 74), 63% were male. The most common diagnosis was acute myeloid leukemia (47.5%), followed by lymphoma (22.5%), acute lymphoblastic leukemia (7.5%) and myelodysplastic syndrome (7.5%). Two patients had undergone previous autologous HCT. At the time of haplo-HCT, 62% of patients were in complete remission, 38% had relapsed/refractory disease. Donor was the child in 58%, sibling in 24% and parent in 18% with an antigen match ratio of 5/10 in 50%, 6/10 in 30%, 7/10 in 12.5%, 8/10 in 7.5%. Mismatch for sex and ABO blood group were seen in 50% and 45%, respectively. Twenty percent of patients received myeloablative conditioning with fludarabine/busulfan/thiotepa, while 80% received reduced-intensity regimen with fludarabine/cyclophosphamide/total body irradiation. The incidence of CRS was 85%, including grade 1-2 CRS in 77.5% and grade 3-4 CRS in 7.5%. Time to the onset of CRS was Conclusions: CRS is commonly observed after PB haplo-HCT and usually manifests with fever within 48 hours of cell infusion. We observed favorable survival outcomes in patients who experienced mild CRS, which may suggest an association between mild CRS and graft-versus-leukemia effect. A minority of patients developed severe CRS requiring intensive care unit support, which was associated with poor OS, high TRM, prolonged hospital stay and increased financial toxicity. Further studies investigating biomarkers that can predict the development of severe CRS might enable the incorporation of CRS prophylaxis (e.g. with tocilizumab) into standard conditioning regimens for PB haplo-HCT. Disclosures Vasu: Boehringer Ingelheim Inc: Membership on an entity's Board of Directors or advisory committees. Mims:Novartis: Consultancy; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Devine:Kiadis Pharma: Consultancy. Jaglowski:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Consultancy.

IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Rα–chain to cells expressing the IL-15Rβγ complex, we postulated that certain IL-15–bearing cells must be required for NK cell homeostasis. Using IL-15WT/WT and IL-15−/− mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11chi dendritic cells (DCs), we demonstrate that ablation of the resting CD11chi DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11chi DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11chi DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.

Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.

The "holy grail" of allogeneic stem cell transplantation is to preserve the graft-versus-tumor (GVT) effect while eliminating graft-versus-host disease (GVHD). Endogenous donor-derived interleukin 15 (IL-15) has been implicated in the pathogenesis of acute GVHD, yet the mechanism by which it impacts this lethal process remains unclear. Using the well-described and clinically relevant C57BL/6 --> B6D2F1 murine model of acute GVHD, we demonstrate that in trans presentation of IL-15 by donor bone marrow-derived cells is required for the rapid onset of acute GVHD. Recipients of IL-15-/- C57BL/6 bone marrow cells show diminished type 1 polarization of T cells, yet there is no decrease in donor T-cell reconstitution. A molecular basis for these findings is provided with the observation that expression of T-bet, the master control gene for type 1 T-cell functions, is necessary for IL-15-mediated acute GVHD lethality. Finally, we demonstrate that in the absence of donor-derived IL-15, the GVT effect is maintained. These findings thus establish a mechanism by which endogenous donor-derived IL-15 impacts the pathobiology of acute GVHD and GVT activity.

There have been substantial advances in understanding the events that regulate gene expression at the cellular and molecular level; however, there has been limited progress integrating this information to understand how biological systems function in vivo. For example, the anti-inflammatory cytokine IL-10 is thought to down-regulate the effects of the pro-inflammatory cytokine IFN-gamma on monocyte activation following LPS stimulation. However, the often-postulated reciprocal regulation of IL-10 gene expression by IFN-gamma has not been studied in vivo. Here we demonstrate that the regulation of IL-10 gene expression has at least two phases following challenge with LPS or a gram-negative organism. In C57BL/6 mice, early IL-10 induction occurs independently of STAT-1, while a delayed active repression of IL-10 gene expression is critically dependent on STAT-1, but only partially dependent upon IFN-alpha/beta and IFN-gamma. This in vivo IL-10 production comes from blood monocytes, but not tissue macrophages, and cannot be reproduced in vitro. This study provides new insights into the regulation of IL-10 following challenge with a gram-negative organism, and highlights the importance of studying these cytokine regulatory pathways in vivo.

CXCL8 (IL-8) is a chemokine with pleiotropic roles in host defense, angiogenesis and tumor metastasis. CXCL8 and its specific receptor, CXCR1, are broadly expressed within the hematopoietic and vascular systems. In an effort to identify novel secreted factors with effects on hematopoietic stem and progenitor cell (HSPC) function, we have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT). The CHT is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p = 0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p = 0.004). These studies did not show whether CXCR1 acted directly on the vascular niche or whether CXCR1 expression in endothelial cells might induce expression of soluble factors or activate circulating cells that then cause expansion of the niche through indirect mechanisms. To address this, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p = 0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as “donors” and WT embryos as “recipients”. The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p = 0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in angiogenesis and HSPC biology and they provide a new example of how innate immune signaling pathways are important for stem cell function. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Citation Format: Bradley W. Blaser, Jessica L. Moore, Elliott Hagedorn, Brian Li, Vera Binder, Owen Tamplin, Leonard I. Zon. CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function [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 B035.

Efforts at harnessing the antitumour activity of natural killer (NK) cells have been investigated for the immunotherapy of human cancer for over two decades. Initial trials, focusing on the use of ex vivo-generated lymphokine activated killer (LAK) cells or activated NK cells, or in vivo cytokine therapy to expand and activate NK cells against autologous tumours, have yielded only modest success. Recent understanding of the means by which NK cells kill target cells through a complex set of activating and inhibitory receptors recognising corresponding ligands on tumour cells has paved the way for the design of improved strategies for NK cell-based immunotherapy. The net balance of activating and inhibitory signals through NK cell receptors determines whether an NK cell becomes activated or not. Successful therapeutic strategies should now focus on manipulating the balance in favour of activating receptor signalling. In the case of autologous cancers, such strategies may include the use of monoclonal antibodies with cytokines to better direct NK cells to their tumour targets through the process of antibody-dependent cellular cytotoxicity (ADCC) or the in vivo blocking of inhibitory interactions between NK receptors (NKRs) and ligands on tumour cells. Alternatively, allogeneic NK cells can be used whenever there is mismatching of inhibitory NK cell receptors and ligands. Finally, methods to modulate expression of NK cell receptors and their ligands on tumour cells by cytokines and other agents should be explored. In this review, the impact of NKR biology on the development of novel strategies for the use of NK cells in the treatment of human cancer is discussed.

The vascular niche is an important regulator of hematopoietic stem and progenitor cell (HSPC) function during development and in response to non-physiologic stress. The zebrafish caudal hematopoietic territory (CHT) is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). We have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT) during late embryogenesis. This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p=0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p=0.004). To understand whether CXCR1 acted directly on the vascular niche or indirectly via secreted factors or circulating cells, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p=0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as "donors" and WT embryos as "recipients". The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p=0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in HSPC biology and they provide a new example of how innate immune signaling pathways are important for interactions between stem and progenitor cells and the niche. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell–mediated lysis of HLA-C–expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3–positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

Murine models of disease are vital to the understanding of pathogenesis and the development of novel therapeutics. We have previously established interleukin (IL)-15 transgenic (tg) mice that demonstrate rapid proliferation of natural killer (NK) and T cells, followed by spontaneous transformation to lethal leukemia. Herein, we have characterized this model, which has many features in common with the aggressive variants of NK and T large granular lymphocyte leukemia (LGLL) in humans. The LGLL blasts are cytolytic and produce IFN-gammaex vivo. Cytogenetic analysis revealed trisomy of chromosome 17 and/or 15. This model should provide opportunities to develop effective standard therapies for this fatal disease.

Natural-killer (NK)-cell dysfunction and IFN-γ deficiencies have been associated with increased incidence of both malignancy and infection. The immunologic basis of NK-cell defects in cancer-bearing hosts has not been extensively studied. Here, we demonstrate that multiple lineages of tumors, including thymoma, breast cancer, colon cancer, and melanoma cell lines, interrupt functional maturation during NK-cell development in the bone marrow. The immature NK cells in the periphery of tumor-bearing mice had impaired IFN-γ production but seemingly normal cytotoxicity. T cells are not involved in this NK maturation arrest, because T-cell depletion did not restore NK-cell development. Moreover, the extent of tumor-cell infiltration into the bone marrow does not correlate with defective NK maturation. Interestingly, the defect was associated with a significant reduction in the IL-15Rα+ cells in the non-T, non-NK compartment of bone marrow cells and restored by overexpression of IL-15. Our data demonstrate that tumor growth can impede functional maturation of NK cells, most likely by interrupting the requisite IL-15 signaling pathway. (Blood. 2006;108:246-252)

Altered mRNA translation is one of the effects exerted by the BCR/ABL oncoprotein in the blast crisis phase of chronic myelogenous leukemia (CML). Here, we report that in BCR/ABL+ cell lines and in patient-derived CML blast crisis mononuclear and CD34+ cells, p210BCR/ABL increases expression and activity of the transcriptional-inducer and translational-regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K or HNRPK) in a dose- and kinase-dependent manner through the activation of the MAPKERK1/2 pathway. Furthermore, HNRPK down-regulation and interference with HNRPK translation-but not transcription-regulatory activity impairs cytokine-independent proliferation, clonogenic potential, and in vivo leukemogenic activity of BCR/ABL-expressing myeloid 32Dcl3 and/or primary CD34+ CML-BC patient cells. Mechanistically, we demonstrate that decreased internal ribosome entry site (IRES)-dependent Myc mRNA translation accounts for the phenotypic changes induced by inhibition of the BCR/ABL-ERK-dependent HNRPK translation-regulatory function. Accordingly, MYC protein but not mRNA levels are increased in the CD34+ fraction of patients with CML in accelerated and blastic phase but not in chronic phase CML patients and in the CD34+ fraction of marrow cells from healthy donors. Thus, BCR/ABL-dependent enhancement of HNRPK translation-regulation is important for BCR/ABL leukemogenesis and, perhaps, it might contribute to blast crisis transformation. (Blood. 2006;107:2507-2516)

Interleukin-2 (IL-2) and IL-15 are structurally related cytokines that share receptor components but display markedly different effects in multiple in vivo model systems. Here we demonstrate that IL-15 but not IL-2 exacerbates xenogeneic graft-versus-host disease (X-GVHD) in severe combined immunodeficient murine recipients of human peripheral-blood lymphocytes (hu-PBL-SCID). Treatment of hu-PBL-SCID mice with IL-15 resulted in rapid fatality, lymphocytic infiltrations in the liver, lung, and spleen consistent with X-GVHD, and a marked expansion of human CD4+ and CD8+ T cells compared with controls. Depletion of human T cells in vivo abrogated the lethality of IL-15 treatment. To our knowledge, these data are the first to demonstrate in vivo activation and expansion of human T lymphocytes in response to IL-15 with concomitant exacerbation of human T-cell-mediated X-GVHD. (Blood. 2005;106:2433-2435)

Interleukin-15 (IL-15) is a pleiotropic proinflammatory cytokine with inefficient posttranscriptional processing. We hypothesized that endogenous IL-15 could affect disease progression in the well-described C57Bl/6 (B6) → (C57Bl/6 × DBA/2) F1 hybrid (B6D2F1) murine model of acute allogeneic graft-versus-host disease (GVHD). B6D2F1 allogeneic recipients received transplants of IL-15-/- B6 bone marrow cells or B6 bone marrow cells expressing a murine IL-15 transgene (IL-15 tg) modified for efficient translation and secretion. Mice that received transplants of IL-15-/- B6 bone marrow cells displayed a significantly longer median survival time (MST) compared with mice that received transplants of wild-type (wt) B6 bone marrow; in contrast, mice that received transplants of IL-15 tg B6 bone marrow cells had a dramatically decreased MST. This decrease in survival was associated with a substantial activation and expansion of effector-memory (CD44highCD62Llow) CD8+ T lymphocytes. Finally, in vivo depletion of either CD4+ or CD8+ T lymphocyte subsets significantly prolonged survival in mice receiving IL-15 tg B6 marrow, while depletion of both CD4+ and CD8+ T cells provided complete protection from acute GVHD. We thus show that acute GVHD is attenuated in the absence of donor bone marrow–derived IL-15 and conclude that donor-derived IL-15 is a critical mediator of T-cell function in acute GVHD.

The mechanism underlying the robust expansion of natural killer (NK) cells during exogenous administration of FL is unknown. Endogenous IL-15 had no impact on the in vivo expansion of NK cell precursors during FL administration but was required for the FL-mediated expansion of mature NK cells in the spleen and blood. Studies performed using in vivo BM chimeras showed that cells derived from hematopoietic precursors (HPC), not stromal cells, provided the endogenous IL-15 required for mature NK cell expansion by FL administration. Exogenous administration of FL significantly increased both CD11b(+)CD11c(-) and CD11b(+)CD11c(+) populations but not their relatively abundant expression of IL-15 or IL-15 receptor alpha on a per cell basis. This increase preceded and correlated with NK cell expansion, the latter of which largely resulted from enhanced survival and proliferation of an existing pool of mature NK cells rather than increased de novo production of NK cells from bone marrow precursors. Finally, in vivo elimination of CD11c+ cells during the course of FL treatment significantly decreased NK cell expansion. In summary, FL administration increases NK cells in vivo by expanding antigen presenting cells which in turn provide the requisite IL-15 to enhance survival and proliferation of mature NK cells.

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome positive (Ph1) acute lymphoblastic leukemia (ALL) are two fatal BCR/ABL-driven leukemias against which the current therapy with Abl kinase inhibitors fails to induce a long-term response, as the majority of patients are either refractory or relapse after a few months of treatment. We recently reported that functional loss of the PP2A tumor suppressor occurs during CML disease progression and that restoration of PP2A activity impairs in vitro and in vivo BCR/ABL leukemogenesis. Here we assessed the therapeutic potential of the PP2A activator FTY720 in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. FTY720 (500 nM-2.5 mM) induces caspase-dependent apoptosis (70–98% annexin V+) and impairs the clonogenic potential (70–95% inhibition) of imatinib/dasatinib-sensitive and -resistant (T315I) p210 and p190 BCR/ABL-expressing myeloid and lymphoid progenitor cell lines (Ph1 K562, 32D-p210BCR/ABL, 32D-p210(T315I)BCR/ABL and BaF3-p190BCR/ABL), respectively, and of primary bone marrow CML-BCCD34+ (n=11) and Ph1 ALLCD34+/CD19+ (n=12) patients cells. Interestingly the cytokine (IL-3 or IL-7)-dependent growth and differentiation of normal CD34+ myeloid and CD34+/CD19+ lymphoid progenitors (n=8) is not affected by FTY720 treatment. Furthermore, pharmacologic doses of FTY720 markedly suppress leukemogenesis in SCID mice (n=13 per group) transplanted with myeloid and lymphoid progenitor cells transformed with p210BCR/ABL and p190BCR/ABL, respectively. In fact, the median survival has not yet been reached in FTY720-treated (10 mg/kg/day) BCR/ABL+ cell-injected mice. Conversely, all of untreated 32D-p210BCR/ABL, 32D-p210BCR/ABL(T315I) and BaF3-p190BCR/ABL leukemic mice died of an overt acute leukemia-like process with a median survival of 4.3, 4.8 and 4.1 weeks, respectively (P

Monokines (i.e. IL-12, IL-18 and IL-15) induce natural killer (NK) cells to produce interferon-γ (IFN-γ), which is critical for monocyte clearance of infectious pathogens and tumor surveillance. To identify new regulators of IFN-γ production we performed oligonucleotide array analysis of unstimulated and IL-12- and IL-18-stimulated NK92 cells. Among the subset of mRNAs differentially regulated in monokine-stimulated cells, we found SET, a potent inhibitor of the protein phosphatase type 2A (PP2A). SET mRNA and/or protein levels were upregulated in IL-12/IL-18- and IL-12/IL-15-stimulated primary human NK cells. Interestingly, the SET protein is also selectively increased in the resting CD56bright NK subset, which is a potent producer of IFN-γ relative to the CD56dim NK subset. To determine whether SET positively regulates IFN-γ production by inhibiting PP2A activity, we employed RNAi and interfered with SET expression in NK92 cells. SET downregulation inhibited IFN-γ secretion by IL-12/IL-18, IL-12/IL-15- or IL-15/IL-18-stimulated NK92 cells. By contrast, ectopic SET expression increased IFN-γ production in monokine-stimulated NK92 and primary human NK cells. Because downregulation of SET augmented PP2A activity in NK92 cells, we sought to investigate whether pharmacologic activation of PP2A inhibits the ability of NK cells to produce IFN-γ. Indeed, suppression of IFN-γ expression and secretion was also observed upon treatment of NK92 and primary NK cells with 1,9-dideoxy-forskolin, a known inducer of PP2A activity. Accordingly, NK cells from mice treated with 1.9-dideoxy-forskolin produced less IFN-γ in response to in vivo monokine stimulation than did NK cells from vehicle-treated mice. Mechanistically, activation of PP2A by SET knock-down or 1,9-dideoxy-forskolin treatment leads to inhibition of ERK1/2, p65RelA and STAT5 activity in monokine-stimulated NK cells. Because these signaling molecules are important for IFN-γ production by monokine-stimulated NK cells, our results strongly suggest that monokine induction of SET expression in NK cells is essential for limiting PP2A activity that, otherwise, would negatively impact the ability of NK cells to produce and release optimal levels of IFN-γ.