Scientific report: Delineating cellular stages and regulation of human NK cell development to improve NK cell-based therapy for cancer (Dnr )

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1 Scientific report: Delineating cellular stages and regulation of human NK cell development to improve NK cell-based therapy for cancer (Dnr ) The main goal of this project focuses on establishing a cellular map of NK cell development and identifying the key pathways that regulate NK lineage restriction, and NK cell expansion and differentiation. Thanks to the generous support from AFA we have completed the studies on identification, purification and characterization of human committed NK cell progenitor. We have identified a novel hematopoietic progenitor with specific and restricted NK cell lineage potential and characterized its genetic profile and its relation to the other early lymphoid progenitors (Renoux et al Immunity 2015, 43: , impact factor 21.6). This work provides an important base for further studies on characterization of NK cell development and identification of critical pathways governing NK lineage commitment and differentiation. We also investigated NK cell compartments in malignant hematopoiesis to address how NK cell development is altered during the onset of myeloproliferative disease. This has been a productive period for the group: 1 paper has been published, 1 manuscript submitted and 1 invited review article has been published. The budget for this project has been used as planned. For the details please see the economic rapport. I. Summary of the obtained data: Proposed specific aim and obtained data for specific aim I and III I. Identification of human committed NK progenitor The early stages of human lymphopoiesis as well as NK cell development are not well studied and the identity of restricted NK cell progenitor (NKP) is unknown. We applied expression of known lymphoid markers as well as cytokine receptors known to be important for NK cell development to identify the human candidate NKP. The Lin - CD123 - CD34 + CD38 + CD45RA + CD7 + CD10 + CD127 - cell population purified from human umbilical cord blood has a robust NK cell potential and lacks ability to generate T, B, ILCs and myeloid cells in vitro at the single cell level. After transplantation into new-born immunedeficient mice, the Lin - CD123 - CD34 + CD38 + CD45RA + CD7 + CD10 + CD127 - cells engrafted and produced mature NK cells clearly detectable in multiple lymphoid tissues. Furthermore, mature NK cells generated from the candidate NKP show cytotoxic activity in the degranulation assay and produce IFN and TNF after activation. The Lin - CD123 - CD34 + CD38 + CD45RA + CD7 + CD10 + CD127 - human NKP has been also found in adult bone marrow and in healthy tonsils as well as in the liver of 6, 8, 10, 12, 16-week; and in the bone marrow of 12, 15 and 16-week old fetuses. The gene expression analysis in NKPs shows up-regulation of NK lineage specific genes while the expression of B and myeloid genes was down regulated. Finally, we also studied developmental relationship between early lymphoid progenitors: LMPP, CLP and NKP; and established that LMPP give rise to both CLP and NKP; CLP can generate only NK progenitors, while NKPs cannot produce CLPs. These data together support that NKP represents a NK lineage restricted progenitor conserved throughout ontogeny placed in the hematopoietic hierarchy downstream CLP and LMPP. (Renoux et al Immunity 2015, 43: , enclosed copy). III. Study the NK compartments in leukemia Summary of obtained data and ongoing studies Our previous work has demonstrated that FLT3 signaling is important for lymphopoiesis and for NK cell development. FLT3 mutations are the most common mutations found in leukemia. Previous studies have shown that NK cells from acute myeloid leukemia (AML) patients have poor cytotoxic functions and reduced expression of killing receptors. Also patients with myelodysplastic syndrome (MDS) show reduced NK cell activity. The transgenic mouse model with activated human FLT3 (FLT3-1

2 ITD) expressed following normal expression pattern provides a clear way to determine the impact of mutated receptor on the defined stages of hematopoiesis. FLT3-ITD mice develop myeloproliferative disease with impaired hematopoiesis: reduced HSC compartment and expanded pool of early myelolymphoid progenitors (Kharazi et al Blood 2011; Mead et al Cell Reports 2013). Analysis of NK cell compartments in FLT3-ITD mice supports that dysregulated FLT3 affects NK cell development and their cytotoxic function. Mice carrying FLT3-ITD mutation have significant reduction in NK cells in all the tissues (Figure 1A), decreased number of NK cell progenitors in bone marrow (Figure 1B), as well as reduced number of mature NK cells (Figure 1C) that are impaired in their ability to kill target tumor cells (Figure 1D). The effects of FLT3-ITD are cell intrinsic as the recipient chimera mice generated after competitive transplantation of cells carrying FLT3-ITD and wild type cells developed the same NK cell phenotype as donor FLT3-ITD mice (Figure 2A-C). Interestingly, at 24 weeks after transplantation the NK cells derived from wild type bone marrow also show the functional impairment supporting that the diseased environment contributes to the NK cell phenotype (Figure 3A-C). Figure 1: Activating FLT3-ITD mutation affects NK cell development in the bone marrow and leads to the reduction of the pool of mature functional NK cells. Figure 1A Figure 1B Figure 1C Figure 1D 2

3 Figure 2: NK cells generated from transplanted FLT3-ITD progenitors are reduced in numbers and impaired in their function. Figure 2 A Figure 2B Figure 2C Figure 3: Progression of myeloproliferative disease from transplanted FLT3-ITD cells affects the generation of NK cells from co-transplanted wild type progenitors. Figure 3A Figure 3B Figure 3C NK cell progenitors and mature NK cells have been purified from FLT3-ITD and control mice and gene expression profiles are currently investigated using Fluidigm. This will establish how dysregulated FLT3 affects the NK lineage regulatory network and will help to uncover the mechanism behind the NK cell deficiency. The comparison of gene profiles of NK cell progenitors from control and FLT3ITD mice will allow identification of novel regulators involved in NK cell development. Other relevant specific aims and obtained data (not initially included in the project plan) Studies on the different NK cell developmental niches and their relationship Although the bone marrow is the main site of NK cell development, the subset of NK cells develops in the thymus. Furthermore, the existence of progenitors with combined T/NK potential, as well as the fact that early T cell progenitors in the thymus maintain the ability to generate NK cells, suggests a 3

4 close developmental relationship between T and NK cells. Since the Notch pathway is the key regulator of T cell development, and it has not been directly studied whether and how Notch impacts NK cell development in vivo, we investigated NK cell compartments in Rbp-Jk deficient mice that lack canonical Notch signaling. The loss of the Notch pathway led to a reduction in early NK cell progenitors and a block in the final stages of NK cell maturation. The conventional NK cells in Rbp-Jkdeleted mice had impaired cytotoxic activity and showed signs of a chronic activation. The conventional NK cells in the thymus of Rbp-Jk deleted mice were expanded, while the thymic NK cells were not affected. Compatible with the reduction in early NK progenitors, and in the pool of mature NK cells, the expression of transcription factors known to regulate the early NK cell developmental stages: Ets1, Ikzf1 and Stat4b and Stat5a, as well as those controlling their later maturation: Eomes, Tcf1 and Mef1 were significantly lower in Rbp-Jk-deleted mice. The changes in NK cell development in the absence of Notch were cell intrinsic, as the chimeric mice generated after transplantation of Rbp-Jk deficient bone marrow cells had the same NK cell phenotype as Rbp-Jk donor mice. These results suggest the involvement of Notch signaling in regulation of multiple NK cell developmental stages. (Chaves et al, manuscript submitted, enclosed copy). Group composition and contributions: Virginie Renoux postdoctoral researcher, responsible for the studies on identification on human NK cell progenitor NKP Patricia Chaves postdoctoral researcher, responsible for studies on NK cell development in mice carrying FLT3-ITD mutation and Rbp-Jk-deleted mice Alya Zriwil PhD student, performed gene expression analyses in both human and mouse NK cell projects Lilian Wittmann research engineer and lab manager, was responsible for breeding the different mouse lines for the projects and provided technical assistance with the experiments (animal breeding, transplantation, tissue isolation) II. Publications and contributions Key publications from Sitnicka group: 1. Cichocki F, Sitnicka E, and Bryceson YT. NK cell development and function plasticity and redundancy unleashed. Invited review, Seminars in Immunology; 2014, 26: Renoux VM, Zriwil A, Peitzsch C, Michaëlsson J, Friberg D, Soneji S, Sitnicka E. Identification of a human natural killer cell lineage restricted progenitor in fetal and adult tissues. Immunity 2015; 43: Manuscripts from Sitnicka group: 1. Chaves P, Zriwil A, Wittmann L, Boukarabila H, Peitzsch C, Jacobsen SE and Sitnicka E. Loss of canonical Notch signaling affects multiple steps in NK cell development. (Manuscript submitted) 4

5 Other publications and manuscripts: 1. Drissen R, Buza-Vidas N, Woll P, Thongjuea S, Gambardella A, Giustacchini A, Manicini E, Zriwil A, Lutteropp M, Grover A, Mead A, Sitnicka E, Jacobsen SEW, Nerlov C. Distinct myeloid progenitor differentiation pathways identified through single cell RNA sequencing. Nature Immunology, in press 2. Zriwil A, Boiers C, Wittmann L, Jacobsen SE, Sitnicka E. Macrophage Colony-stimulating Factor marks and regulates a fetal myeloid-primed B cell progenitor in mouse. Blood, invited resubmission 3. Kristiansen TA, Jaensson Gyllenbäck E, Zriwil A, Björklund T, Daniel JA, Sitnicka E, Soneji S, Bryder D, Yuan J. Cellular barcoding links B-1a B cell potential to a reversible fetal hematopoietic stem cell state at the clonal level. Immunity, invited resubmission 4. Singh T, Colberg JK, Sarmiento L, Johansson JK, Chaves P, Ganic E, Mazur M, Hansen L, Holmberg D, Sitnicka E, Cilio CM, Artner I. Dysregulation of MafB expression in islet cells causes aberrant cytokine production, viral propagation and islet inflammation. Manuscript Future perspective We are grateful for the generous support from AFA that allowed us to identify a progenitor restricted to the NK lineage. This novel finding will provide a basis for a new study focused on identification of the critical pathways governing human NK cell development and establishing means for NK cell expansion. Furthermore, these results will help us to uncover mechanisms behind the NK cell disorders and to understand how the disrupted cytokine signaling pathways and malignant transformation affect NK cell development. The newly identified progenitor could be applied as a potential tool for screening the patients with immune deficiencies and to follow their recovery after therapy. 5