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1
Kaur, Kawaljit, Angie Perez Celis, and Anahid Jewett. "Natural Killer Cell-Secreted IFN-γ and TNF-α Mediated Differentiation in Lung Stem-like Tumors, Leading to the Susceptibility of the Tumors to Chemotherapeutic Drugs." Cells 14, no. 2 (January 10, 2025): 90. https://doi.org/10.3390/cells14020090.
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We demonstrate that natural killer (NK) cells induce a higher cytotoxicity against lung cancer stem-like cells (hA549) compared to differentiated lung cancer cell lines (H292). The supernatants from split-anergized NK cells (IL-2 and anti-CD16 mAb-treated NK cells) induced differentiation in hA549. Differentiated lung cancer cell line (H292) and NK cells differentiated hA549 expressed reduced NK cell-mediated cytotoxicity but expressed higher sensitivity to chemotherapeutic drugs. This finding validated our previous reports demonstrating that the levels of tumor killing by NK cells and by chemotherapeutic drugs correlate directly and indirectly, respectively, with the stage and levels of tumor differentiation. We also demonstrate the role of IFN-γ and TNF-α in inducing tumor differentiation. NK cells’ supernatants or IFN-γ and TNF-α-induced tumor differentiation was blocked when we used antibodies against IFN-γ and TNF-α. Therefore, IFN-γ and TNF-α released from NK cells play a significant role in differentiating tumors, resulting in increased susceptibility of tumors to chemotherapeutic drugs. We also observed the different effects of MHC-class I antibodies in CSCs vs. differentiated tumors. Treatment with anti-MHC-class I decreased NK cell-mediated cytotoxicity in hA549 tumors, whereas it increased NK cell-mediated cytotoxicity when differentiated tumors were treated with antibodies against MHC-class I.
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Persyn, Eva, Sigrid Wahlen, Laura Kiekens, Sylvie Taveirne, Wouter Van Loocke, Els Van Ammel, Filip Van Nieuwerburgh, et al. "TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality." International Journal of Molecular Sciences 23, no. 19 (September 26, 2022): 11345. http://dx.doi.org/10.3390/ijms231911345.
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The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.
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Vargas, Claudia L., Jennifer Poursine-Laurent, Liping Yang, and Wayne M. Yokoyama. "Development of thymic NK cells from double negative 1 thymocyte precursors." Blood 118, no. 13 (September 29, 2011): 3570–78. http://dx.doi.org/10.1182/blood-2011-06-359679.
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Abstract The differentiation of natural killer (NK) cells and a subpopulation of NK cells which requires an intact thymus, that is, thymic NK cells, is poorly understood. Previous in vitro studies indicate that double negative (CD4−CD8−, DN) thymocytes can develop into cells with NK cell markers, but these cells have not been well characterized. Herein, we generated and characterized NK cells differentiating from thymic DN precursors. Sorted DN1 (CD44+CD25−) CD122−NK1.1− thymocytes from Rag1−/− mice were adoptively transferred into Rag1−/−Ly5.1 congenic mice. After intrathymic injection, donor-derived cells phenotypically resembling thymic NK cells were found. To further study their differentiation, we seeded sorted DN1 CD122−NK1.1− thymocytes on irradiated OP9 bone marrow stromal cells with IL-15, IL-7, Flt3L, and stem cell factor. NK1.1+ cells emerged after 7 days. In vitro differentiated NK cells acquired markers associated with immature bone marrow–derived NK cells, but also expressed CD127, which is typically found on thymic NK cells. Furthermore, we found that in vitro cells generated from thymic precursors secreted cytokines when stimulated and degranulated on target exposure. Together, these data indicate that functional thymic NK cells can develop from a DN1 progenitor cell population.
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Vitale, Chiara. "Plasticity of NK-cell differentiation." Blood 117, no. 13 (March 31, 2011): 3482–83. http://dx.doi.org/10.1182/blood-2011-01-327965.
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Freud, Aharon G., Akihiko Yokohama, Brian Becknell, Melissa T. Lee, Hsiaoyin C. Mao, Amy K. Ferketich, and Michael A. Caligiuri. "Evidence for discrete stages of human natural killer cell differentiation in vivo." Journal of Experimental Medicine 203, no. 4 (April 10, 2006): 1033–43. http://dx.doi.org/10.1084/jem.20052507.
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Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.
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Grzywacz, Bartosz, Nandini Kataria, Niketa Kataria, Bruce R. Blazar, Jeffrey S. Miller, and Michael R. Verneris. "Natural killer–cell differentiation by myeloid progenitors." Blood 117, no. 13 (March 31, 2011): 3548–58. http://dx.doi.org/10.1182/blood-2010-04-281394.
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Abstract Because lymphoid progenitors can give rise to natural killer (NK) cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that rare human CD34+ hematopoietic progenitors develop into NK cells in vitro in the presence of cytokines (interleukin-7, interleukin-15, stem cell factor, and fms-like tyrosine kinase-3 ligand). Adding hydrocortisone and stromal cells greatly increases the frequency of progenitor cells that give rise to NK cells through the recruitment of myeloid precursors, including common myeloid progenitors and granulocytic-monocytic precursors to the NK-cell lineage. WNT signaling was involved in this effect. Cells at more advanced stages of myeloid differentiation (with increasing expression of CD13 and macrophage colony-stimulating factor receptor [M-CSFR]) could also differentiate into NK cells in the presence of cytokines, stroma, and hydrocortisone. NK cells derived from myeloid precursors (CD56−CD117+M-CSFR+) showed more expression of killer immunoglobulin-like receptors, a fraction of killer immunoglobulin–like receptor-positive–expressing cells that lacked NKG2A, a higher cytotoxicity compared with CD56−CD117+M-CSFR− precursor-derived NK cells and thus resemble the CD56dim subset of NK cells. Collectively, these studies show that NK cells can be derived from the myeloid lineage.
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Lee, Jiwon, Suk Hyung Lee, Mira Jeong, and Inpyo Choi. "The effects of tumor necrosis factor-alpha on in vitro differentiation of natural killer cells (138.13)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 138.13. http://dx.doi.org/10.4049/jimmunol.182.supp.138.13.
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Abstract Natural killer (NK) cells are differentiated from hematopoietic stem cells (HSCs) in bone marrow. The differentiation of NK cells is regulated by various factors including soluble growth factors and transcription factors. Here, we demonstrated that tumor necrosis factor-α (TNF-α) is a positive regulator of NK cell differentiation. HSC-derived precursor NK (pNK) cells were further differentiated into mature NK (mNK) cells in the presence of IL-15 in vitro. The potential role of TNF-α on NK cell maturation was evaluated in the presence or absence of IL-15. TNF-α itself induced the expression of NK1.1 and CD122 of mature NK cells and its effects were significantly augmented in the presence of IL-15. IFN-γ production was maximal when NK cells were matured in the combination of IL-15 and TNF-α. Moreover mRNA expression of several transcription factors including T-bet and GATA-3 was increased during TNF-α -mediated in vitro NK cell differentiation. Overall, these data indicate that TNF-α regulates NK differentiation by increasing transcription factors which are crucial for NK maturation.
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Holmes, Tim D., Ram Vinay Pandey, Eric Y. Helm, Heinrich Schlums, Hongya Han, Tessa M. Campbell, Theodore T. Drashansky, et al. "The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation." Science Immunology 6, no. 57 (March 12, 2021): eabc9801. http://dx.doi.org/10.1126/sciimmunol.abc9801.
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Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.
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Sánchez, M. J., M. O. Muench, M. G. Roncarolo, L. L. Lanier, and J. H. Phillips. "Identification of a common T/natural killer cell progenitor in human fetal thymus." Journal of Experimental Medicine 180, no. 2 (August 1, 1994): 569–76. http://dx.doi.org/10.1084/jem.180.2.569.
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The phenotypic similarities between natural killer (NK) and T cells have led to the hypothesis that these distinctive lymphocyte subsets may be developmentally related and thus may share a common progenitor (Lanier, L. L., H. Spits, and J. H. Phillips, 1992. Immunol. Today. 13:392; Rodewald, H.-R., P. Moingeon, J. L. Lurich, C. Dosiou, P. Lopez, and E. L. Reinherz. 1992. Cell. 69:139). In this report, we have investigated the potential of human CD34+ triple negative thymocytes ([TN] CD3-, CD4-, CD8-) to generate both T cells and NK cells in murine fetal thymic organ cultures (mFTOC) and in vitro clonogenic assays. CD34+ TN thymocytes, the majority of which express prominent cytoplasmic CD3 epsilon (cytoCD3 epsilon) protein, can be divided into high (CD34Bright) and low (CD34Dim) surface expressing populations. CD34Bright TN thymocytes were capable of differentiating into T and NK cells when transferred into mFTOC, and demonstrated high NK cell clonogenic capabilities when cultured in interleukin (IL)-2, IL-7, and stem cell factor (SCF). Likewise, CD34Bright TN thymocyte clones after 5 d in culture were capable of generating NK and T cells when transferred into mFTOC but demonstrated clonogenic NK cell differentiation capabilities when maintained in culture with IL-2. CD34Dim TN thymocytes, however, possessed only T cell differentiation capabilities in mFTOC but were not expandable in clonogenic conditions containing IL-2, IL-7, and SCF. No significant differentiation of other cell lineage was detected in either mFTOC or in clonogenic assays from CD34+ TN thymocytes. These results represent the first definitive evidence of a common T/NK cell progenitor in the human fetal thymus and delineate the point in thymocyte differentiation where T and NK cells diverge.
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Cavazzana-Calvo, M., S. Hacein-Bey, G. de Saint Basile, C. De Coene, F. Selz, F. Le Deist, and A. Fischer. "Role of interleukin-2 (IL-2), IL-7, and IL-15 in natural killer cell differentiation from cord blood hematopoietic progenitor cells and from gamma c transduced severe combined immunodeficiency X1 bone marrow cells." Blood 88, no. 10 (November 15, 1996): 3901–9. http://dx.doi.org/10.1182/blood.v88.10.3901.bloodjournal88103901.
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Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2), IL-7, and IL-15 cytokines, which share gamma c receptor subunit, in NK cell differentiation, we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF), IL-2, and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells, while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors, it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL-15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients.
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Eisman, Shira, Batya Koenigsberg, Frederique van den Haak, Luis Pedroza, Everardo Hegewisch-Solloa, Michael Shannon, and Emily Mace. "The role of CXCR4-CXCL12 in the development and migration of human natural killer cells." Journal of Immunology 212, no. 1_Supplement (May 1, 2024): 1152_4422. http://dx.doi.org/10.4049/jimmunol.212.supp.1152.4422.
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Abstract Natural killer (NK) cells play a critical role in controlling viral infection and malignancy and undergo differentiation from multipotent progenitors in secondary lymphoid tissue. Despite dependence on the microenvironment for differentiation, little is known about the cell-cell interactions that promote NK cell precursor trafficking and maturation in tissue. We have identified the chemokine receptor CXCR4 and its ligand CXCL12 as regulators of the cellular interactions between human NK cell precursors and stromal cells that support NK cell differentiation. Using in-vitro NK cell differentiation, we found that treatment of CD34+ precursors with CXCR4 antagonist AMD3100 leads to a lower efficiency of NK cell generation. Since previous work demonstrates the association of cell migration with NK cell development, we interrogated the effect of AMD3100 on NK cell migration. Using live cell imaging we found that AMD3100 treatment of NK cells or progenitor cells results in altered migratory properties and decreased arrest of cells on CXCL12+ stromal cells. Additional experiments using high resolution microscopy and flow cytometry suggest that integrins are interacting with CXCR4 in NK cells. Our data suggests that the CXCR4-CXCL12 interaction is critical for the migration and proper development of NK cells. Future studies will focus on the necessity for CXCR4-CXCL12 in specific key transitions in development and interrogate the cell biological mechanisms of this process.
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Soderquest, Katrina, Nick Powell, Carmelo Luci, Nico van Rooijen, Andrés Hidalgo, Frederic Geissmann, Thierry Walzer, Graham M. Lord, and Alfonso Martín-Fontecha. "Monocytes control natural killer cell differentiation to effector phenotypes." Blood 117, no. 17 (April 28, 2011): 4511–18. http://dx.doi.org/10.1182/blood-2010-10-312264.
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Abstract Natural killer (NK) cells play a major role in immunologic surveillance of cancer. Whether NK-cell subsets have specific roles during antitumor responses and what the signals are that drive their terminal maturation remain unclear. Using an in vivo model of tumor immunity, we show here that CD11bhiCD27low NK cells migrate to the tumor site to reject major histocompatibility complex class I negative tumors, a response that is severely impaired in Txb21−/− mice. The phenotypical analysis of Txb21-deficient mice shows that, in the absence of Txb21, NK-cell differentiation is arrested specifically at the CD11bhiCD27hi stage, resulting in the complete absence of terminally differentiated CD11bhiCD27low NK cells. Adoptive transfer experiments and radiation bone marrow chimera reveal that a Txb21+/+ environment rescues the CD11bhiCD27hi to CD11bhiCD27low transition of Txb21−/− NK cells. Furthermore, in vivo depletion of myeloid cells and in vitro coculture experiments demonstrate that spleen monocytes mediate the terminal differentiation of peripheral NK cells in a Txb21- and IL-15Rα–dependent manner. Together, these data reveal a novel, unrecognized role for Txb21 expression in monocytes in promoting NK-cell development and help appreciate how various NK-cell subsets are generated and participate in antitumor immunity.
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Risdon, G., TA Moore, V. Kumar, and M. Bennett. "Inhibition of murine natural killer cell differentiation by dehydroepiandrosterone." Blood 78, no. 9 (November 1, 1991): 2387–91. http://dx.doi.org/10.1182/blood.v78.9.2387.2387.
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Abstract Dehydroepiandrosterone (DHEA) is a naturally occurring steroid. We have previously shown that dietary DHEA (0.45% wt/wt) inhibits murine lymphopoiesis but not myelopoiesis. To assess the effect of DHEA on stages of natural killer (NK) cell differentiation, lethally irradiated mice fed DHEA or not were infused with 10(6) or 20 x 10(6) syngeneic bone marrow cells (BMC) as a source of transplantable NK cell progenitors. The differentiation of progenitor cells to lytic NK cells was assessed by the ability to clear radiolabeled YAC-1 tumor cells from the lungs. DHEA-fed recipients of 10(6) or 20 x 10(6) BMC failed to generate NK activity. Because NK progenitor cells are believed to differentiate into interleukin-2 (IL-2)-responsive precursor cells before maturation, BMC from recipient mice were cultured with IL-2 and the generation of NK cells was assessed. DHEA feeding prevented the generation of IL-2-responsive precursor cells in recipients of 10(6) BMC, but this inhibition was overcome in recipients of 20 x 10(6) BMC. To evaluate the capacity of stem cells to generate NK progenitor cells in DHEA-fed mice, the ability of marrow cells from primary recipients to generate NK activity in irradiated secondary recipients was determined. The production of NK progenitors was inhibited 20-fold. Thus, DHEA appears to inhibit the generation of NK progenitors from more primitive stem cells, the differentiation of progenitors into IL-2- responsive precursors cells and the maturation of IL-2-responsive precursor cells into mature NK cells.
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Risdon, G., TA Moore, V. Kumar, and M. Bennett. "Inhibition of murine natural killer cell differentiation by dehydroepiandrosterone." Blood 78, no. 9 (November 1, 1991): 2387–91. http://dx.doi.org/10.1182/blood.v78.9.2387.bloodjournal7892387.
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Dehydroepiandrosterone (DHEA) is a naturally occurring steroid. We have previously shown that dietary DHEA (0.45% wt/wt) inhibits murine lymphopoiesis but not myelopoiesis. To assess the effect of DHEA on stages of natural killer (NK) cell differentiation, lethally irradiated mice fed DHEA or not were infused with 10(6) or 20 x 10(6) syngeneic bone marrow cells (BMC) as a source of transplantable NK cell progenitors. The differentiation of progenitor cells to lytic NK cells was assessed by the ability to clear radiolabeled YAC-1 tumor cells from the lungs. DHEA-fed recipients of 10(6) or 20 x 10(6) BMC failed to generate NK activity. Because NK progenitor cells are believed to differentiate into interleukin-2 (IL-2)-responsive precursor cells before maturation, BMC from recipient mice were cultured with IL-2 and the generation of NK cells was assessed. DHEA feeding prevented the generation of IL-2-responsive precursor cells in recipients of 10(6) BMC, but this inhibition was overcome in recipients of 20 x 10(6) BMC. To evaluate the capacity of stem cells to generate NK progenitor cells in DHEA-fed mice, the ability of marrow cells from primary recipients to generate NK activity in irradiated secondary recipients was determined. The production of NK progenitors was inhibited 20-fold. Thus, DHEA appears to inhibit the generation of NK progenitors from more primitive stem cells, the differentiation of progenitors into IL-2- responsive precursors cells and the maturation of IL-2-responsive precursor cells into mature NK cells.
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Mikhailova, V. A., D. O. Bazhenov, K. L. Belyakova, S. A. Selkov, and D. I. Sokolov. "DIFFERENTIATION OF NK CELLS. A LOOK THROUGH THE PRISM OF TRANSCRIPTION FACTORS AND INTRACELLULAR MESSENGERS." Medical Immunology (Russia) 21, no. 1 (January 24, 2019): 21–38. http://dx.doi.org/10.15789/1563-0625-2019-1-21-38.
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All lymphoid cells are referred to as an innate or adaptive immunity unit in terms of the mechanisms of performing immune reactions. The functional activity of natural killer (NK) cells is not associated with pre-activation processes resulting from contact with antigen, rearrangement of antigen-recognition receptor genes, and clonal proliferation. In this regard, NK cells are traditionally referred to as cells of innate immunity. Previously, it was believed that NK cells represent the only population of innate immunity lymphoid cells, but, more recently, there has been increasing evidence in the literature concerning existence of different populations of these cells, thus serving a basis for isolating a common cluster called Innate Lymphoid Cells (ILC). According to the ILC classification, NK cells are classified as the first group of innate lymphoid cells according to their overall functional characteristics, as well as contribution of the T-bet transcription factor to their differentiation. Complexity, multistage and partially nonlinear character of NK cell differentiation are associated with influence of the cellular microenvironment, consistent expression of transcription factors and activation of various intracellular signaling pathways in NK cells. The review considers positioning of NK cells in the ILC classification, the main transcription factors involved in NK cell differentiation. The authors are seeking for generalization of the major routes of intracellular signal transmission in NK cells depending on their activation by cytokines located in the cellular microenvironment and affecting NK cells. The decidual NK cells during pregnancy represent a special object of NK cell differentiation. Stromal cells, trophoblast cells and macrophages are present in the decidua, in addition to NK cells. The review concerns a special case of microenvironmental effects upon expression of transcription factors and activation of NK intracellular messengers, while considering trophoblast cells an example of such influences. The recently discovered variety of NK cells, induced by the microenvironment in the course of their differentiation, requires further study.
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Montaldo, Elisa, Chiara Vitale, Francesca Cottalasso, Romana Conte, Timor Glatzer, Paolo Ambrosini, Lorenzo Moretta, and Maria Cristina Mingari. "Human NK cells at early stages of differentiation produce CXCL8 and express CD161 molecule that functions as an activating receptor." Blood 119, no. 17 (April 26, 2012): 3987–96. http://dx.doi.org/10.1182/blood-2011-09-379693.
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Abstract Human natural killer (NK) cell development is a step-by-step process characterized by phenotypically identified stages. CD161 is a marker informative of the NK cell lineage commitment, whereas CD56, CD117, and CD94/NKG2A contribute to define discrete differentiation stages. In cells undergoing in vitro differentiation from CD34+ umbilical cord blood (UCB) progenitors, LFA-1 expression allowed to discriminate between immature noncytolytic CD161+CD56+LFA-1− and more differentiated cytolytic CD161+CD56+LFA-1+ NK cells. CD161+CD56+LFA-1− NK cells produce large amounts of CXCL8 after phorbol myristate acetate (PMA) or cytokine treatment. Remarkably, CXCL8 mRNA expression was also detected in fresh stage III immature NK cells isolated from tonsils and these cells expressed CXCL8 protein on PMA stimulation. Within in vitro UCB-derived CD161+CD56+LFA-1− NK cells, CXCL8 release was also induced on antibody-mediated cross-linking of NKp44 and CD161. Such unexpected activating function of CD161 was confined to the CD161+CD56+LFA-1− subset, because it did not induce cytokine release or CD107a expression in CD161+CD56+LFA-1+ cells or in mature peripheral blood NK cells. Anti-CXCL8 neutralizing antibody induced a partial inhibition of NK cell differentiation, which suggests a regulatory role of CXCL8 during early NK cell differentiation. Altogether, these data provide novel information that may offer clues to optimize NK cell maturation in hematopoietic stem cell transplantation.
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Huyghe, Matthias, Christophe Desterke, Jusuf Imeri, Nathan Belliard, Ali G. Turhan, Annelise Bennaceur Griscelli, and Frank Griscelli. "Abstract 2778: Influence of the differentiation strategy on the phenotypic and genotypic features of iPSC derived NK cells." Cancer Research 84, no. 6_Supplement (March 22, 2024): 2778. http://dx.doi.org/10.1158/1538-7445.am2024-2778.
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Abstract The iPSC-derived NK (iNK) cells offer new perspectives to produce large-scale of homogeneous immunotherapeutic cellular products and open the way towards manufacturing off-the-shelf cancer immunotherapies. Numerous strategies and protocols of differentiation of iPSCs toward NK lineage have been published in the last decade. Nevertheless, the embryological development of the NK cells and the impact of the differentiation strategy on the activity of the NK cells are still not clearly understood. Furthermore, very few studies have compared different NK differentiation strategies from iPSC. this study, we compared two previously reported protocols for NK cell generation from the same iPSC line, one feeder-based and the other feeder-free. In the first protocol, 3D hematopoietic differentiation was followed by lymphoid differentiation, using an OP9 DLL4 expressing feeder cell line. The second protocol employed a clinical-grade approach, utilizing the “spin embryoid body” method to generate primitive hematopoietic progenitors. Subsequently, these progenitors were differentiated towards the NK lineage in a feeder-free environment. Hematopoietic progenitors produced by these protocols have distinct phenotypes, suggesting variations in their maturity levels. Furthermore, the protocols have different kinetics of differentiation revealed by CD56 and CD7 phenotypes. Although, both protocols were able to efficiently generate functional and mature NK cells, characterized by the expression of activating and maturity ligands but with a distinct transcriptomic profile. iNK cells generated using feeders exhibit a more pronounced inflammatory profile with the expression of hallmark NFKB and STAT3 signaling pathways. In contrast, feeder-free iNK cells have a more proliferative profile as evidenced by increased expression of cell cycle factors such as E2F or Myc. Feeder-based iNK cells exhibit increased expression of transcription factors involved in NK cell differentiation, such as GATA2, STAT1, and IRF1. These transcriptional variances are reinforced by superior functionality observed in feeder-based NK cells compared to feeder-free NK cells, evidenced by increased degranulation (p-value < 0.0001) and cytotoxicity (p-value = 0.0004) at a 1:2 (effector-to-target) ratio against the K562 cell line. Our findings indicate that, despite the advantages of the feeder-free strategy, the feeder-based protocol remains more efficient and produces iPSC-derived NK cells with an enhanced cytotoxic profile in vitro. Our comprehensive transcriptional analysis offers significant insights into the biological and functional properties of iNK cells. This information has the potential to oncover novel mechanisms for NK differentiation strategies from iPSCs. Citation Format: Matthias Huyghe, Christophe Desterke, Jusuf Imeri, Nathan Belliard, Ali G. Turhan, Annelise Bennaceur Griscelli, Frank Griscelli. Influence of the differentiation strategy on the phenotypic and genotypic features of iPSC derived NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2778.
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Lachota, Mieszko, Daniel Alfredo Palacios, Dennis Clement, Eivind Heggernes Ask, Hanna Julie Hoel, Merete Thune Wiiger, Marianna Vincenti, Magdalena Winiarska, Radoslaw Zagozdzon, and Karl-Johan Malmberg. "Innate-like Chemokine Receptor Profile and Migratory Behaviour By Terminally Differentiated and Educated NK Cells." Blood 136, Supplement 1 (November 5, 2020): 24–25. http://dx.doi.org/10.1182/blood-2020-140944.
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Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.
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Koo, G. C., F. J. Dumont, M. Tutt, J. Hackett, and V. Kumar. "The NK-1.1(-) mouse: a model to study differentiation of murine NK cells." Journal of Immunology 137, no. 12 (December 15, 1986): 3742–47. http://dx.doi.org/10.4049/jimmunol.137.12.3742.
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Abstract The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.
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Béziat, Vivien, Darragh Duffy, Stéphanie Nguyen Quoc, Magali Le Garff-Tavernier, Julie Decocq, Béhazine Combadière, Patrice Debré, and Vincent Vieillard. "CD56brightCD16+NK Cells: A Functional Intermediate Stage of NK Cell Differentiation." Journal of Immunology 186, no. 12 (May 9, 2011): 6753–61. http://dx.doi.org/10.4049/jimmunol.1100330.
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Hidalgo, Laura, Víctor G. Martínez, Jaris Valencia, Carmen Hernández-López, Miriam N. Vázquez, José R. Nuñez, Agustín G. Zapata, Rosa Sacedón, Alberto Varas, and Angeles Vicente. "Expression of BMPRIA on human thymic NK cell precursors: role of BMP signaling in intrathymic NK cell development." Blood 119, no. 8 (February 23, 2012): 1861–71. http://dx.doi.org/10.1182/blood-2011-07-370650.
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Abstract The bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34+CD1a−BMPRIA+ precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34+ cells mostly differentiate into functional CD56+ natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15–responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.
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Carotta, Sebastian, Swee Heng Milon Pang, Stephen L. Nutt, and Gabrielle T. Belz. "Identification of the earliest NK-cell precursor in the mouse BM." Blood 117, no. 20 (May 19, 2011): 5449–52. http://dx.doi.org/10.1182/blood-2010-11-318956.
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Abstract Natural killer (NK) cells are generated in the bone marrow (BM) from lymphoid progenitors. Although several different maturation states of committed NK cells have been described, the initial stages of NK-cell differentiation from the common lymphoid progenitor are not well understood. Here we describe the identification of the earliest committed NK-cell precursors in the BM. These precursors, termed pre-pro NK cells, lack the expression of most canonical NK cell–specific surface markers but express the transcription factor inhibitor of DNA binding 2 and high levels of the IL-7 receptor. In vitro differentiation studies demonstrate that pre-pro NK cells are committed to NK-cell lineage and appear to be upstream of the previously identified NK-cell progenitor population.
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Giardina, SL, JD Coffman, HA Young, SJ Potter, JL Frey, JR Ortaldo, and SK Anderson. "Association of the expression of an SR-cyclophilin with myeloid cell differentiation." Blood 87, no. 6 (March 15, 1996): 2269–74. http://dx.doi.org/10.1182/blood.v87.6.2269.bloodjournal8762269.
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The role of a 150-kD SR-cyclophilin (NK-TR1) in monocyte differentiation was investigated. Using an antipeptide monoclonal antibody, we have detected NK-TR1 in human peripheral blood monocytes and HL-60 cells. Unstimulated monocytes showed a low intracellular level of NK-TR1 protein that increased over 3 days of lipopolysaccharide + interferon-gamma treatment, consistent with the kinetics of monocyte differentiation. Normal HL-60 cells also had a low level of NK-TR1 protein, and exposure to 1.25% dimethyl sulfoxide (DMSO) resulted in a marked transient increase in expression that returned to basal levels before the development of granulocyte differentiation-associated biochemical changes. Phorbol myristate acetate, a promoter of monocytic differentiation in HL-60 cells, also caused a significant increase in NK-TR1 over basal levels. Transfection of a vector expressing NK-TR1 antisense RNA into HL-60 cells suppressed DMSO-mediated growth arrest. In addition, the development of a more mature phenotype, as measured by expression of CD16, and the ability to reduce nitroblue tetrazoleum dye was inhibited in transfectants when compared with controls. These results are consistent with the hypothesis that the NK-TR1 gene product is required for the progression towards a mature differentiated phenotype.
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Grzywacz, Bartosz J., Nandini Kataria, Jeffrey S. Miller, and Michael R. Verneris. "Stromal Cells Support a Myeloid Pathway of Human NK Cell Differentiation." Blood 110, no. 11 (November 16, 2007): 1336. http://dx.doi.org/10.1182/blood.v110.11.1336.1336.
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Abstract Natural killer (NK) cells belong to the lymphocyte lineage; however a myeloid origin has been debated in the past based on nascent experimental evidence. We studied the in vitro development of human NK cells from UCB-derived CD34+ cells following culture with cytokines (IL15, IL7, SCF, FLT3L, IL3) on a murine fetal stromal cell line EL08.1D2 (Blood, 2006; 108: 3824–3833). We investigated the differential requirement of CD34+ subsets for stromal cell support. Limiting dilution experiments showed that CD34+ cells negative for phenotypic markers of NK commitment (CD7, CD161, integrin B7, CD122, CD45RA) absolutely require stromal cells and/or addition of hydrocortisone (HC) to differentiate into functional NK cells. Without stromal cells or HC those progenitors give rise to myeloid lineage cells, but not NK cells. Thus, we hypothesized that stromal cells could instruct myeloid precursors to convert to the NK lineage. Indeed, CD56+ cells generated in stroma supported cultures frequently co-express CD33 and CD13. To determine whether myeloid cells developing from CD34+ cells after 2–3 wk cultures could give rise to NK cells, we FACS sorted the CD56−CD33+CD13high and CD56−CD14+ populations. Such CD33+CD13high and CD14+ cells express macrosialin (CD68) and acquire lyzozyme (by FACS), confirming their myeloid characteristics. Sorted cells cultured further in cytokines alone (IL15, IL7, SCF, FLT3L) did not give rise to NK cells. However, in the presence of cytokines, stromal cells and HC, NK cells were generated. To exclude the possibility of NK cell contamination, CD33+CD13high and CD14+ cells were isolated from cultures of CD34+ cells in conditions not supportive of NK cell development (GM-CSF, IL3, FLT3L, SCF, without stroma, IL15 or IL7). Such cells gave the same results as above (i.e., NK cells developed only with stroma and HC). In additional studies, a fraction (∼16%) of CFU-GM colonies isolated from methylocellulose cultures could generate NK cells only in the presence of stromal cells, HC and cytokines, but not cytokines alone. As more of a definitive marker of the monocytic lineage, we used the surface expression of M-CSF receptor (CD115) on hematopoietic precursors. CD56−CD117+CD115+ and CD56−CD117+CD115− fractions were FACS sorted from 2–3 wk cultures of CD34+ cells. While both populations could differentiate into NK cells, only the CD115+ monocytic precursors required stromal cells. Quantitatively the CD117+CD115− cells were the main source of NK cells in this culture system. Notably the NK cells derived from CD115+ precursors were remarkably different, showing significantly higher expression of Killer Immunoglobulin-like Receptors (KIR: CD158a, CD158b and CD158e) than their CD115− derived counterparts (52% vs 15% KIR+, n=3, p=0.002). With respect to the repertoire of HLA-specific inhibitory receptors, NK cells derived from monocytic precursors resemble the dominant fraction of peripheral blood NK cells, including potentially alloreactive NK cells (KIR+CD94/NKG2A−). Collectively we present evidence that NK cells can be derived from developmental intermediates of the monocytic lineage and this differentiation pathway is dependent upon interaction with stroma. Our data indicate that the developmental trajectory shapes the pattern of inhibitory receptor expression on mature NK cells. Such findings have bearing on our understanding of NK cell biology, post transplant NK cell reconstitution and could explain the paucity of recognized immature NK cell leukemias coinciding with the occurrence of AML variants with NK specific antigen expression.
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Kaur, Kawaljit, Anna Karolina Kozlowska, Paytsar Topchyan, Meng-Wei Ko, Nick Ohanian, Jessica Chiang, Jessica Cook, et al. "Probiotic-Treated Super-Charged NK Cells Efficiently Clear Poorly Differentiated Pancreatic Tumors in Hu-BLT Mice." Cancers 12, no. 1 (December 24, 2019): 63. http://dx.doi.org/10.3390/cancers12010063.
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Background and Aims: We have previously demonstrated that the stage of differentiation of tumors has profound effect on the function of NK cells, and that stem-like/poorly differentiated tumors were preferentially targeted by the NK cells. Therefore, in this study we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stem-like/undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics. The phenotype, growth rate and metastatic potential of pancreatic tumors differentiated by the NK cells (NK-differentiated) or patient derived differentiated or stem-like/undifferentiated pancreatic tumors were investigated. Methods: Pancreatic tumor implantation was performed in NSG and hu-BLT mice. Stage of differentiation of tumors was determined using our published criteria for well-differentiated tumors exhibiting higher surface expression of MHC- class I, CD54, and PD-L1 (B7H1) and lower expression of CD44 receptors. The inverse was seen for poorly-differentiated tumors. Results: Stem-like/undifferentiated pancreatic tumors grew rapidly and formed large tumors and exhibited lower expression of above-mentioned differentiation antigens in the pancreas of NSG and hu-BLT mice. Unlike stem-like/undifferentiated tumors, NK-differentiated MP2 (MiaPaCa-2) tumors or patient-derived differentiated tumors were not able to grow or grew smaller tumors, and were unable to metastasize in NSG or hu-BLT mice, and they were susceptible to chemotherapeutic drugs. Stem-like/undifferentiated pancreatic tumors implanted in the pancreas of hu-BLT mice and injected with super-charged NK cells formed much smaller tumors, proliferated less, and exhibited differentiated phenotype. When differentiation of stem-like tumors by the NK cells was prevented by the addition of antibodies to IFN-γ and TNF-α, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs. Greater numbers of immune cells infiltrated the tumors of NK-injected and AJ2-probiotic bacteria-fed mice. Moreover, increased IFN-γ secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected and AJ2 fed mice. Tumor-induced decreases in NK cytotoxicity and IFN-γ secretion were restored/increased within PBMCs, spleen, and bone marrow when mice received NK cells and were fed with AJ2. Conclusion: NK cells prevent growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.
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Choi, Inpyo, Won Sam Kim, and Suk Ran Yoon. "Tanshinones increase natural killer cell maturation via activating p38 phosphorylation (P4464)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 52.49. http://dx.doi.org/10.4049/jimmunol.190.supp.52.49.
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Abstract The maturation of natural killer cells is regulated by various growth and nuclear transcription factors. Tanshinones, such as cryptotanshinone (CTS) and tanshinone IIA (TS), are the major lipophilic constituents extracted from the rhizome of Salvia miltiorrhiza Bunge. Here, we demonstrated that tanshinones are positive regulators for NK cell maturation. The tanshinones augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells. Tanshinones also increased IFN-gamma production in NK cells in the presence of IL-15. They also induced the expression of several transcription factors, including Id2, GATA3, T-bet, and Ets-1. Additionally, tanshinones increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, the p38 inhibitor SB203580 blocked the developmental effects of the tanshinones and suppressed Id2, T-bet, and Ets-1 expression during NK cell differentiation. These results suggest that tanshinones significantly increased IL-15-induced NK cell differentiation via enhancing the p38 phosphorylation and the expression of transcription factors.
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Liu, Xuxiang, Jibin Zhang, Yunfei Shi, Kunal Shetty, Can Kucuk, Esra Esmeray, and Wing C. Chan. "PRDM1 Promotes Primary Human Circulating CD56 dim NK-Cell Differentiation." Blood 142, Supplement 1 (November 28, 2023): 1177. http://dx.doi.org/10.1182/blood-2023-180323.
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Background: Natural killer (NK) cells are innate lymphocytes that mount immune responses against viral infection and malignant cells. Circulating NK-cells can be generally divided into two groups, CD56 bright and CD56 dim, with the latter constituting ~90% of NK-cells in peripheral blood. While CD56 bright NK-cells are considered less mature and capable of producing more cytokines, CD56 dim population represents more differentiated cells with greater cytotoxicity. Transcription factor (TF) regulatory circuits were proposed for CD56 bright and CD56 dim NK-cells, indicating that TCF1/LEF1 and BACH2 are important in the former population and that Blimp1 and MAF are master TFs in the latter group. Blimp1 (encoded by PRDM1) has been previously found to regulate terminal differentiation of B-cells and CD8 + T-cells. In NK-cells, deletion of PRDM1 has been shown to promote cell growth and survival in vitro, and its loss of function was commonly detected in NK-cell lymphomas. Here, we employed a multi-omics approach to investigate the role of PRDM1 in the differentiation of human primary NK-cells. Methods: We used CRISPR-Cas9 system to knock out PRDM1 in primary human NK-cells isolated from PBMCs of healthy donors and performed whole transcriptome sequencing. ChIP-seq was employed to investigate the global binding spectrum of PRDM1 in primary human NK-cells cultured with K562-mbIL21-Cl9 feeder cells or with IL-2 alone. We also performed integrative genomics analyses to examine the transcriptomic regulation of NK-cell differentiation by PRDM1. Results: Gene set enrichment analyses (GSEA) demonstrated that PRDM1 KO resulted in enrichment of genes highly expressed in CD56 bright cells and depletion of genes upregulated in CD56 dim NK-cells. Specifically, we found that PRDM1 deficient NK-cells expressed CD56 bright NK-cell master TFs at higher levels, such as TCF7, RUNX2, and MYC. In contrast, TFs that play an important role in more differentiated CD56 dim NK cells were downregulated in PRDM1-KO cells, including IKZF3, MAF, and TBX21. Notably, BCL11B, a TF that has been reported to promote canonical and adaptive NK-cell differentiation, was significantly downregulated along with its target gene ZBTB16 when PRDM1 was deleted. Moreover, genes encoding cytotoxic molecules such as PRF1 and GZMB were also repressed in PRDM1-KO NK-cells. Together, these results indicate that PRDM1-KO NK-cells closely resembled the less mature CD56 bright NK-cells than the CD56 dim terminally differentiated counterpart. ChIP-seq analysis of PRDM1 revealed that many of the differentially expressed genes (DEGs) between PRDM1 KO and WT cells were directly bound by PRDM1. When integrated with publicly available ATAC-seq datasets, we found that some of these PRDM1 bound sites showed differential chromatin accessibility between CD56 bright and CD56 dim NK-cells. To explore the transcriptional program regulated by PRDM1, we utilized a regulated system to re-express PRDM1 in KHYG1, an NK-cell lymphoma cell line that is sensitive to PRDM1 overexpression. RNA-seq analysis showed that the DEGs between KHYG1 with short-term re-expression of PRDM1 and control cells overlapped with DEGs between PRDM1 KO vs WT NK-cells. These genes included BCL11B, MAF, PRF1, and GZMB. Therefore, PRDM1 directly regulates genes that are crucial in NK-cell differentiation. Furthermore, we detected upregulation of memory T-cell or progenitor exhausted T-cell signature genes in PRDM1 KO NK-cells compared with WT, including TCF7, SELL, CCR7, and IL7R. Consistent with this, GSEA analysis demonstrated enrichment of TCF1 + progenitor exhausted T cell or memory T cell upregulated genes in PRDM1 deleted NK-cells, suggesting that PRDM1 KO NK-cells may share some features with memory T-cells or progenitor exhausted T cells. Importantly, MYB, which was found to be essential in progenitor exhausted T cells, was also a direct target of PRDM1 and was upregulated in PRDM1 KO NK-cells. GSEA also showed that PRDM1 depletion in NK-cells led to upregulation of genes highly expressed in mast cell, Th1 cell, B cell, or CD8 + T cell compared to NK cells, which may indicate a role of PRDM1 in maintaining lineage commitment. Conclusions: Our findings collectively show that PRDM1 may be a master TF that regulates human NK-cell differentiation and functions through direct transcriptional regulation of key target genes.
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Qiao, Wenhua, Peng Dong, Hui Chen, and Jianmin Zhang. "Advances in Induced Pluripotent Stem Cell-Derived Natural Killer Cell Therapy." Cells 13, no. 23 (November 29, 2024): 1976. http://dx.doi.org/10.3390/cells13231976.
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Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of killing virus-infected cells and/or cancer cells. The commonly used NK cells for therapeutic applications include primary NK cells and immortalized NK cell lines. However, primary NK cell therapy faces limitations due to its restricted proliferation capacity and challenges in stable storage. Meanwhile, the immortalized NK-92 cell line requires irradiation prior to infusion, which reduces its cytotoxic activity, providing a ready-made alternative and overcoming these bottlenecks. Recent improvements in differentiation protocols for iPSC-derived NK cells have facilitated the clinical production of iPSC-NK cells. Moreover, iPSC-NK cells can be genetically modified to enhance tumor targeting and improve the expansion and persistence of iPSC-NK cells, thereby achieving more robust antitumor efficacy. This paper focuses on the differentiation-protocols efforts of iPSC-derived NK cells and the latest progress in iPSC-NK cell therapy. Additionally, we discuss the current challenges faced by iPSC-NK cells and provide an outlook on future applications and developments.
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Nabekura, Tsukasa, and Lewis L. Lanier. "Antigen-specific expansion and differentiation of natural killer cells by alloantigen stimulation." Journal of Experimental Medicine 211, no. 12 (November 3, 2014): 2455–65. http://dx.doi.org/10.1084/jem.20140798.
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Natural killer (NK) cells provide important host defense against microbial pathogens and can generate a population of long-lived memory NK cells after infection or immunization. Here, we addressed whether NK cells can expand and differentiate after alloantigen stimulation, which may be important in hematopoietic stem cell and solid tissue transplantation. A subset of NK cell in C57BL/6 mice expresses the activating Ly49D receptor that is specific for H-2Dd. These Ly49D+ NK cells can preferentially expand and differentiate when challenged with allogeneic H-2Dd cells in the context of an inflammatory environment. H-2Dd is also recognized by the inhibitory Ly49A receptor, which, when coexpressed on Ly49D+ NK cells, suppresses the expansion of Ly49D+ NK cells. Specificity of the secondary response of alloantigen-primed NK cells was defined by the expression of activating Ly49 receptors and regulated by the inhibitory receptors for MHC class I. Thus, the summation of signals through a repertoire of Ly49 receptors controls the adaptive immune features of NK cells responding to allogeneic cells.
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Mazzurana, Luca, Marianne Forkel, Anna Rao, Aline Van Acker, Efthymia Kokkinou, Tamaki Ichiya, Sven Almer, Charlotte Höög, Danielle Friberg, and Jenny Mjösberg. "Suppression of Aiolos and Ikaros expression by lenalidomide reduces human ILC3–ILC1/NK cell transdifferentiation." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 187.10. http://dx.doi.org/10.4049/jimmunol.202.supp.187.10.
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Abstract The Ikaros family of transcription factors (TFs) are important regulators of lymphocyte function. However, their roles in human innate lymphoid cell (ILC) function remain unclear. Here, we found that Ikaros (IKZF1) is expressed by all ILC subsets, including NK cells, in blood, tonsil and gut, while Helios (IKZF2) is preferentially expressed by ILC3 in tonsil and gut. Aiolos (IKZF3) followed the expression pattern of T-bet and Eomes, being predominantly expressed by ILC1 and NK cells. Differentiation of IFN-γ-producing ILC1 and NK cells from ILC3 by IL-1β plus IL-12-stimulation was associated with upregulation of T-bet and Aiolos. Selective degradation of Aiolos and Ikaros by lenalidomide suppressed ILC1 and NK cell differentiation and expression of ILC1 and NK cell-related transcripts (LEF1, PRF1, GRZB, CD244, NCR3, IRF8). In line with reduced ILC1/NK cell differentiation we observed an increase in the expression of the ILC3-related TF Helios, as well as ILC3 transcripts (TNFSF13B, IL22, NRP1 and RORC) and in the frequency of IL-22 producing ILC3 in cultures with IL-1β and IL-23. These data suggest that suppression of Aiolos and Ikaros expression inhibits ILC1 and NK cell differentiation while ILC3 function is maintained. Hence, our results open up for new possibilities in targeting Ikaros family TFs for modulation of type 1/3 immunity in inflammation and cancer.
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Roeven, Mieke WH, Jeanette Cany, Frans Maas, Arwa Kohela, Jansen Joop, Nicole MA Blijlevens, Nicolaas PM Schaap, and Harry Dolstra. "The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1 Stimulates Expression of NK Cell Related Transcription Factors, Thereby It Facilitates Generation of Highly Functional NK Cells in Vitro." Blood 124, no. 21 (December 6, 2014): 3833. http://dx.doi.org/10.1182/blood.v124.21.3833.3833.
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Abstract Introduction Allogeneic stem cell transplantation (SCT) can be a curative treatment for hematological malignancies. The therapeutic effectiveness is attributed to the graft-versus-tumor (GVT) effect, mediated by alloreactive T cells and natural killer (NK) cells. Although T cells can induce a potent GVT effect, they can also induce graft-versus-host disease (GVHD), causing high morbidity and mortality. Interestingly, after non-myeloablative allogeneic SCT, early NK cell repopulation has been associated with decreased relapse rates, without increasing GVHD incidence, illustrating a possible role for donor NK cell adoptive transfer after allogeneic SCT. However, isolation of sufficient numbers of activated NK cells from donor origin is challenging. Recently, it has been described that antagonizing the aryl hydrocarbon receptor (AhR) using the antagonist StemReginin1 (SR1) promotes expansion of human hematopoietic stem cells. Furthermore, AhR turned out to regulate differentiation of multiple immune cells like dendritic cells, regulatory T cells, γδ T cells, and also NK cells. Therefore, we investigated if SR1 could enhance NK cell generation in a cytokine-based culture system. Methods CD34+ hematopoietic progenitor cells (HPCs) were isolated using immunomagnetic beads from G-CSF mobilized aphaeresis material. These HPCs were expanded during 14 days in medium containing SCF, Flt3L, TPO, IL-7 and IL-15 and subsequently differentiated into NK cells using IL-15 and IL-2. HPC-NK cell expansions were performed with or without SR1. RNA was collected from the cultures weekly and expression of NK cell related genes was analyzed using qPCR. After 35 days, HPC-NK cells were assessed for their cytolytic functions against acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines and primary AML blasts. In addition, expression levels of typical NK-activating receptors and differentiation markers were analyzed by flow cytometry. Results Interestingly, SR1 induced expression of TOX, ID2, EOMES, GATA3 and SH2D1B, which are important factors involved in early and late NK cell differentiation. In addition, SR1 improved the expansion, differentiation, and functionality of the NK cells generated. In the presence of SR1, we were able to expand PB-derived HPCs up to 1000-fold in 5 weeks. The SR1-generated HPC-NK cell products contained 80 – 92% NK cells, which expressed high levels of activating NKG2D and natural cytotoxicity receptors. Furthermore, functional analysis showed marked degranulation and cytokine release upon co-culture with AML and MM cell lines and efficient lysis of primary AML blasts at low NK-target ratios. Conclusion Addition of the AHR antagonist SR1 in our culture system induces expression of transcription factors involved in NK cell differentiation and thereby facilitates the generation of high numbers of functional NK cells from G-CSF mobilized CD34+ progenitor cells. These NK cells hold great promise for future donor NK cell-mediated therapy after allogeneic SCT. Disclosures No relevant conflicts of interest to declare.
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Sohlberg, Ebba, Aline Pfefferle, Eivind Heggernes Ask, Astrid Tschan-Plessl, Benedikt Jacobs, Herman Netskar, Susanne Lorenz, et al. "Perturbed NK-cell homeostasis associated with disease severity in chronic neutropenia." Blood 139, no. 5 (February 3, 2022): 704–16. http://dx.doi.org/10.1182/blood.2021013233.
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Abstract Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.
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Sullivan, Ryan P., Jeffrey W. Leong, Stephanie E. Schneider, Rizwan Romee, Veronika Sexl, Riccardo Dalla-Favera, and Todd A. Fehniger. "Mir-15/16 Antagonizes Myb To Control Natural Killer Cell Differentiation and Maturation." Blood 122, no. 21 (November 15, 2013): 17. http://dx.doi.org/10.1182/blood.v122.21.17.17.
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Abstract Introduction Natural Killer (NK) cells are lymphocytes that are important for early host defense against infectious pathogens and malignant transformation. NK cells differentiate from the CLP in the bone marrow, where they are identified by markers such as CD56 and NKp46 in humans, and NK1.1, CD122, and NKp46 in mice. NK cells further mature in the periphery, and this maturation is essential for NK cell function, as both NK cell cytotoxicity and IFN-g production are dependent upon maturation. NK cell maturation is distinguished by surface marker transitions, including CD56bright to CD56dim in humans, and loss of CD27 expression in mice. However, the factors controlling NK cell differentiation and maturation are incompletely understood. We hypothesized that the transcription factor Myb had a role in this process, due to its high expression in immature NK cells and subsequent loss upon maturation. miRNAs are a family of small RNA molecules that control a wide variety of cellular processes via binding to target sites in the 3'UTR of messenger RNAs and downregulate protein production. The miR-15/16 family is very highly expressed in NK cells, and directly targets the 3'UTR of Myb. We hypothesized that a miR-15a/16-1KO mouse would have NK cell-intrinsic alterations in Myb levels, and would serve as a model of Myb upregulation. Here, we use lentiviral overexpression in primary human and mouse NK cells, as well as an in vitro human NK cell differentiation system, to demonstrate that Myb has critical roles in the NK cell differentiation and maturation processes. Furthermore, we generate a novel mouse model of miR-15/16 deficiency, and show that miR-15/16 is critically important for the regulation of Myb levels, and disruption of miR-15/16 prevents appropriate NK cell maturation. Results and Conclusions In order to investigate the role of Myb in NK cells, we transduced human NK cells, and cultured them in vitro. After 5 days of culture, GFP+ NK cells overexpressing Myb remained CD56bright (84±3 v. 6±2%, p<0.01), whereas NK cells expressing GFP only had differentiated to CD56dim (16±2 v. 94±3%, p<0.001). Mouse CD27+ NK cells were transduced with the same viruses, and adoptively transferred and allowed to mature for 7 days in their new hosts. 0% of NK cells overexpressing Myb matured to CD27-, while 11% of GFP only matured, and 22% of NK cells with knockdown of Myb matured to CD27-. Thus, cells overexpressing Myb have a block in maturation, and Myb downregulation is essential for complete NK cell maturation. To further investigate the role of Myb, we lentivirally transduced and cultured CD34+ progenitors in NK cell differentiation conditions. We found that cells overexpressing Myb had an increased percentage of immature CD56bright NK cells, which arose with more rapid kinetics (91±8 v. 28±16%, p<0.001 at day 14) [Fig. 1]. However, at later time points, cells overexpressing Myb failed to differentiate from CD56bright to the more mature CD56dim NK cells (8±6 v. 64±11%, p<0.01 at day 21). In contrast, CD34 cells transduced with an shRNA directed against Myb, differentiated to CD56dim NK cells more rapidly than control cells (90±7 v. 65±11, p<0.05 at day 21). Therefore, Myb drives initial NK cell differentiation, but prevents final maturation of NK cells. We found that Myb is a direct target of miR-15/16, as overexpression of miR-15/16 reduces the signal of luciferase fused to the 3'UTR of Myb by 50% (p<0.001), while a sponge directed against miR-15/16 increases signal by 40% (p<0.001). Therefore, we generated a novel mouse model of NK cell-specific miR-15a/16-1 knockout driven by NKp46 (Ncr1), and confirmed that Myb expression was increased in miR-15a/16-1KO NK cells (9-fold in CD27+ NK cells, p<0.05). No early differentiation phenotype was observed, because Cre is expressed later, after NK cell lineage determination. In contrast, these mice lacked mature NK cells (31±4 v 62±6 %CD27- of splenic NK, p<0.01, Fig. 2). Additionally, miR-15a/16-1 overexpression in human CD34+ cells recapitulates the phenotype of Myb knockdown, establishing a direct link between miR-15/16 and Myb [Fig. 1]. Therefore, miR-15/16 controls Myb expression in a cell-intrinsic manner, and thereby directs NK cell differentiation and maturation. Disclosures: No relevant conflicts of interest to declare.
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Hackett, J., M. Bennett, and V. Kumar. "Origin and differentiation of natural killer cells. I. Characteristics of a transplantable NK cell precursor." Journal of Immunology 134, no. 6 (June 1, 1985): 3731–38. http://dx.doi.org/10.4049/jimmunol.134.6.3731.
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Abstract To study the origin and differentiation of natural killer (NK) cells, we developed an assay for the transplantable precursor of NK(YAC-1) cells present in the bone marrow. Mice were depleted of endogenous NK(YAC-1) cells by injection of anti-asialo GM1 antibody, followed by lethal whole body irradiation. Normal syngeneic bone marrow cells were transplanted into such pretreated mice. Regeneration of NK(YAC-1) activity in the recipient mice was monitored by two different assays: the ability of spleen cells to lyse YAC-1 cells in vitro and the ability to clear i.v. injected, 125IUdR-labeled YAC-1 cells from the lungs. With both assays, a dose-response relationship between the number of bone marrow cells injected and the degree of NK(YAC-1) activity generated could be demonstrated. However, the lung clearance assay appeared superior because the NK regeneration could be detected earlier and with lower numbers of injected marrow cells. With this assay, several characteristics of the NK precursors and their differentiation could be defined. 1) The generation of mature, lytic NK cells from their transplantable precursor requires an intact "marrow microenvironment" in the recipient mice, because differentiation failed to occur in mice rendered osteopetrotic by estradiol treatment. 2) The NK(YAC-1) precursors lack the surface antigens (NK-2.1, asialo GM1, Qa-5, Thy-1) that are characteristically seen on mature NK cells. 3) The NK-precursors could be eliminated from the bone marrow with anti-Qa-2 or anti-H-2 antisera + complement, indicating that these two antigens are expressed on the precursors. The relationship between NK(YAC-1) precursors and multipotent myeloid stem cells (CFU-S) was investigated by utilizing W/Wv and Sl/Sld mutant mice. Bone marrow cells of W/Wv anemic mice, although markedly deficient in CFU-S, have a normal frequency of NK(YAC-1) precursors. Sl/Sld mice that lack a suitable microenvironment for the development of CFU-S allowed normal differentiation of NK(YAC-1) precursors when transplanted with normal bone marrow cells. Together, these data suggest that multipotent myeloid progenitor cells, as defined by the CFU-S assay, and the NK(YAC-1) precursors are not closely related.
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Miller, JS, KA Alley, and P. McGlave. "Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor." Blood 83, no. 9 (May 1, 1994): 2594–601. http://dx.doi.org/10.1182/blood.v83.9.2594.2594.
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Abstract We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34–7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.
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Miller, JS, KA Alley, and P. McGlave. "Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor." Blood 83, no. 9 (May 1, 1994): 2594–601. http://dx.doi.org/10.1182/blood.v83.9.2594.bloodjournal8392594.
Full textAbstract:
We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34–7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.
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Guimont-Desrochers, Fanny, Geneviève Boucher, Zhongjun Dong, Martine Dupuis, André Veillette, and Sylvie Lesage. "Redefining interferon-producing killer dendritic cells as a novel intermediate in NK-cell differentiation." Blood 119, no. 19 (May 10, 2012): 4349–57. http://dx.doi.org/10.1182/blood-2011-11-395954.
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Abstract The cell lineage origin of IFN-producing killer dendritic cells (IKDCs), which exhibit prominent antitumoral activity, has been subject to debate. Although IKDCs were first described as a cell type exhibiting both plasmacytoid DC and natural killer (NK) cell properties, the current view reflects that IKDCs merely represent activated NK cells expressing B220, which were thus renamed B220+ NK cells. Herein, we further investigate the lineage relation of B220+ NK cells with regard to other NK-cell subsets. We surprisingly find that, after adoptive transfer, B220− NK cells did not acquire B220 expression, even in the presence of potent activating stimuli. These findings strongly argue against the concept that B220+ NK cells are activated NK cells. Moreover, we unequivocally show that B220+ NK cells are highly proliferative and differentiate into mature NK cells after in vivo adoptive transfer. Additional phenotypic, functional, and transcriptional characterizations further define B220+ NK cells as immediate precursors to mature NK cells. The characterization of these novel attributes to B220+ NK cells will guide the identification of their ortholog in humans, contributing to the design of potent cancer immunotherapies.
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Mitchell, Birgitta, Maritza Gonzalez, Jared Manning, and Gerald J. Spangrude. "Opposing Effects of Toll-Like Receptor Ligands and Ascorbic Acid On T and Natural Killer Cell Development From Lymphoid Progenitor Cells." Blood 114, no. 22 (November 20, 2009): 1491. http://dx.doi.org/10.1182/blood.v114.22.1491.1491.
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Abstract Abstract 1491 Poster Board I-514 Introduction: A complete understanding of lymphocyte development, particularly factors driving T and natural killer (NK) cell differentiation from progenitor cells, remains an elusive goal in medicine. T and NK cells are key regulators in the defense against infections and malignancies and play a direct causative role in autoimmune diseases and graft-versus-host disease. The OP9-DL1 stromal line is an important tool in the in vitro study of lymphocyte development. Lymphocyte progenitors (KLS,Thy1.1-) harvested from adult murine bone marrow and seeded on this stromal line can be followed through stages of maturation by immunophenotyping. We observed that addition of stem cell factor (SCF), contaminated with lipopolysaccharide (LPS) through its production in E. coli, was particularly effective at promoting NK cell development in the OP9-DL1 culture system. Toll-like receptors, an important component of anti-microbial defense by the innate immune response, recognize LPS and other microbial products. Toll-like receptor ligands (TLR-L) have been shown to enhance NK cell proliferation, however an effect on NK cell differentiation from progenitor cells has not been established. A separate set of experiments led us to hypothesize that ascorbic acid (vitamin C) promotes T cell differentiation. We therefore designed experiments to evaluate the differential effects of TLR-L and ascorbic acid on NK and T cell development from lymphoid progenitors co-cultured with OP9-DL1 stromal cells. Methods: Lymphocyte progenitor cells (KLS,Thy1.1-) were sorted from adult mouse bone marrow and 1000-2000 progenitor cells were seeded per well in a 24 well plate coated with OP9-DL1 stroma. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/ml), and SCF (100 ng/ml) plus one of 5 different TLR-L (TLR1/2, TLR3, TLR4, TLR5, and a crude LPS preparation that likely contains a number of TLR-L), with or without addition of a stabilized form of ascorbic acid. Cells were passaged, counted and re-seeded with fresh media and supplements twice a week over a 30-day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, T cell receptor beta chain and T cell receptor gamma-delta chains. NK cells were evaluated for the presence of NKp46, NK1.1, and DX5. Results: We observed robust cell expansion, inhibited somewhat by addition of ascorbic acid. The inhibitory effect of ascorbate on expansion was most pronounced in the culture condition lacking TLR-L. T cell differentiation was markedly advanced by the addition of ascorbic acid in the absence of TLR-L, with the majority of cells co-expressing CD4/CD8 and TCRB/CD3. The addition of different TLR-Ls inhibited T cell differentiation, and this inhibition was partially rescued by addition of ascorbic acid. NK cell differentiation, defined as co-expression of NKp46 and NK1.1, was two to three-fold greater with the addition of TLR1/2, TLR4, TLR5, and crude LPS compared to cultures lacking TLR-L addition. In each of these conditions, NK cell differentiation was markedly inhibited by addition of ascorbic acid. Conclusions: Our data supports the hypothesis that both T and NK cell progenitors require Notch signaling for differentiation. In our in vitro model, differentiation of one lineage at the expense of the other can be manipulated with addition of TLR-L or ascorbic acid. Addition of bacterial TLR-L promotes NK cell differentiation at the expense of T cell differentiation; an effect that is partially overcome with the addition of ascorbic acid. The addition of ascorbic acid promotes robust T cell differentiation, and inhibits significant NK cell differentiation in all conditions. The ability of ascorbic acid to promote T cell differentiation appears to dominate over TLR-L promotion of NK lineage differentiation. Further work will include microarray to evaluate these effects at a genetic level. These findings will contribute to our understanding of the immune response under normal and pathologic conditions, and further a model both for study and ex vivo expansion of immune cells for therapeutic use. Disclosures: No relevant conflicts of interest to declare.
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Koo, G. C., C. L. Manyak, J. Dasch, L. Ellingsworth, and L. D. Shultz. "Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice." Journal of Immunology 147, no. 4 (August 15, 1991): 1194–200. http://dx.doi.org/10.4049/jimmunol.147.4.1194.
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Abstract Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.
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Park, Il-Kyoo, Chiara Giovenzana, Tiffany L. Hughes, Jianhua Yu, Rossana Trotta, and Michael A. Caligiuri. "The Axl/Gas6 Pathway Is Required for Human Natural Killer Cell Development." Blood 110, no. 11 (November 16, 2007): 2305. http://dx.doi.org/10.1182/blood.v110.11.2305.2305.
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Abstract Natural killer (NK) cells play an important role in host defense against microbial infection and tumors. Previous studies showed that IL-15 is essential for NK cell differentiation in vitro and in vivo. However the molecular mechanisms by which IL-15 is able to drive NK differentiation remain poorly understood. Here we show that blocking interaction between the receptor tyrosine kinase Axl and its ligands (Gas6 and protein S) by either soluble Axl/immunoglobulin Fc fusion protein (Axl-Fc) or warfarin, a vitamin K inhibitor, diminished the number and percentage of CD3−CD56+ NK cells differentiated from CD34+ human hematopoietic progenitors (HPCs) in blood and secondary lymphoid tissues in the presence of IL-15. Axl-Fc or warfarin increased HPC apoptosis, reduced the frequency of NK precursors, and resulted in impaired IFN-γ production. Mechanistically, in human CD34+ HPCs, Axl-Fc significantly inhibited IL-15-induced signaling events, such as phosphorylation of STAT5 and ERK. Taken together, our results suggest that the Axl/Gas6 pathway is important for IL-15-induced differentiation of NK cells from human CD34+ HPCs.
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Sohlberg, Ebba, Aline Pfefferle, Eivind Heggernes Ask, Astrid Tschan-Plessl, Benedikt Jacobs, Suzanne Lorenz, Stephan Meinke, et al. "Systems-Level Analysis of the Immune Repertoire in Neutropenia Reveal Arrested NK Cell Differentiation and Exhaustion." Blood 136, Supplement 1 (November 5, 2020): 24–25. http://dx.doi.org/10.1182/blood-2020-141981.
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Neutrophils are innate cells that have been suggested to play a critical role in terminal differentiation of NK cells. Whether this is a direct effect or a consequence of global immune changes with effects on NK cell homeostasis remains unknown. Here, we used high-resolution flow and mass cytometry to examine NK cell repertoires in 64 neutropenic patients and 27 healthy age- and gender-matched controls. A subgroup of neutropenic patients had lower frequencies and absolute numbers of NK cells, yet increased frequencies of CD56bright among NK cells (Figure 1A-C). Moreover, their CD56dim compartment was characterized by a block in differentiation, with a relative lack of NKG2A-CD57+KIR+ NK cells. In line with the differentiation arrest, no expansion of adaptive NK cells could be detected in CMV-seropositive patients from this subgroup. Furthermore, CD56dim NK cells showed increased frequencies of Ki-67+, Tim-3+ and TIGIT+ cells suggestive of activation and exhaustion (Figure 1D). The systemic imprint in the NK cell repertoire was associated with a blunted tumor target cell response with inefficient killing and a lower proportion of degranulating CD56dim cells (Figure 1E). RNA sequencing of the NK cell compartment further revealed that the differentiation arrest was linked to increased expression of transcription factors and genes involved in proliferation and cytokine signaling (Figure 1F). Serum protein profiling of 264 proteins showed upregulation of pathways related to apoptosis and cell turnover, as well as immune regulation and inflammation including higher levels of IL-10, IL-18 and IL-27 in these patients (Figure 1G-H). Notably, the majority of patients with perturbed NK cell compartment exhibited high-grade neutropenia, overall suggesting that the profoundly altered NK cell homeostasis was tightly connected to the severity of their underlying etiology (Figure 1I). Disclosures Meinke: XNK Therapeutics AB: Consultancy. Palmblad:Roche Sweden Inc: Speakers Bureau; Chieti Canada Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties.
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Huntington, Nicholas D., Nicolas Legrand, Nuno L. Alves, Barbara Jaron, Kees Weijer, Ariane Plet, Erwan Corcuff, et al. "IL-15 trans-presentation promotes human NK cell development and differentiation in vivo." Journal of Experimental Medicine 206, no. 1 (December 22, 2008): 25–34. http://dx.doi.org/10.1084/jem.20082013.
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The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16+KIR+ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56hiCD16−KIR− to CD56loCD16+KIR−, and finally to CD56loCD16+KIR+. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.
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Chakraborty, Damayanti, M. A. Karim Rumi, and Michael Soares. "NK cells, hypoxia and trophoblast cell differentiation." Cell Cycle 11, no. 13 (July 2012): 2427–30. http://dx.doi.org/10.4161/cc.20542.
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Mailliard, Robbie B., Sean M. Alber, Hongmei Shen, Simon C. Watkins, John M. Kirkwood, Ronald B. Herberman, and Pawel Kalinski. "IL-18–induced CD83+CCR7+ NK helper cells." Journal of Experimental Medicine 202, no. 7 (October 3, 2005): 941–53. http://dx.doi.org/10.1084/jem.20050128.
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In addition to their cytotoxic activities, natural killer (NK) cells can have immunoregulatory functions. We describe a distinct “helper” differentiation pathway of human CD56+CD3− NK cells into CD56+/CD83+/CCR7+/CD25+ cells that display high migratory responsiveness to lymph node (LN)–associated chemokines, high ability to produce interferon-γ upon exposure to dendritic cell (DC)- or T helper (Th) cell–related signals, and pronounced abilities to promote interleukin (IL)-12p70 production in DCs and the development of Th1 responses. This helper pathway of NK cell differentiation, which is not associated with any enhancement of cytolytic activity, is induced by IL-18, but not other NK cell–activating factors. It is blocked by prostaglandin (PG)E2, a factor that induces a similar CD83+/CCR7+/CD25+ LN-homing phenotype in maturing DCs. The current data demonstrate independent regulation of the “helper” versus “effector” pathways of NK cell differentiation and novel mechanisms of immunoregulation by IL-18 and PGE2.
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Yin, Jie, Jianmei W. Leavenworth, Yang Li, Qi Luo, Huafeng Xie, Xinhua Liu, Shan Huang, et al. "Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): 15988–93. http://dx.doi.org/10.1073/pnas.1521740112.
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Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27me3) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122+ NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.
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Colucci, Francesco, Claire Soudais, Eleftheria Rosmaraki, Lesley Vanes, Victor L. J. Tybulewicz, and James P. Di Santo. "Dissecting NK Cell Development Using a Novel Alymphoid Mouse Model: Investigating the Role of the c-abl Proto-Oncogene in Murine NK Cell Differentiation." Journal of Immunology 162, no. 5 (March 1, 1999): 2761–65. http://dx.doi.org/10.4049/jimmunol.162.5.2761.
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Abstract NK lymphocytes participate in both innate and adaptive immunity by their prompt secretion of cytokines including IFN-γ, which activates macrophages, and by their ability to lyse virally infected cells and tumor cells without prior sensitization. Although these characteristics of NK cells are well documented, little is known about the genetic program that orchestrates NK development or about the signaling pathways that trigger NK effector functions. By crossing NK-deficient common γ-chain (γc) and recombinase activating gene (RAG)-2 mutant mice, we have generated a novel alymphoid (B−, T−, and NK−) mouse strain (RAG2/γc) suitable for NK complementation in vivo. The role of the c-abl proto-oncogene in murine NK cell differentiation has been addressed in hemopoietic chimeras generated using RAG2/γc mice reconstituted with c-abl−/− fetal liver cells. The phenotypically mature NK cells that developed in the absence of c-abl were capable of lysing tumor targets, recognizing “missing self,” and performing Ab-dependent cellular cytotoxicity. Taken together, these results exclude any essential role for c-abl in murine NK cell differentiation in vivo. The RAG2/γc model thereby provides a novel approach to establish a genetic map of NK cell development.
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Colucci, Francesco, and James P. Di Santo. "The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells." Blood 95, no. 3 (February 1, 2000): 984–91. http://dx.doi.org/10.1182/blood.v95.3.984.003k40_984_991.
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Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit−/− (W/W) mice. Although c-kit−/−NK cells were generated inW/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)+ cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit+ NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.
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Yoshimori, Mayumi, Miwako Nishio, Ayaka Ohashi, Megumi Tateishi, Ayaka Mimura, Naomi Wada, Minori Saito, Norio Shimizu, Ken-Ichi Imadome, and Ayako Arai. "Interferon-γ Produced by EBV-Positive Neoplastic NK-Cells Induces Differentiation into Macrophages and Procoagulant Activity of Monocytes, Which Leads to HLH." Cancers 13, no. 20 (October 12, 2021): 5097. http://dx.doi.org/10.3390/cancers13205097.
Full textAbstract:
Epstein–Barr virus (EBV)-positive T- or NK-cell neoplasms show progressive systemic inflammation and abnormal blood coagulation causing hemophagocytic lymphohistiocytosis (HLH). It was reported that inflammatory cytokines were produced and secreted by EBV-positive neoplastic T- or NK-cells. These cytokines can induce the differentiation of monocytes into macrophages leading to HLH. To clarify which products of EBV-positive neoplastic T- or NK-cells have effects on monocytes, we performed a co-culture assay of monocytes with the supernatants of EBV-positive T- or NK-cell lines. The expression of differentiation markers, the phagocytosis ability, and the mRNA expression of the inflammatory cytokines of THP-1, a monocytic cell line, clearly increased after culturing with the supernatants from EBV-NK-cell lines. Co-culturing with the supernatants promoted the expression of CD80 and CD206 as well as M1 and M2 macrophage markers in human monocytes. Co-culturing with the supernatants of EBV-NK-cell lines significantly enhanced the procoagulant activity and the tissue factor expression of monocytes. Interferon (IFN)-γ was elevated extremely not only in the supernatant of EBV-NK-cell lines but also in the plasma of EBV-positive NK-cell neoplasms patients accompanying HLH. Finally, we confirmed that IFN-γ directly enhanced the differentiation into M1-like macrophages and the procoagulant activity of monocytes. Our findings suggest that IFN-γ may potentially serve as a therapeutic target to regulate HLH in EBV-positive NK-cell neoplasms.
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Hackett, J., M. Tutt, M. Lipscomb, M. Bennett, G. Koo, and V. Kumar. "Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells." Journal of Immunology 136, no. 8 (April 15, 1986): 3124–31. http://dx.doi.org/10.4049/jimmunol.136.8.3124.
Full textAbstract:
Abstract Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.
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Nakano, Saori, Akira Niwa, Yohko Kitagawa, Hidefumi Hiramatsu, and Megumu K. Saito. "IL-4 Acts at an Early Fate-Determining Junction in Hematopoiesis to Induce NK Cell Subsets Expressing Endogenous CD16." Blood 142, Supplement 1 (November 28, 2023): 1. http://dx.doi.org/10.1182/blood-2023-188768.
Full textAbstract:
Human NK cells are known to contain subsets of CD56 +CD16 + and CD56 +CD16 - cells. The former directly attack target cells by an antibody-dependent cellular cytotoxicity (ADCC) various organs in the body, whereas the latter are mainly found in lymph nodes and activate inflammatory and immune responses by secreting IFNγ. However, despite their importance in the biological defence systems, whether and how these subsets emerge is not yet fully understood. In particular, several recent studies have challenged the conventional linear differentiation model which positioned CD56 +CD16 - cells as progenitors of CD56 +CD16 + cells. However, during the stepwise fate specification from immature multipotent progenitors to NK cells, it is still unknown at which stage the final CD16-expressing capacity is determined and what stimuli are involved in this decision. Through cell fate tracking in human HPC-derived differentiation, present study has demonstrated that fate specification into distinct progenitor cells specific for the two subsets occurs at a stage before the cells have yet to express CD56. Furthermore, it showed that IL-4 has a positive effect, both in bona fide and PSC-derived hematopoiesis, on the fate decision to progenitor cells from which CD56 +CD16 + NK cells emerge. While IL-4 was previously known to act on NK cells after CD56 expression, the present results show that IL-4 is also involved in future fate decisions at a more earlier progenitor cell stage than previous reports. We first identified three CD34 -CD56 - NK progenitor cell fractions, which were clearly distinguished due to the intensity of CD117 expression (CD34 -CD56 -CD117 ++, CD34 -CD56 -CD 117 +, CD34 -CD56 -CD117 -) in the differentiation pathway from lineage-negative cord blood progenitors. Tracking the cell fate of each fraction showed that they all produced CD56 + NK cells, but that CD16-expressing cells emerged predominantly from the CD34 -CD56 -CD117 + and CD34 -CD56 -CD117 - fractions at a rate of 15-30%. In contrast, NK cells originating from the CD34 -CD56 -CD117 ++ fraction hardly expressed CD16. Based on expression profile comparisons, we next identified IL-4 as a factor involved in progenitor cell fate decisions. IL-4 stimulation during early stage differentiation increased the CD34 -CD56 -CD117 + and CD34 -CD56 -CD117 - fractions, and conversely reduced the CD34 -CD56 -CD117 ++ cells. As a result, not only was the emergence efficiency of CD16-expressing NK cells increased, but their cytotoxic potential was also enhanced. During this process, IL-4 enhanced EOMES expression, which was in common with previous reports on T cells. Finally, we investigated whether NK cells derived from human pluripotent stem cells (hPSCs) also develop along the same trajectory as bona fide hematopoietic cells. Using a refined system of our previously reported NK cell differentiation cultures, we found that, as in cord blood, many hPSC strains produced all of three progenitor cell fractions, and both CD16 + and CD16 - NK cells appeared at the end of culture. In contrast, some strains produced only CD16 - NK cells, which were nearly deficient in the CD34 -CD56 -CD117 + fraction on the way of differentiation. Interestingly, stimulation with IL-4 significantly reduced the proportion of CD34 -CD56 -CD117 ++ progenitor cells from those strains (52.8±14.4% to 10.4 ±4.7%), which instead also produced CD34 -CD56 -CD117 + progenitors (13±3.4% to 68.1 ±8.3%) from which around 15% of CD16 + NK cells were eventually generated (Figure). Our results support a branching model in human NK cell differentiation, no matter whether they are derived from cord blood or hPSCs. We found that IL-4 stimulation accelerates specification into the progenitors capable of producing CD16 + NK cells in the future at a much earlier stage than previously thought. Furthermore, we showed that IL-4 could correct strain-dependent differentiation propensities of hPSCs to stably induce functional NK cells with high ADCC activity.