Human ILC1s target leukemia stem cells and control development of AML

doi:10.21203/rs.3.rs-2319959/v1

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Human ILC1s target leukemia stem cells and control development of AML

https://doi.org/10.21203/rs.3.rs-2319959/v1

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Innate lymphocytes can mediate cancer immunosurveillance and protect against disease. We have demonstrated that mouse type I innate lymphoid cells (ILC1s) can contribute to controlling the growth of acute myeloid leukemia (AML). However, the functional roles of human ILC1s in AML remain largely undefined. Here, we found that the ILC1s in patients with AML are impaired while a high expression of the ILC1 gene signature is associated with better overall survival in AML. By directly interacting with leukemia stem cells (LSCs), human ILC1s can eliminate LSCs via production of IFNγ and block LSC differentiation into M2 macrophage-like, leukemia-supporting cells through TNF. Collectively, these effects converge to limit leukemogenesis in vivo. We also identified Lin⁻CD127⁺CD161⁻CRTH2⁻CD117⁻ cells as the human ILC1 subset. The use of umbilical cord blood (UCB) CD34⁺ hematopoietic stem cells to generate CD161⁻ ILC1s could allow for a readily available supply of ILC1s to be produced for human adoptive transfer studies. Together, our findings provide evidence that targeting human ILC1s may be a promising therapeutic approach for prolongation of disease-free survival in AML.

Biological sciences/Immunology/Innate immune cells/Innate lymphoid cells

Health sciences/Diseases/Cancer/Cancer stem cells

Acute myeloid leukemia (AML) is a devastating disease and the median 5-year survival is 40–45% for patients younger than age 65 treated with standard chemotherapy¹. Although allogeneic stem cell transplantation has shown to be curative in some cases, the treatment-related mortality and the risk for disease relapse due to the possible persistence of leukemia stem cells (LSCs) remain relatively high. Therefore, safer and more effective novel therapeutic approaches are needed to improve clinical outcomes of patients with AML. The innate lymphoid cell (ILC) plays a critical role in mediating immune responses, regulating tissue homeostasis, and inflammation^2–7. We recently reported that mouse ILC1s contribute to the control of AML by eliminating LSCs and inhibiting their differentiation into myeloid blasts⁸. Functional impairment of mouse ILC1s in AML leads to the outgrowth of LSCs and disease relapse⁸. However, the potent anticancer properties and mechanisms of human ILC1s in AML remain to be further explored. Our study suggests that human ILC1s are clinically relevant in halting progression of AML and have the potential to be manufactured ex vivo for the successful treatment of AML.

We have reported that the higher IFNγ levels produced by ILC1s isolated from mice can induce apoptosis of LSCs⁸. However, most of the mechanistic results were demonstrated in mouse ILC1s. Whether ILC1s control LSC fate also in the human AML remains largely undefined. Following analysis of ILC1s in the blood of patients with AML at disease onset, we observed a highly significant reduction of total ILC1s among lineage-negative cells (Lin⁻, defined as depletion of CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FceRI, and CD56 positive cells) relative to healthy donors (Fig. 1A). However, among total ILCs (defined as Lin⁻CD127⁺) in the same patient population, there was a significant enrichment of the ILC1 subset relative to healthy donors (Fig. 1B). Further, the IFNγ⁺ functional ILC1s were significantly reduced in the patients with AML compared to healthy donors (Fig. 1C). Together, these data suggest that ILC1s in patients with new onset AML are impaired in their frequency among Lin⁻ cells and in their production of IFNγ. Using The Cancer Genome Atlas (TCGA) database, we identified a significant positive correlation between ILC1s and leukemia blast and/or LSC signatures (R = 0.25, p = 0.00086) (Fig. 1D). Analysis of 53 AML cases from TCGA showed that AML patients with high ILC1 gene signature had a significantly prolonged overall survival compared to AML patients with a low ILC1 gene signature (Fig. 1E). Collectively, these data suggest that the functional roles of human ILC1s become dysregulated in the context of AML, and a high level of human ILC1s correlates with more favorable clinical outcomes in AML.

To investigate the impact of human ILC1s (defined as Lin⁻CD127⁺CD161⁺CRTH2⁻CD117⁻, hereafter referred to as CD161⁺ ILC1s or ILC1s) on human LSCs (CD34⁺CD38⁻ cells), we isolated LSCs from the peripheral blood of patients with AML and cocultured them with human ILC1s isolated from the peripheral blood of healthy donors at a ratio of one ILC1 to four LSCs (1:4), or with recombinant human (rh) IFNγ or rhTNF for 3 days. We observed that compared to the control group of LSCs alone, coculture of LSCs with ILC1s or with rhIFNγ, but not with rhTNF, significantly decreased the CD34⁺CD38⁻ cell fraction (Fig. 2A). The percentage and absolute number of CD34⁺CD38⁻ cells were reversed in an identical coculture of ILC1s with LSCs that was treated with anti-IFNγ and compared to coculture of ILC1s with LSCs alone (Fig. 2B), indicating that ILC1−produced IFNγ eliminated LSCs. To understand whether ILC1s can affect the differentiation of LSCs into the non-LSC fractions, we sorted CD34⁺CD38⁻ cells and cocultured them with ILC1s at the 1:4 ratio of ILC1s to LSCs for 3 days. By Wright-Giemsa staining, we observed that ILC1s blocked the differentiation of CD34⁺CD38⁻ cells into macrophage-like leukemia-supporting cells (Fig. 2C) that were previously reported to support the growth of leukemic cells rather than inhibiting them^9–11. Flow cytometry of the differentiated cells showed that some express CD11b and CD206 (CD11b⁺CD206⁺) with an M2 phenotype⁹, and the population, equivalent to macrophage-like leukemia-supporting cells, was found to be significantly decreased in the presence of ILC1s (Fig. 2D). When TNF neutralizing antibody was added to the culture of ILC1s with LSCs, the ability of ILC1s to block the differentiation of LSCs decreased modestly but significantly, as evidenced by partial recovery of the otherwise decreased CD11b⁺CD206⁺ population in the presence of the neutralizing TNF antibody and ILC1s (Fig. 2D). A similar effect was not seen in the presence of neutralizing IFNg. This indicates that ILC1-derived TNF rather than IFNγ at least partially contributes to suppressing the differentiation of LSCs into M2 macrophage-like leukemia-supporting cells. The colony-forming unit (CFU) assay further confirmed that ILC1s impede the differentiation of human LSCs into leukemia-supporting M2 macrophages, as indicated by decreased numbers of total colonies and granulocyte-macrophage progenitor (CFU-GM) colonies after coculture with ILC1s compared to culture without ILC1s, while there was no significant difference in the number of granulocyte (CFU-G) colonies (Fig. 2E and 2F). Collectively, these data indicate that both IFNγ and TNF produced by ILC1s contribute to the control of human CD34⁺CD38⁻ LSC development in vitro, the former eliminated it while the latter blocked its differentiation into leukemia-supporting M2 macrophages. We also performed the in vivo transplantation experiment, in which human CD34⁺CD38⁻ cells and human ILC1s were co-injected i.v. into NOD.Cg-Prkdc^scid Il2rg^tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice expressing human hIL3, hGM-CSF (CSF2) and hSCF (KITLG), three cytokines that support the stable engraftment of myeloid lineages¹². On each of days 1–7, the mice received an intraperitoneally (i.p.) injection of human IL-15. We found that the injection of human ILC1s from healthy individuals reduced the LSC engraftment into mice and suppressed the progression of AML, as evidenced by the significantly decreased number of CD45⁺CD33⁺ blast cells, the significantly decreased number of CD34⁺CD38⁻ LSCs, and the significantly prolonged survival of mice, all compared to mice that did not receive injection of ILC1s (Fig. 2G-2J). These data suggest that human ILC1s can suppress the differentiation and development of human LSCs in vivo. Taken together, these findings demonstrate that ILC1s play a positive role against AML and provide a rationale for using ILC1s as a cellular-based therapy to prolong disease-free survival in AML.

ILC1s have been considered CD161-expressing cells. However, there is no evidence that isolation and ex vivo expansion of Lin⁻CD161⁺ ILC1s from AML patients or healthy donors results in a homogeneous population of ILC1s. Through extensive flow cytometric analyses, we found that ILC1s were heterogeneous in the peripheral blood of humans using ILC1-specific surface markers (Lin, CD127, CRTH2, and CD117) that were reported previously^13,14 and a combination of CD161 antibody (Extended data Fig. 1A). The heterogeneous ILC1s included the conventional CD161⁺ ILC1s and Lin⁻CD127⁺CD161⁻CRTH2⁻CD117⁻cells (hereafter referred to as CD161⁻ ILC1s). In our hands, the percentage of CD161⁻ ILC1s was higher than CD161⁺ ILC1s among total ILCs isolated from the blood of healthy donors (Extended data Fig. 1A). We further expanded the total ILCs on either OP9-DL1 or DL4-transfected OP9 (hereafter referred to as DL1 and DL4) stromal cells in the presence of IL-2, IL-7, and IL-15 and found that there were far fewer CD161⁺ ILC1s and a far greater number of CD161⁻ ILC1s regardless of whether they were co-cultured with DL1 or DL4 stromal cells (Extended data Fig. 1B-1E). The CD161⁻ ILC1s were further identified by the expression of IFNγ, Eomes, and T-bet (Extended data Fig. 1F and 1G), indicative of an ILC1 lineage. Using the same method recently reported for derived ILCs from umbilical cord blood (UCB) CD34⁺ hematopoietic stem cells (HSCs)¹⁵, we obtained ILC1s that were almost entirely CD161⁻, as demonstrated by the phenotype, including the expression of IFNγ, Eomes, and T-bet (Extended data Fig. 1H-1J). A nearly 700-fold increase from UCB CD34⁺ cells to CD161⁻ ILC1s (> 97% purity) was observed on day 28 (Extended data Fig. 1K). The cytotoxicity against LSCs was comparable between CD161⁻ ILC1s differentiated from UCB CD34⁺ HSCs and the conventional CD161⁺ ILC1s isolated from peripheral blood mononuclear cells (PBMCs; Extended data Fig. 1L). Because ILC1s are a minute population in PBMCs and it is very difficult to expand and produce adequate numbers of fresh total ILCs ex vivo for therapeutic use, these results pave the way for developing a novel adoptive cell transfer therapy, using UCB CD34⁺ HSCs to generate a readily available, allogeneic, and off-the-shelf supply of CD161⁻ ILC1s for treating diseases such as AML.

Acknowledgments

This work was supported by grants from the National Institutes of Health (CA210087, CA265095, and CA163205 to M.A.C.; NS106170, AI129582, CA247550, CA264512, CA266457 and CA223400 to J.Y.) and the Leukemia and Lymphoma Society (1364-19 to J.Y.). The authors regret that it was not possible to include many interesting studies in the field due to limited space. Images were created with BioRender.com and GraphPad Prism 9.

Author contributions

Conception and design: Z.L., J.Y., and M.A.C.

Development of methodology: Z.L., R.M., H.T.

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z.L., R.M., H.T., G.M.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z.L. and J.Z.

Writing, review, and/or revision of the manuscript: Z.L., R.M., T.B., J.Y., and M.A.C.

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.Z.

Study supervision and funding acquisition: J.Y. and M.A.C.

All authors discussed the results and commented on the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Kantarjian, H et al. Acute myeloid leukemia: current progress and future directions. Blood Cancer J 41. doi: 10.1038/s41408-021-00425-3 (2021).
Cortez, V. S., Robinette, M. L. & Colonna, M. Innate lymphoid cells: new insights into function and development. Current opinion in immunology 32, 71-77, doi:10.1016/j.coi.2015.01.004 (2015).
de Weerdt, I. et al. Innate lymphoid cells are expanded and functionally altered in chronic lymphocytic leukemia. Haematologica 101, e461-e464, doi:10.3324/haematol.2016.144725 (2016).
Kansler, E. R. et al. Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies. Nat Immunol 23, 904-915, doi:10.1038/s41590-022-01213-2 (2022).
Weizman, O. E. et al. ILC1 Confer Early Host Protection at Initial Sites of Viral Infection. Cell 171, 795-808.e712, doi:10.1016/j.cell.2017.09.052 (2017).
Nabekura, T., Riggan, L., Hildreth, A. D., O'Sullivan, T. E. & Shibuya, A. Type 1 Innate Lymphoid Cells Protect Mice from Acute Liver Injury via Interferon-γ Secretion for Upregulating Bcl-xL Expression in Hepatocytes. Immunity 52, 96-108.e109, doi:10.1016/j.immuni.2019.11.004 (2020).
Nixon, B. G. et al. Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity. Sci Immunol 7, eabi8642, doi:10.1126/sciimmunol.abi8642 (2022).
Li, Z. et al. ILC1s control leukemia stem cell fate and limit development of AML. Nat Immunol 23, 718-730, doi:10.1038/s41590-022-01198-y (2022).
Al-Matary, Y. S. et al. Acute myeloid leukemia cells polarize macrophages towards a leukemia supporting state in a Growth factor independence 1 dependent manner. Haematologica 101, 1216-1227, doi:10.3324/haematol.2016.143180 (2016).
Mussai, F. et al. Acute myeloid leukemia creates an arginase-dependent immunosuppressive microenvironment. Blood 122, 749-758, doi:10.1182/blood-2013-01-480129 (2013).
Miari, K. E., Guzman, M. L., Wheadon, H. & Williams, M. T. S. Macrophages in Acute Myeloid Leukaemia: Significant Players in Therapy Resistance and Patient Outcomes. Frontiers in cell and developmental biology 9, 692800, doi:10.3389/fcell.2021.692800 (2021).
Wunderlich, M. et al. AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3. Leukemia 24, 1785-1788, doi:10.1038/leu.2010.158 (2010).
Trabanelli, S. et al. CD127+ innate lymphoid cells are dysregulated in treatment naive acute myeloid leukemia patients at diagnosis. Haematologica 100, e257-260, doi:10.3324/haematol.2014.119602 (2015).
Goc, J. et al. Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer. Cell 184, 5015-5030.e5016, doi:10.1016/j.cell.2021.07.029 (2021).
Hernández, D. C. et al. An in vitro platform supports generation of human innate lymphoid cells from CD34(+) hematopoietic progenitors that recapitulate ex vivo identity. Immunity 54, 2417-2432.e2415, doi:10.1016/j.immuni.2021.07.019 (2021).
Tang, Z., Kang, B., Li, C., Chen, T. & Zhang, Z. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res 47, W556-w560, doi:10.1093/nar/gkz430 (2019).
Roan, F. & Ziegler, S. F. Human Group 1 Innate Lymphocytes Are Negative for Surface CD3ε but Express CD5. Immunity 46, 758-759, doi:10.1016/j.immuni.2017.04.024 (2017).
Paczulla, A. M. et al. Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion. Nature 572, 254-259, doi:10.1038/s41586-019-1410-1 (2019).
Walter, R. B., Appelbaum, F. R., Estey, E. H. & Bernstein, I. D. Acute myeloid leukemia stem cells and CD33-targeted immunotherapy. Blood 119, 6198-6208, doi:10.1182/blood-2011-11-325050 (2012).
Song, J. et al. Requirement of RORα for maintenance and antitumor immunity of liver-resident natural killer cells/ILC1s. Hepatology, doi:10.1002/hep.32147 (2021).

Human samples

Peripheral blood samples were obtained from healthy donors and patients with AML at the City of Hope National Medical Center (COHNMC). Mononuclear cells were isolated using Ficoll separation. LSCs were sorted using a BD FACSAria™ Fusion (BD Biosciences). ILCs were isolated using EasySep™ Human Pan-ILC Enrichment Kit (STEMCELL) or were sorted using a BD FACSAria™ Fusion. Umbilical cord blood leukopaks were obtained from StemCyte, Inc (Baldwin Park, Californai). CD34⁺ cells were isolated using the CD34 MicroBead Kit (Miltenyi Biotec). All healthy donors and patients with AML signed an informed consent form. Sample acquisition was approved by the Institutional Review Board at the COHNMC.

Cells and cell culture

OP9-DL1 and DL4 were maintained in DMEM GlutaMAX media supplemented with 20% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 µM β-Mercaptoethanol. Human LSCs were cultured in StemSpan™ Serum-Free Expansion Medium II with penicillin (100 U/mL), streptomycin (100 mg/mL), stem cell factor (SCF, 20 ng/ml), thrombopoietin (TPO, 20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Human ILC1s were cultured in MEMα GlutaMAX media. Media was supplemented with 10% human AB serum, IL-2 (500 IU/ml), IL-7 (20 ng/ml), and IL-15 (20 ng/ml). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2.

Flow cytometry

ILC1s from human peripheral blood were identified using surface staining with a live/dead cell viability cell staining kit and the following monoclonal antibodies: lineage (FITC-anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD20, anti-CD33, anti-CD34, anti-CD203c, anti-FceRI), CD56 (FITC, AF700 or BV421-anti-CD56), CD127 (APC-anti-CD127), CRTH2 (PE-Cy7-anti-CRTH2), and c-Kit (BV711-anti-c-Kit). Human LSCs were identified by lineage (FITC-anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD19, anti-CD20, anti-CD11b, anti-CD56, and anti-CD235a), CD45 (BV510-anti-CD45), CD34 (PE-anti-CD34), and CD38 (BV605- or PE-Cy7-anti-CD38). To examine intracellular cytokine production, intracellular staining for IFN-γ, Eomes, and T-bet was performed using a Fix/Perm kit (eBiosciences), followed by staining with a BV421-anti-IFN-γ antibody, an APC-anti-T-bet antibody, or BUV395-Eomes, respectively. All analyses were performed on a Fortessa X-20 flow cytometer (BD Biosciences) and sorting was performed using a BD FACSAria™ Fusion.

Survival analysis and correlation analysis

Survival analysis and pairwise correlation analysis of gene expression signatures were performed using GEPIA2¹⁶, based on TCGA and GTEx databases. There were 53 AML tumor samples included in the survival analysis. We established gene expression signatures for ILC1s and leukemia blasts and/or LSCs based on previous human studies^13,17−20. For each signature gene set, AML cohort was divided into high and low expression groups by median value (50% cut-off). ILC1: IFNG, TNFA, TRAIL, CD49A, NKP46, and RORA. Leukemia cell (leukemia blasts / LSCs): CD34, CD33, CD133, CD7, and CD13. Survival analyses were performed with log-rank Mantel-Cox test. Pairwise correlation analyses of gene expression signatures were performed with two-tailed Pearson correlation test.

LSCs and ILC1s in vitro co-culture assay

Human LSCs from patients with AML were labeled with 5 mM CellTrace™ Violet dye (CTV) and co-cultured with or without ILC1s (Lin⁻CD127⁺CRTH2⁻CD117⁻) isolated from the peripheral blood of healthy donors in the presence of human IL-12 (10 ng/ml) and IL-15 (100 ng/ml). For coculture assays with cytokines and antibodies, human LSCs were cocultured with or without IFNγ (10 ng/ml), TNF (10 ng/ml), anti-IFNγ antibody (10 µg/ml), or anti-TNF (10 µg/ml) antibody. For all co-culture assays, after three days of co-culture, cells were harvested and analyzed using flow cytometry. Annexin V and 7-amino-actinomycin D (7-AAD, BD Biosciences) were used to identify dead cells following the manufacturers' instructions.

In vitro colony-forming unit assay

Human LSCs were isolated from the blood of patients with AML and were cocultured with or without ILC1s isolated from the blood of healthy donors for 3 days. Cells were then plated into human methylcellulose complete media (R&D, HSC003) supplied with recombinant human SCF (50 ng/ml), human recombinant IL-3 (10 ng/ml), IL-6 (10 ng/ml), recombinant human GM-CSF (10 ng/ml), and recombinant human EPO (3 IU/ml). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 for 10–14 days. Colony numbers were counted using a microscope (Zeiss AxioCam 702).

In vivo LSC transplantation assay

For the human LSC engraftment experiment, 0.5×10⁴ human LSCs were isolated from peripheral blood or bone marrow cells of patients with AML and then transplanted via tail vein injection into sublethally irradiated (200 cGy) 6–8-week-old NOD.Cg-Prkdc^scidIl2rg^tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) purchased from the Jackson laboratory. All mice were maintained by the Animal Resource Center of COH. Mouse care and experimental procedures were performed in accordance with federal guidelines and protocols approved by the Institutional Animal Care and Use Committee at City of Hope. One day later, 5×10⁴ human ILC1s isolated from the peripheral blood of human healthy donors were injected via the tail vein into these mice. Recombinant human IL-15 (2 µg/mouse) was intraperitoneally injected into recipient mice daily for 7 days. Engraftment of human CD45⁺CD33⁺ and CD34⁺CD38⁻ cells in the blood of mice was monitored at 3 weeks.

In vitro ILC1 induction from umbilical cord blood (UCB) CD34⁺ hematopoietic stem cells

CD34⁺ cells from UCBs were sorted by FACS and were plated onto approximately 80–90% confluent OP9-DL1 or OP9-DL4 stromal cell monolayers in MEMα GlutaMAX media (Thermo Fisher Scientific). Media was supplemented with 10% human AB serum, and SCF (20 ng/ml), Flt3-L (20 ng/ml), IL-7 (20 ng/ml), IL-15 (20 ng/ml), and IL-3 (5 ng/ml) at the first week. One week later, IL-3 cytokine was removed from the media. Two weeks later, Flt3-L was reduced to 5 ng/ml. Media and cytokines were refreshed every 3–4 days by replacing half of the media containing 1× concentration of cytokines. At the end of weeks 1, 2, and 3, cells were replated onto fresh OP9-DL1 or OP9-DL4 onto six-well plates.

Statistical analysis

For continuous endpoints, Student’s t test was used to compare two independent conditions and one-way ANOVA models were used to compare three or more independent conditions. For survival data, survival functions were estimated by the Kaplan–Meier method and compared by log-rank tests. All tests were two-sided. P values were adjusted for multiple comparisons by Holm’s procedure. Data are presented as mean ± SD. Prism software v.8 (GraphPad, CA, USA) and SAS v.9.4 (SAS Institute. NC, USA) were used to perform statistical analyses. The p-values are represented as: * <0.05, ** <0.01, *** <0.001, and **** <0.0001

There is NO Competing Interest.

LietalExtendeddataFigure.pdf
Extended data Figure 1 In vitro identified and induced CD161⁻ILC1s. (A) Total ILCs were isolated from peripheral blood mononuclear cells (PBMCs) using EasySep™ Human Pan-ILC Enrichment Kit. Percentage of CD161⁺ and CD161⁻ ILCs within Lin⁻ (Lin: surface CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD56, CD203c, CD123, FcεRIα, viability marker) cells. (B) Schematic of culture conditions. Total ILCs were isolated from PBMCs and cultured on either DL1-transfected or DL4-transfected OP9 (hereafter referred to as DL1 and DL4) stromal cells in the presence of IL-2 (500 IU/ml), IL-7 (20 ng/ml), and IL-15 (20 ng/ml) at day 7 and day 14. (C) Representative flow cytometry plots of percentage (upper) and statistics (bottom) of CD161⁺ cells on day 7 and day 14. (D) Representative flow cytometry plots of percentage (upper) and bar graphs of statistics (bottom) of ILC1s, ILC2s, and ILC3s within Lin⁻CD161⁺cells on day 7 and day 14. (E) Representative flow cytometry plots of percentage (upper) and statistics (bottom) of ILC1s, ILC2s, and ILC3s within Lin⁻CD161⁻ cells on day 7 and day 14. (F) Representative expression of IFN-γ within Lin⁻CD161⁻ ILC1s at day 14, after PMA/Iono stimulation. (G) Representative expression of T-bet and Eomes within CD161⁻ILCs on day 14. (H) Schematic of culture conditions. CD34⁺ cells were isolated from UCBs and cultured on either DL1 and DL4 stromal cells in the presence of Flt3-L (20 ng/ml), SCF (20 ng/ml), IL-3 (5 ng/ml), IL-7 (20 ng/ml), and IL-15 (20 ng/ml) at day 14. (I) Percentage of CD161⁺ and CD161⁻ ILC1s, ILC2s, and ILC3s within Lin⁻ cells. (J) Representative expression of IFN-γ, T-bet, and Eomes within CD161⁻ILC1s at day 14. (K) The fold change of harvested ILC1s vs. pre-seeded CD34⁺ cells isolated from UCB and cultured with OP9-DL1 cells in the presence of Flt3-L, SCF, IL-7, IL-15, and IL-3 (20 ng/ml for all cytokines except for IL-3 with 5 ng/ml). (L) Human LSCs from peripheral blood of patients with AML were labeled with 5 mM CellTrace™ Violet dye (CTV) and co-cultured with or without various numbers of CD161⁺ILC1s isolated from the peripheral blood (PBMCs) of healthy donors or CD161⁻ILC1s differentiated from UCB-derived CD34⁺HSCs. Representative flow cytometry plots (left) and statistics of (right) the percentages of apoptotic cells identified by Annexin V and DAPI in human LSCs (n = 3 individual). Data are shown as the means ± s.d., compared among multiple groups with repeated measures by linear mixed models.
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Human ILC1s target leukemia stem cells and control development of AML

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