Patent Application
20240082398
Acute myeloid leukemia (AML) usually occurs following the successive acquisition of mutations in hematopoietic stem and progenitor cells. AML is a highly heterogeneous and aggressive malignancy characterized by morphologic and chromosomal aberrations with high mortality despite treatment. With the exception of those undergoing hematopoietic stem cell transplantation (HSCT), only a small fraction of patients are cured of the disease1. However, older adult patients are often ineligible for HSCT due to co-morbid conditions2. Due to the persistence of leukemia stem cells (LSCs) following standard chemotherapy, relapse is all but certain and highly fatal; 5-year overall survival is only 40-45% in pediatric and younger adult patients (<40 years of age) and 5-15% in older AML patients (>60 years of age)3-5. How immune cells interact with leukemia stem cells (LSCs) to prevent AML relapse is largely unknown. Therefore, identifying and exploiting the underlying mechanisms to prevent AML relapse is of significance and is an unmet medical need.
Type I innate lymphoid cells (ILC1s) play a critical role in regulating inflammation and immunity in mammalian tissues. However, their functional roles in cancer immunity and immunotherapy are less defined. This application is based in part on the surprising discovery that isolated ILC1s induce leukemia stem cell (LSC; Lin−Sca-1+c-Kit+) apoptosis, promote LSC differentiation into Lin−Sca-1+c-Kit−non-leukemic cells, suppress LSC differentiation into Lin−Sca-1−c-Kit+leukemia progenitor cells, and thereby block differentiation into terminal myeloid blasts. Without being bound by theory, ILC1s produce abundant interferon-γ (IFN-γ), particularly when stimulated by tumor cells, and ultimately suppress leukemogenesis. Also without being bound by theory, inhibition of JAK-STAT and PI3K-AKT signaling pathways in LSCs decrease the anti-leukemic effects of ILC1s. As described herein, inter alia, ILC act as anti-cancer immune cells suitable for immunotherapy. In some aspects, the ILC1s are used to treat a cancer or leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndromes (MDS)). Thus, the use of ILC1s provides a previously unknown strategy to treat cancer (e.g., AML) and/or prevent relapse of the disease.
ILC1s play critical roles in inflammation and in the early anti-viral response40,41. However, the role of ILC1s in preventing and/or promoting cancer, including AML, has not been explored42. In particular, it is unknown whether ILC1s suppress or promote cancer development. Described herein are in vitro studies as well as three different mouse models that show that the progression of AML can be controlled by ILC1s. Without being bound by theory, this is accomplished by ILC1 directly interacting with LSCs. ILC1s play dual roles in regulating LSCs, particularly in AML: 1) ILC1s induce apoptosis of LSCs; and 2) ILC1s suppress differentiation of LSCs into leukemia progenitor cells, facilitate differentiation of LSCs into non-leukemic cells, and block differentiation of LSCs into myeloid blasts. Without being bound by theory, IFN-γ mediates ILC1-induced effects on LSCs via both the JAK-STAT and PI3K-AKT signaling pathways.
As shown herein, high concentration of normal murine ILC1s induced leukemia stem cell (LSC) apoptosis. At a lower concentration, ILC1s prevented LSCs from differentiating into leukemia progenitors and promoted their differentiation into non-leukemic cells, thus blocking the production of terminal myeloid blasts. Without being bound by theory, these effects are mediated by ILC1s' ability to produce interferon-γ after cell-cell contact with LSCs. ILC1s also displayed to suppress leukemogenesis in vivo, and thus in some embodiments, disclosed herein are methods of suppress leukemogenesis comprising administering to a patient in need thereof a therapeutically effect amount of a composition comprising a population of ICL1s. In some embodiments, disclosed herein are methods of using a population of ILC1s described herein to prolong relapse-free survival in AML, prevent relapse of AML, and/or reduce the chance of relapse of AML.
AML is a highly heterogeneous and aggressive malignancy. The most commonly used therapies are chemotherapy followed by allogeneic stem cell transplantation. However, among patients who relapse, there exists a small population of leukemia-initiating cells or LSCs that ultimately proved resistant to therapy4,43. Thus, developing novel approaches to targeting LSCs offers a potential strategy to prolong relapse-free survival of AML patients. Chemotherapy and targeted therapy (e.g., tyrosine kinases inhibitors including the Food and Drug Administration (FDA)-approved drugs midostaurin and gilteritinib) can kill leukemic blasts but may also enrich LSCS44,45. As described herein, ILC1s act directly on LSCs, resulting in reduced progression of AML in vivo. Thus, expanding ILC1 cells ex vivo during times of remission or combining expanded ILC1s with an FDA-approved drug that enriches LSCs, may have a positive impact on prolonging relapse-free survival of AML patients. The methods described herein can be used alone or in combination with other treatments and methods used and known in the art to treat AML, ameliorate a symptom of AML, prolong relapse-free survival in AML, prevent or reduce the chance of relapse of AML, or kill or reduce LSCs or leukemic blasts.
IFN-γ plays important roles in anti-viral and anti-tumor immunity and has been used clinically to treat several diseases46. However, IFN-γ-based therapies have at least two limitations in the clinic that preclude routine use for the treatment of cancer patients. The first limitation is that IFN-γ cannot be delivered into local tumor sites and subsequently achieve effective concentrations in the TME (tumor microenvironment) without causing significant toxicities47-49; the second limitation is that IFN-γ is rapidly cleared from the blood after intravenous administration, further limiting the ability to achieve effective local concentrations. These disadvantages in the clinical use of IFN-γ necessitate the development of alternative methods to ensure its effectiveness in the local milieu of the marrow while limiting its toxicity. Thus, in some embodiments, the methods described herein increase the IFN-γ concentration in the TME.
Described herein, inter alia, are methods of treating AML by utilizing a cell-based source of IFN-γ to target LSCs. Although ILC1s are a minute cell population, they express abundant IFN-γ, especially when they interact with tumor cells in the TME. ILC1s also express high levels of chemokine receptors including CXCR3 and CXCR6, the receptors for CXCL9-11 and CXCL16, respectively, that are expressed by AM cells41,50. Without being bound by theory, these receptor-ligand interactions may help recruit ILC to the bone marrow or tumor sites, where the majority of LSCs reside51. Also described herein, ILC1s rapidly and persistently produce IFN-γ locally (e.g. within the TME) after contacting LSCs or more mature tumor cells, yielding sufficient doses of the cytokine to target and kill AML blasts52. Also described herein, ILC1s induce apoptosis and differentiation of LSCs within the TME. Moreover, ILC1s are associated with reducing severe progression of graft-versus-host disease (GVHD) after allogeneic HSCT in AML patients53. This suggests that ILC can control AML in different layers and at different settings through their multifaceted roles.
In some embodiments, provided herein are methods to rapidly and reproducibly expand ILC and the use of ILC for application as a cellular therapy (e.g., prolong relapse-free survival in AML patients who achieve complete remission but may carry quiescent LSCs, especially for patients ineligible for HSCT).
The IFN-γ signaling pathway is associated with several biological responses and plays an important role in innate and adaptive immunity. It can not only induce apoptosis of tumor cells51, but also activate immune cells, two processes that are crucial for defending against cancer46,55. IFN-γ induces PD-L1 expression in tumor cells including AML blast cells56 and immune cells57,58; it regulates PD-L1 expression mainly through the JAK1/2-STAT1/3-IRF1 axis in melanoma cells59. As described herein, ILC1s and recombinant IFN-γ block differentiation of LSCs into leukemia progenitor cells. The action of IFN-γ on tumors, tumor stem cells, and immune cells can induce PD-L1 expression, which can block T cell responses to tumor cells and their stem cells60, differentiation of cancer stem cells, and activation of immune cells61. The use of IFN-γ should consider all of these effects, the ability of an anti-PD-L1 antibody to block the adverse effects of IFN-γ-upregulated PD-L1. In some embodiments, the methods described herein can be sued alone or in combination with IFN-γ, cells that produce this cytokine, or mimetics thereof. In some embodiments, the methods described herein (e.g., a method of treating AML using ILC1s) can be combined with administering anti-PD-L1 antibody. The innovative methods described herein (e.g. leukemia treatment) may bring new hope to patients with AML, especially relapsed older patients who otherwise may live only for several months.
Disclosed herein, inter alia, are compositions comprising ILC1s to treat AML and regulate LSCs by inducing apoptosis, inhibiting LSC differentiation into leukemia progenitors cells, promoting LSC differentiation into a non-leukemic lineage, blocking differentiation into myeloid blasts, and increasing and prolonging IFN-γ concentrations in the TME. Also described herein is are methods of treatment comprising ILC1 cell therapy (e.g., to prolong relapse-free survival of patients diagnosed with AML).
Described herein, inter alia, are method of preparing isolated ILC1 cells, methods of preparing ex vivo expanded ILC1 cells (i.e., human ILC1 cells), and compositions comprising each. In some embodiments, described herein is a method comprising:
In some embodiments, the population of ILC1s are human. In some embodiments, the population of ILC1s are from a mouse or other mammal. In some embodiments, the population of ILC1s are isolated from blood, peripheral blood, or peripheral blood mononuclear cells (PBMCs) and are autologous to patient that is to be administered the cells. In some embodiments, the population of ILC1s comprise 30%, 40% 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% ILC1s. In some embodiments, the population of ILC1s comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% ILC1s. In some embodiments, the population of ILC1s comprise cells selected from:
In some embodiments the population of cells comprises ILC1s that are:
In some embodiments, the population of ILC1s is contacted with at least one of IL-2, IL-12, IL-15, or IL-7 (preferably human IL-2, IL-12, IL-15, or IL-7). In some embodiments, the isolated population of ILC1s is co-cultured with feeder cells. In some embodiments, the feeder cells comprise 721.221 cells or K562 cells. In some embodiments, the ILC1:feeder cell ratio is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1.
Also described herein is an isolated population of ILC1 cells, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells are selected from:
Also described herein are compositions comprising a population of isolated ILC1s, a population of ex vivo expanded ILC1s, or a population of ILC prepared by any of the methods described herein.
Provided herein, inter alia, is a method of treating a cancer or leukemia, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC1s prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of killing, eliminating, or reducing cancer cells, leukemia cells, leukemia stem cells (LSCs), leukemia progenitor cells, myeloid blasts, or cells expressing CXCL9-11 or CXCL16, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of reducing or ameliorating a symptom associated with a cancer or leukemia, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of inhibiting or reducing leukemogenesis, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC1s prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of inhibiting or reducing differentiation of LSCs into leukemia progenitor cells or myeloid blasts, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of promoting or increasing differentiation of LSCs to non-leukemic cells, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of prolonging relapse-free survival, preventing relapse, or decreasing the risk of relapse in a cancer or leukemia patient, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC prepared by any of the methods described herein, a composition described herein, or a composition comprising any of the populations of ILC1 described herein.
Provided herein, inter alia, is a method of increasing prolonging INF-γ concentration or prolonging INF-γ presence in a tumor microenvironment, the method comprising administering to a subject in need thereof a population of isolated ILC1s, a population of ex vivo expanded ILC1s, a population of ILC1s prepared by any of the methods described herein, a composition described herein, or a composition comprising any of population of ILC1 described herein.
In some embodiments, the isolated ILC or ex vivo expanded ILC are human. In some embodiments, the isolated ILC1s or ex vivo expanded ILC1s are autologous or allogenic. In some embodiments, the autologous ILC1s are isolated from the patient during remission or any cancer free time. In some embodiments, the population of isolated ILC1s or ex vivo expanded ILC1 cells or a composition described herein is administered in single or repeat dosing. In some embodiments, an effective amount of the population of isolated ILC or ex vivo expanded ILC1 cells or a composition described herein is administered.
In some embodiment, the population of isolated ILC or ex vivo expanded ILC1 cells or a composition described herein is administered locally or systemically. In some embodiments, the population of isolated ILC1s or ex vivo expanded ILC1s or a composition described herein is infused or administered intravenously, locally or directly injected, injected into tumor microenvironment, or administered intratumorally. In some embodiments, at least one symptom of a cancer or leukemia is reduced, ameliorated, or relieved. In some embodiments, the leukemia is any of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndromes (MDS). In some embodiments, the population of isolated ILC1s or ex vivo expanded ILC1 cells or a composition described herein is administered before remission, during remission, or during relapse. In some embodiments, the population of isolated ILC1s or ex vivo expanded ILC1 cells or a composition described herein is administered before, after, or in combination with one or more of IFN-γ (or a nucleic acid encoding IFN-γ), a cytokine (or a nucleic acid encoding a cytokine), IL-15 (or a nucleic acid encoding IL-15), an anti-PD-L1 antibody or a PD-L1 inhibitor, an anti-PD-1 antibody or a PD-1 inhibitor, a chemotherapy, a kinase inhibitor (e.g., midostaurin and gilteritinib), or radiation therapy.
Also described herein are ILC1s harboring a recombinant nucleic acid molecule encoding a protein of interest.
For example, the recombinant nucleic acid can encode human IL-15 (Gene ID: 3600; GenBank® Accession: NP 000576.1). For example, it can encode amino acids 1-162, 30-162, 49-162 or a functional portion thereof of SEQ ID NO: 1
For example the recombinant nucleic acid can encode human IL-12 (IL-12 subunit A: Gene ID: 3592; GenBank® Accession: NP 000873 and IL-12 subunit B Gene ID: 3593; GenBank® Accession: NM 002187.2). For example, it can encode amino acids 1-253, 57-253 or a functional portion thereof of SEQ ID NO: 2
and amino acids 1-328, 23-328 or a functional portion thereof of SEQ ID NO: 3).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
X-axis represents the rank ordering (ILC1s vs. Ctrl) of all genes (Ctrl means no treatment). (D) shows GSEA plots showings enrichment of some target genes in LSCs after co-cultured with IFN-γ. The X-axis represents the rank ordering (IFN-γ vs. Ctrl) of all genes. (E-F) are heatmaps showing RNA differential expression of downstream genes of IFN-γ. All data are shown as mean±SD. P values were calculated by either one-way ANOVA or student's t test. *p<0.05, **p<0.01, ****p<0.0001.
untreated Ctrl). Left panel shows signaling pathways downregulated in LSCs. Right panel shows signaling pathways upregulated in LSCs (n=3). (C) Gene Set Enrichment Analysis (GSEA) plots showing enrichment of selected target genes in LSCs co-cultured with ILC1s. The rank orders (ILC1 vs. Ctrl) of all the genes (n=3) are shown on the X-axis. d-f, Differential expression of RNA from Akt3 (D), Jak2 (E), and Stat1, and Stat2 (F) genes. Results are expressed as means compared with the Ctrl (n=3). (G-H) Mouse LSCs labeled with CTV were treated with or without the indicated JAK and AKT inhibitors for 30 min and then co-cultured with or without WT or IFN-γ−/− ILC1s in the presence of IL-12 (10 ng/ml) and IL-15 (100 ng/ml) for 3 days. Flow cytometry plots (G) and statistics of absolute cell numbers (H) of Lin−Sca-1+c-Kit+, Lin−Sca-1−c-Kit+, Lin−Sca-1+c-Kit−, and Lin−Sca-1−c-Kit− cells are shown (n=3). Data are presented as mean±s.d.; P values were calculated by one-way ANOVA models. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. NS, not significant.
injected into C57BL/6J mice. Twenty-one days later, the production of IFN-γ and TNF-α by ILC1s from the liver (D), bone marrow (E), and spleen (F) of those normal mice or mice with AML are shown (n=5). (G) GSEA plot shows the relative abundance of genes involved in the TNF-α-NF-κB signaling pathways in liver ILC isolated from mice with AML or normal mice (n=3). All data (D-F) are shown as mean±s.d.; P values were calculated by Student's t test. *P<0.05; **P<0.01; NS, not significant.
Lin−CD56−CD127+c-Kit−CRTH2−. (F) Gating strategy for flow cytometry analysis of human LSCs. Lineage markers: CD2, CD3, CD4, CD8, CD14, CD16, CD19, Mac-1, CD56, and CD235a. Human LSCs were defined as Lin−CD45dimCD34+CD38−.
Innate lymphoid cells (ILCs) are a heterogeneous population of non-B and non-T lymphocytes that originate from the common lymphoid progenitor (CLP) and lack antigen-specific receptors. ILCs can be classified into three groups based on the unique cytokines that they produce and the transcription factor signatures that drive their differentiation: group 1 ILCs (comprised of natural killer [NK] cells and type I innate lymphoid cells [ILC1s]), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s)6. ILC1s, which usually reside in the liver, produce the cytokines IFN-γ, granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-α, and TNF-related apoptosis inducing ligand (TRAIL), and express T-BET but lack expression of EOMES. ILC2s produce the cytokines IL-4, IL-5, and IL-13 and express the transcription factor GATA3. ILC3s produce the cytokines IL-22 and IL-17A and express the retinoic acid-related orphan receptor γt (RORγt) transcription factor7,8. Recent studies reported that ILCs, especially ILC2s9,10 and ILC3s11-13, play a key role in antivirus or antimicrobial immune response, tumor surveillance, and tumorigenesis. However, no available studies have elucidated the interaction of ILC1s with tumor cells, in particular cancer stem-like cells or LSCs, and the relevance of this interaction to anti-tumor response in patients with a cancer such as AML.
In the present study, the inventors discovered that ILC1s target LSCs in AML. They discovered that ILC1s isolated from normal mice or healthy humans induce LSC apoptosis, mainly via secretion of IFN-γ, while in AML, these multifaceted functions of ILCs were impaired. They performed a series of functional and mechanistic studies to characterize the important roles that ILC1s play in inhibiting LSC differentiation into leukemia progenitor cells, blocking differentiation into terminal myeloid blasts, and as a result, suppressing leukemogenesis.
This work demonstrated that ILC1s isolated from normal mice or healthy humans induced LSC apoptosis. Further, normal ILC1s target LSCs to suppress leukemogenesis by preventing their differentiation into leukemia progenitor, thus blocking their differentiation into terminal myeloid blasts. Without being bound by theory, these effects occurred via the production of interferon-γ by ILC1s. Moreover, ILC produced more IFN-γ than NK cells through the receptors DNAM-1 and IL-7R interacting with LSCs. Because these functions are impaired in AML, ILC1s can no longer effectively target LSCs, which can then differentiate into leukemia cells. Collectively, these data define an essential protective role for ILC1s in AML: inducing apoptosis and targeting differentiation of LSCs.
The methods described herein include methods for the treatment of disorders associated with cancer or leukemia. In some embodiments, the disorder is a cancer or leukemia (e.g., Acute lymphocytic leukemia (ALL), Acute myeloid leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronic myeloid leukemia (CML), Hairy cell leukemia (HCL), or Myelodysplastic syndromes (MDS)). Generally, the methods include administering a therapeutically effective amount of ILC1s as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer or leukemia. ILC1 treatment results in elimination, killing, or reducing cancer or leukemia cells; thus, ILC1 treatment can result in a reduction in or prevention of relapse of the cancer or leukemia and a prolonged survival or prolonged relapse-free survival. Administration of a therapeutically effective amount of a composition described herein for the treatment of a condition associated with cancer or leukemia will result in decreased cancer or leukemia cells, increased IFN-γ (e.g., in the tumor micro environment (TME), and/or prolong survival.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
The following materials and methods were used in the Examples set forth herein.
Peripheral blood (PB) and bone morrow samples from healthy and AML individuals were obtained from donors at the City of Hope National Medical Center (COHNMC). Mononuclear cells were isolated using Ficoll separation. Lin−CD34+CD38− cells were sorted by Aria Fusion III. Lin−CD45dimCD34+CD38− cells were sorted using a BD FACSAria™ Fusion (BD Biosciences). All patients with AML and healthy donors signed an informed consent form. Sample acquisition was approved by the Institutional Review Boards at the COHNMC.
C57BL/6J, Rag2−/−γc−/−, MllPTD/WT/Flt3ITD/ITD, IL-15 transgenic, IFN-γ−/− and TNF-α−/− mice were maintained by the Animal Resource Center of City of Hope. 8 to 12-week-old Rag2−/− 665 c−/− or C57BL/6J mice of both sexes were used as recipients for AML cell transplantation. MllPTD/WT/Flt3ITD/ITD mice of both sexes were used as donor mice. Mouse care and experimental procedures were performed in accordance with federal guidelines and protocols approved by the Institutional Animal Care and Use Committee at the City of Hope.
C57BL/6J (B6, CD45.2), Rag2−/− γc−/−, TNF-α−/− and CD45.1 (B6.SJL-PtprcaPepcb/BoyJ) were purchased from the Jackson Laboratory. MllPTD/WT: Flt3ITD/ITD mice 24 and IL-15 transgenic mice34 on the B6 background were generated as described previously. All mice were maintained by the Animal Resource Center of COH. Six- to twelve-week-old CD45.2 and CD45.1 mice of both sexes were used as recipients for AML cell transplantation; MllPTD/WT: Flt3ITD/ITD mice with AML of both sexes were used as donor mice. 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.
Human LSCs were cultured in StemSpan™ SFEM II (Stem cell, USA) with penicillin (100 U/mL) and streptomycin (100 mg/mL). Stem cell factor (SCF, 20 ng/ml), thrombopoietin (TPO, 20 ng/ml), erythropoietin (EPO, 20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Mouse LSCs were cultured in IMDM with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), SCF (20 ng/ml), TPO (20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Human and mouse ILC1s or NK cells were cultured in RPMI 1640 with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), IL-12 (10 ng/ml), and IL-15 (100 ng/ml). Mouse AML cell lines (C1498) were cultured in RPMI 1640 with 10% FBS, penicillin (100
U/mL) and streptomycin (100 mg/mL). Cultures were incubated at 37° C. in a humidified atmosphere of 5% CO2. All cell lines are from American Type Culture Collection (ATCC). All cytokines are from PeproTech.
ILC1s from human peripheral blood were identified by a surface stain including a live/dead cell viability cell staining kit (Invitrogen) and the following monoclonal antibodies: lineage (FITC-conjugated 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 conjugated anti-CD56), CD127 (APC-conjugated anti-CD127), CRTH2 (PE-Cy7-conjugated anti-CRTH2), and c-Kit (PE-conjugated anti-c-Kit). ILC from mice were identified by a surface stain and the following monoclonal antibodies: lineage (PE-Cy7-conjugated anti-CD3 and anti-CD19), NK1.1 (BV510-conjugated anti-NK1.1), NKp46 (BV421, FITC or AF647-conjugated anti-NKp46), CD49b (BUV395 or PE-conjugated anti-CD49b), and CD49a (BV711-conjugated anti-CD49a). Human LSCs were identified by lineage (FITC-conjugate anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD19, anti-CD20, anti-Mac-1, anti-CD56, and anti-CD235a), CD45 (BV510-conjugated anti-CD45), CD34 (BV510-conjugate anti-CD34), and CD38 (BV605 conjugated anti-CD38). Mouse LSCs were identified by lineage (PE-Cy7 conjugated anti-CD3, anti-CD19, anti-B220, anti-Ly6G/C, anti-Mac-1, anti-CD11b, and anti-Ter119), Sca-1 (PE-CF594 or BV510-conjugate anti-Sca-1), and c-Kit (BV711-conjugated anti-c-Kit). Mouse long-term hematopoietic stem cells (LTHSCs), short-term hematopoietic stem cells (STHSCs), and multipotent progenitors (MPP) 1 and 2 were identified by lineage, Sca-1, c-Kit, Flt3 (APC-conjugated anti-Flt3), CD150 (PE-conjugated anti-CD150), and CD48 (FITC or BV786-conjugated anti-CD48). The expression of CD155 and CD112 on mouse LSCs was identified by APC-conjugated anti-CD155 and BV786-conjugated anti-CD112, respectively. The expression of DNAM-1 and IL-7R on mouse ILC1s was identified by BV421-conjugated anti-DNAM-1 and PerCP-Cy5.5-conjugated anti-IL-7R, respectively. The expression of CD45.1 and CD45.2 were identified by BV605-conjugated-anti-CD45.1 and APC/Fire™ 750- or FITC- conjugated-anti-CD45.2, respectively. Human ILC1s were gated by Lin−CD56−CD127+CRTH2−c-Kit−. Mouse ILC1s were gated by Lin−NK1.1+NKp46+CD49b−CD49a+. Mouse NK cells were gated by Lin−NK1.1+NKp46+CD49b+CD49a−. Human LSCs were gated by Lin−CD45dimCD34+CD38−. Mouse LSCs were gated by Lin−Sca-1+c-Kit+. Mouse LTHSCs were gated by Lin−Sca-1+c-Kit+Flt3−CD150+CD48−. Mouse STHSCs were gated by Lin−Sca-1+c-Kit+Flt3−CD150−CD48−. Mouse MPPls were gated by Lin−Sca-1+c-Kit+Flt3−CD150−CD48+. Mouse MPP2s were gated by Lin−Sca-1+c-Kit+Flt3−CD150+CD48+. Myeloid cells were gated by Mac-1+Gr-1+. To examine intracellular cytokine production, mouse ILC1s or NK cells co-cultured with or without LSCs were stimulated by IL-12 (10 ng/ml) and IL-15 (100 ng/ml) or IL-7 (100 ng/ml) for 4 h or 12 h in the presence of BD GolgiPlug™. Human ILC were gated by Lin−CD56−CD127+CRTH2−c-Kit−. Mouse ILC1s were gated by Lin− NK1.1+NKp46+CD49b−CD49a+. Human LSCs were gated by Lin−CD34+CD38−. Mouse LSCs were gated by Lin−Scal-1+c-Kit+. Intracellular staining for TNF-α or IFN-γ was performed using a Fix/Perm kit (eBiosciences), followed by staining with an AF700-conjugated anti-TNF-α antibody or a BV786-conjugated anti-IFN-γ antibody, respectively. All analyses were performed on a Fortessa X-20 flow cytometer (BD Biosciences) and sorting was performed using Aria Fusion III instruments (BD Biosciences) or a BD FACSAria™ Fusion (BD Biosciences).
To isolate ILC or NK cells from mouse liver, we washed harvested liver and pressed it through a 100 μm mesh to make single cells, which were washed once with PBS. The cells were re-suspended in 40% Percoll (Sigma-Aldrich) and then gently overlaid on 70% Percoll, followed by centrifugation according to the manufacturer's instructions. Mononuclear cells (MNCs) were collected from the interphase and washed twice with PBS. The washed MNCs were stained with anti-CD3, anti-CD19, anti-NK1.1, anti-NKp46, anti-CD49b, and anti-CD49a antibodies. Thirty minutes later, the cells were washed 3 times and then sorted using BD FACSAria™ Fusion.
To isolate ILC1s from human peripheral blood, we diluted blood cone samples 1:1 with phosphate-buffered saline (PBS). We layered the blood on the top of Ficoll-Paque (GE Healthcare), and centrifuged it according to the manufacturer's instructions. The mononuclear cell fraction was aspirated and washed with PBS, and then the red blood cells were lysed. The mononuclear cells were stained with lineage (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD20, anti-CD33, anti-CD34, anti-CD203c, anti-FceRI, and anti-CD56), anti-CD127, anti-CRTH2, and anti-c-Kit antibodies. Thirty minutes later, the cells were washed 3 times and then sorted using BD FACSAria™ Fusion.
A total of 2,000 LSCs from AML patients labeled with CTV were co-cultured with different numbers of human ILC1s supplemented with human IL-12 (10 ng/ml) and IL-15 (100 ng/ml). After 3 days of co-culture, cells were harvested and analyzed by flow cytometry. 7-AAD was used to identify dead cells. For mouse LSCs co-culture assay, 2,000 LSCs from MllPTD/WT/Flt3ITD/ITD mice labeled with CTV were co-cultured with different numbers of mouse ILC1s supplemented with mouse IL-12 (10 ng/ml) and IL-15 (100 ng/ml). After 3 days of co-culture, cells were harvested and analyzed by flow cytometry. 7-AAD was used to identify the dead cells. For the co-culture of LSCs and ILC1s using the transwell co-culture system, 2,000 human or mouse LSCs were seeded in the lower chamber while different numbers of human or mouse ILC1s were seeded in the upper chamber. After 3 days, cells were harvested and analyzed by flow cytometry. 7-AAD was used to identify the dead cells. For co-culture assay with cytokines and antibodies, 2,000 human or mouse LSCs were co-cultured with different doses of human or mouse TNF-α, IFN-γ, anti-TNF-α (10 μg/ml) Ab, or anti-IFN-γ Ab (10 μg/ml). Three days after the co-culture, cells were harvested and analyzed by flow cytometry. 7-AAD was used to identify the dead cells.
For mouse LSC co-culture assays, LSCs from MllPTD/WT: Flt3ITD/ITD mice with AML were labeled with 5 mM CellTrace Violet (CTV, Thermo Fisher Scientific, USA) and co-cultured in the presence of mouse IL-12 (10 ng/ml) and IL-15 (100 ng/ml) with various numbers of ILC1s or NK cells isolated from liver of normal mice or mice with AML. For human LSC co-culture assays, LSCs from patients with AML were labeled with 5 mM CTV and co-cultured in the presence of human IL-12 (10 ng/ml) and IL-15 (100 ng/ml) with various numbers of ILC1s isolated from peripheral blood of healthy donors or patients with AML. For co-culture of LSCs and ILC1s in the Transwell co-culture system, LSCs were seeded in the lower chamber of a 96-well Transwell plate, while varying numbers of mouse ILC1s were seeded in the upper chamber. For co-culture assays with cytokines and antibodies, mouse or human LSCs were co-cultured with various doses of mouse TNF-α (0.25 μg/ml, 0.5 μg/ml, 0.75 μg/ml, and 1 μg/ml), mouse IFN-γ (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 0.25 μg/ml, 0.5 μg/ml, 0.75 μg/ml, and 1 μg/ml), anti-TNF-α (10 μg/ml) antibody, or anti-IFN-γ antibody (10 μg/ml). For all co-culture assays, cells were harvested after 3 days and analyzed using flow cytometry. 7-amino-actinomycin D (7-AAD, BD Biosciences) was used to identify dead cells following the manufacturers' instructions. Cell images were taken by microscope (ZEISS).
For the LSC differentiation assay, 1,000 LSCs were isolated from MllPTD/WT/Flt3ITD/ITD mice and then were co-cultured with or without 500 ILC1s isolated from normal mouse livers for 1 to 4 days. LSCs were isolated from MllPTD/WT: Flt3ITD/ITD mice with AML and co-cultured with or without ILC1s isolated from liver of normal mice or MllPTD/WT: Flt3ITD/ITD mice with AML for 1 to 4 days in the presence or absence of anti-TNF-α (10 μg/ml) antibody or anti-IFN-γ antibody (10 μg/ml)Cells were harvested and analyzed by flow cytometry.
In all the transplantation experiments, recipient mice were placed on sulfatrim-based food (5053/.025% Tri/.1242% Sulf ½ IRR; Catalogue number: 5W8F; TestDiet, Richmond, IN) post-transplantation to avoid any infection/toxicity-associated with irradiation. 1×106 support bone marrow cells depleted of NK1.1+NKp46+ cells from IL-15 transgenic mice were transplanted by tail vein injection with 30,000 LSCs that were obtained from MllPTD/WT/Flt3ITD/ITD transgenic mice into lethally (900 cGy, 96 cGy/min, γ-rays) irradiated 6- to 10-week-old B6.SJL (Ly5.1) or C57BL/6 (CD45.2) recipient mice. Next, WT or IFN-γ ILC1s, which were purified from C57BL/6 mice, were injected by tail vein injection (30,000 cells/mouse) into these mice. In some experiments, 0.5 μg per mice animal-free recombinant murine IFN-γ were intraperitoneally injected into recipient mice for 10 days. The number of white blood cells (WBCs), neutrophils and monocytes were monitored every 3 weeks. In Rag2−/− γc−/− mice experiments, 30,000 LSCs were transplanted into 200 cGy irradiated 6- to 10-week-old Rag2−/− γc−/− mice, followed by multiple injection of ILC1s. The number of WBCs, neutrophils and monocytes were monitored every 3 weeks. Leukemic mice were euthanized by CO2 inhalation when they showed signs of systemic illness.
0.5×106 bone marrow cells from CD45.1 WT mice or bone marrow cells isolated from IL-15 transgenic mice (CD45.2) depleted of NK1.1+ NKp46+ cells were transplanted by i.v injection with 3×104 LSCs obtained from MllPTD/WT: Flt3ITD/ITD mice with AML into lethally (900 cGy, 96 cGy/min, 665 -rays) irradiated 6- to 12-week-old C57BL/6J (CD45.1) recipient mice. Next, WT or IFN-γ−/− ILC1s (CD45.2), which were purified from WT or IFN-γ−/− C57BL/6J mice, were injected via i.v. into recipient mice (3×104 cells/mouse). In some experiments, animal-free recombinant murine IFN-γ (0.5 μg/mouse) was i.p. injected into recipient mice daily for 7 days. For all transplantations, the numbers of WBCs, LSCs, or immature blast cells in peripheral blood were counted at the indicated times using Element HT5 hematology analyzer and flow cytometry. (Peripheral blood was also collected for making blood smear slides). Blood smear slides were stained with Wright-Giemsa (Polysciences). Leukemic mice were euthanized using CO2 inhalation when they showed signs of systemic illness.
In Vivo HSC Transplantation Assay In all transplantation experiments, recipient mice were fed with sulfatrim-based food (Catalogue number: 5W8F; TestDiet, Richmond, IN) post-transplantation to avoid any infection/toxicity associated with irradiation. 3×104 HSCs were isolated from bone marrow cells of normal CD45.2 mice and i.v. co-injected with 5×105 CD45.1 bone marrow cells (as support cells) into lethally irradiated (900 cGy) 6- to 12-week-old C57BL/6J (CD45.1) recipient mice. One day later, 3×104 ILC1s (CD45.2) isolated from the liver of normal mice were i.v. injected into these recipient mice. The LSKs, Lin−Sca-1−c-Kit+ cells, Lin−Sca-1+c-Kit− cells, STHSCs, LTHSCs, MPP1, MPP2, Mac-1+Gr-1+ cells, and WBCs derived from donor mice were analyzed 3 weeks post HSC transplantation using Element HT5 hematology analyzer (Heska, USA) and flow cytometry (BD Biosciences).
ILC1s were co-cultured with LSCs at a ratio of 1:1 or 1:2 for 6 h. Next, 100 μl of Caspase-Glo 3/7 reagent was added to each well. Plates were then shaken at 300 rpm for 1 min, incubated for 60 min at room temperature, and then read on a luminometer (Promega, Glomax). Background luminescence was determined with 100 μl of culture medium without cells and subtracted before fold changes were calculated.
Mouse ILC1s or NK cells were sorted from the liver of normal mice or mice with AML and then were co-cultured with or without LSCs for 12 h in the presence of IL-12 (10 ng/ml) plus IL-15 (100 ng/ml).
For stimulation by anti-DNAM-1 or anti-IL-7R neutralizing antibody or isotype IgG control, mouse ILC1s or NK cells were sorted from the liver of normal mice and co-cultured with or without an anti-DNAM-1 (10 μg/ml) or anti-IL-7R neutralizing antibody (10 μg/ml) at 5% CO2 and 37° C. in RPMI-1640 culture medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), IL-12 (10 ng/ml), and IL-15 (100 ng/ml). Thirty minutes later, LSCs were added at an equal ratio to some of the cultures of the ILC1s or NK cells and then co-cultured for 12 h. For stimulation with recombinant mouse IL-7, mouse ILC1s or NK cells were sorted from the liver of normal mice and then were treated with or without recombinant mouse IL-7 (100 ng/ml) for 12 h at 5% CO2 and 37° C. in RPMI-1640 culture medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), IL-12 (10 ng/ml), and IL-15 (100 ng/ml).
For all of the above stimulation assays, BD GolgiPlug™ was added to the cultures 4 h before cells were collected. Then cells were harvested, washed, and stained for surface molecules and intracellular IFN-γ. Percentages of IFN-γ+ ILC1s or NK cells were calculated by flow cytometry.
In vivo, NK cells and ILC1s were depleted by i.p. injection with 200 μg/mouse anti-mouse NK1.1 antibody (clone PK136; BioXcell, USA); NK cells alone were depleted by i.p. injection with 40 μl/mouse anti-asialo-GM1 antibody (clone Poly21460; BioLegend, USA). To maintain the depletion, the same injections were given on days 7, 14, and 21.
For mouse ILC1 RNA-sequencing, mouse ILC1s were sorted from the liver of normal mice or mice with AML using BD FACSAria™ Fusion. For LSC RNA-sequencing, 2,000 mouse LSCs sorted from MllPTD/WT: Flt3ITD/ITD mice with AML were co-cultured with 1,000 ILC or treated with 10 ng/ml IFN-γ for 3 days; then the LSCs were re-sorted using BD FACSAria™ Fusion. Total RNA was isolated from ILC1s or LSCs using a miRNeasy mini kit (QIAGEN). PolyA RNA-seq was performed in the Integrative Genomics Core of City of Hope National Medical Center. SMART-Seq® Ultra Low Input RNA Kit for Sequencing-v4 (Takara Bio) was used for getting double-strand cDNA from each sample with 2 ng of input total RNA. The resulting cDNA was sheared using a Covaris LE220 sonicator. The sheared DNA was used for to prepare a sequencing library, using a KAPA HyperPrep Kit. The final libraries were quantified using the Qubit Assay Kit (Thermo Fisher Scientific) and Bioanalyzer (Agilent). Sequencing was performed using the single-read mode of 51 cycles of read1 and 7 cycles of index read with V4 reagents on a Hiseq 2500 system (Illumina). Real-time analysis (RTA) 2.2.38 software was used to process the image analysis and base calling.
For quantitative (q)RT-PCR and regular PCR analyses, RNA was isolated from 1,000 cells using a miRNeasy mini kit (QIAGEN) and reverse-transcribed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TAKARA). qPCR reactions were run on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) using SYBR Green reagents (Thermofisher). Values were normalized to 18s rRNA expression levels. qPCR analysis was conducted to assess the expression of mouse Bak1 (Forward: 5′-CAGCTTGCTCTCATCGGAGAT-3′, Reverse: 5′-GGTGAAGAGTTCGTAGGCATTC-3′), human Bak1 (Forward: 5′-GTTTTCCGCAGCTACGTTTTT-3′, Reverse:5′-GCAGAGGTAAGGTGACCATCTC-3′), and 18S rRNA (Forward: 5′-GTAACCCGTTGAACCCCATT-3′; Reverse: 5′ -CCATCCAATCGGTAGTAGCG-3′). Regular PCR reactions to determine the expression of mouse Il7 (Forward: 5′-TTCCTCCACTGATCCTTGTTCT-3′, Reverse: 5′-AGCAGCTTCCTTTGTATCATCAC-3′) were performed on a ProFlex PCR System (Applied Biosystems) using 2xMyTaq Red Mix (Meridian Bioscience).
In Vitro Kinase Inhibitor Experiments
LSCs isolated from spleen of MllPTD/WT: Flt3ITD/ITD mice with AML were treated with the JAK2 inhibitor AZD1480 (10 nM), the JAK1/2/3 inhibitor decernotinib (VX-509, 10 nM), or the AKT inhibitor afuresertib (10 nM) for 30 min. Then LSCs were co-cultured with ILC1s isolated from liver of WT or IFN-γ−/− mice labeled with CTV at a ratio of 4:1, or treated with IFN-γ (10 ng/ml). Three days later, cells were harvested and analyzed using flow cytometry.
Cell supernatants were collected and analyzed for cytokine content by ELISA according to the manufacturer's protocols. LSCs isolated from the peripheral blood of patients with AML were co-cultured with the ILC1 s isolated from healthy donors or patients with AML in the presence of IL-12 (10 ng/ml) and IL-15 (100 ng/ml) for 3 days. Levels of IFN-γ in culture supernatants were measured using the human IFN-γ Quantikine ELISA Kit (Cat# DIF50C, R&D). Samples for each condition were assayed in three duplicates.
In vitro the Colony Forming Cell (CFC) Assay
1000 LSCs were obtained from MllPTD/WT/Flt3ITD/ITD mouse spleens and co-cultured with or without 500 ILC1s for 3 days. Cells were then plated into mouse methylcellulose complete media (R&D, HSC007) supplied with human transferrin (200 μg/ml), recombinant human insulin (10 μg/ml), recombinant human SCF (50 ng/ml), murine recombinant IL-3 (10 ng/ml), IL-6 (10 ng/ml) and recombinant mouse Epo (5 IU/ml). Cultures were incubated at 37° C. in a humidified atmosphere of 5% CO2 for 10-14 days. Colony numbers were counted.
LSCs were obtained from MllPTD/WT: Flt3ITD/ITD mouse spleen and co-cultured with or without WT, IFN-γ−/− or TNF-α−/− ILC1s for 3 days. Cells were then plated into mouse methylcellulose complete medium (R&D, HSC007) supplied with human transferrin (200 μg/ml), recombinant human insulin (10 μg/ml), recombinant human SCF (50 ng/ml), murine recombinant IL-3 (10 ng/ml), IL-6 (10 ng/ml), and recombinant mouse EPO (5 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
mRNA Isolation and qPCR
Total mRNA was isolated using the RNeasy mini kit (QIAGEN) according to manufacturer's instructions. mRNA purity and quantity were determined with NanoDrop (Thermo Scientific) before RT-PCR and RNA-seq analysis. For RT-PCR, mRNA samples were reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Scientific).
2,000 mouse LSCs were co-cultured with 1,000 ILC1s or treated with 1 ng/ml IFN-γ for 3 days, and then the LSCs were sorted using BD FACSAria Fusion. Total RNA of LSCs were isolated using miRNeasy mini kit (QIAGEN). SMART-Seq® Ultra Low Input RNA Kit for Sequencing-v4 was used for generating amplified double strand cDNA from each sample with 2 ng of input total RNA according to the manufacturer's protocol. The resulting double-stranded cDNA sheared with Covaris LE220 with the setting of DNA fragment size of 200 bp peak. The sheared DNA was used for sequencing library preparation by using KAPA HyperPrep Kits. The final libraries were quantified with qubit and bioanalyzer. The sequencing was performed with the single read mode of 51 cycles of read1 and 7 cycles of index read with V4 reagents on Hiseq2500. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis and base calling.
1,000 LSCs isolated from spleen of MllPTD/WT/Flt3ITD/ITD mice were treated with decernotinib (VX-509, 10 μM), AZD1480 (10 μM), or afuresertib (10 nM) for 30 min. Then 500 mouse ILC1s isolated from liver of WT or IFN-γ−/− mice labeled by CTV or IFN-γ (10 ng/ml) were cocultured with LSCs. Three days later, cells were harvested and analyzed by flow cytometry.
Prism software v.8 (GraphPad, CA, USA) was used to perform statistical analysis. Two group comparisons were performed with a two-tailed Student's t-test; multiple group comparisons were performed with a one-way ANOVA test with a multiple comparisons option. For Kaplan-Meier survival curve analysis, the comparisons were performed using a log-rank (Mantel-Cox) test. 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 repeated measures over time, linear mixed models were used to account for the variance and covariance structure. Mouse survival was 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. For RNA-seq analysis, sequencing reads were trimmed from sequencing adapters using Trimmomatic74 and polyA tails using FASTPT75, and then mapped back to the mouse genome (mm10) using STAR (v. 020201)76. The gene-level count table was created by HTSeq (v.0.6.0)77 and normalized by the TMM78 method. General linear models based on negative binomial distributions (R package “EdgeR”) were used to compare gene expression levels between two specific cell types. Genes with an FDR-adjusted p-value less than 0.05 and a fold change (FC) greater than 1.5 (upregulated) or less than 0.7 (downregulated) were considered as differentially expressed genes (DEG). Pathway and gene set enrichment analyses were performed using the GSEA79,80 program, which runs the GSEAPreranked algorithm on a ranked list of genes. 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
This example investigates the function of ILC1s in AML, or in cancer in general, which is largely unknown. Using mouse models of decreased production of IFN-γ and TNF-α in mice with AML compared to control mice show the function of ILC1s isolated from the liver was impaired (
To investigate if ILC1s have an adverse effect on the genesis of AML, we conducted cell lysis analyses on AML cells after exposure to ILC1s. Sorted ILC1s from the livers of normal mice were co-cultured for 3 days with splenic LSCs (Lin -Sca-1+c-Kit+ cells)20,21 isolated from the MllPTD/WT/Flt3ITD/ITD AML mouse model, previously generated and characterized by our group . Surprisingly, LSCs were lysed by ILC1s (
Murine leukemia stem cells (LSCs or Lin−Sca-1+c-Kit+ cells) are found mainly in bone marrow (BM) and spleen in AML20,21. Since ILC mainly reside in the liver, to investigate whether LSCs also reside in the liver of AML mice, we isolated LSKs from the liver of normal mice and MllPID/WT: Flt3ITD/ITD mice with AML22 and then i.v. injected them into immunodeficient Rag2−/−γc−/− mice. We observed that all immunodeficient Rag2−/−γc−/− mice injected with LSKs isolated from the liver of normal mice lived, while all immunodeficient Rag2−/−γc−/− mice injected with LSKs isolated from the liver of mice with AML died, suggesting that LSCs are present in the liver of mice with AML (
Using a mouse model of AML (C1498 cells i.v. injected into C57BL/6J mice)18, we noted that the function of ILC (Lin−NK1.1+NKp46+CD49b−CD49a+) (
ILC1s, which lack cytolytic activity, primarily function as immunoregulatory cells via their secretion of cytokines such as IFN-γ and TNF-α23. To determine whether their production of either cytokine affects leukemogenesis, ILC1s and LSCs were co-culture in the presence of neutralizing antibodies against IFN-γ or TNF-α. In both mouse (
To determine whether cell-cell contact is required for induction of LSC apoptosis by ILC1s, ILC with LSCs were co-cultured using a transwell, in which ILC1s and LSCs were seeded in the upper and lower chambers, respectively. After three days of co-culture, ILC1s did not induce LSC apoptosis when separated by the transwell chamber (
The co-culture experiment was repeated using normal ILC1s and LSCs in the presence of neutralizing antibodies against IFN-γ or TNF-α. In that mouse experiment, neutralizing IFN-γ, but not TNF-α, prevented ILC1s from mediating the death of LSCs (
Initiation and differentiation of LSCs into leukemia progenitor cells drives the progression of AML24,25. This example investigates the effects of ILC1s on the process of AML cell differentiation. LSCs isolated from the spleen of MllPTD/WT/Flt3ITD/ITD AML mice were co-cultured with ILC1s isolated from the livers of mice for 1, 2, 3, and 4 days. On days 2, 3, and 4, the percentage of Lin−Sca-1−c-Kit+ leukemia progenitor cells (LS−K+ cells) was significantly lower in the group co-cultured with ILC compared to the group cultured without ILC (
ILC1s inhibit differentiation of LSCs into LS−K+ leukemia progenitor cells while promoting differentiation of LSCs into non-leukemic LS+K− cells. To determine how ILC1s inhibit differentiation of LSCs into LS−K+ leukemia progenitor cells and promote differentiation into non-leukemic LS+K− cells, neutralizing antibodies against
IFN-γ and TNF-a were added to the ILC1—LSC co-culture. The IFN-γ neutralizing antibody blocked both ILC1-mediated suppression of LSC differentiation into LS−K+ leukemia progenitor cells and induction of LSC differentiation into non-leukemic LS+K− cells (
To investigate the role of IFN-γ produced by ILC1s to mediate these effects on LSCs, LSCs were incubated with recombinant murine IFN-γ. Similar to the ILC1-LSC co-culture, recombinant murine IFN-γ blocked differentiation of LSCs into LS−K+ leukemia progenitor cells and facilitated differentiation of LSCs into non-leukemic LS+K− cells (
Experiments assessed the effects of ILC1s on LSC differentiation. For this purpose, we co-cultured LSCs isolated from the spleen of MllPTD/WT: Flt3ITD/ITD mice with AML with ILC1s isolated from the liver of normal mice for 4 days. The ratio of ILC1s: LSCs was 1:4, which was lower than in the apoptosis assay. On days 3 and 4, the percentages and absolute numbers of LSCs were higher, whereas the percentages and the absolute numbers of Lin−Sca-1−c-Kit+ leukemia progenitor cells (LS−K+ cells) were significantly lower in the group co-cultured with ILC1s compared to the group co-cultured without ILC1s (
To determine if ILC1s regulate LSC differentiation through cell-cell contact (as thought to be critical for LSC apoptosis), we separated LSCs and ILC1s in a transwell chamber. As expected, the percentages of LSCs, LS−K+ leukemia progenitor cells, and LS+K− non-leukemic cells varied between LSCs cultured with and without 20 ILC1s (
LSCs are capable of differentiating into normal myeloid cells and malignant blasts28-30. To determine whether ILC1s affect LSCs differentiation into terminal myeloid blast cells, LSCs were co-cultured with ILC1s for 1, 2, 3, and 4 days. ILC1s significantly inhibited LSC differentiation into terminal myeloid blasts, as shown by reduced populations of cells expressing macrophage-1 antigen (Mac-1) and the myeloid differentiation antigen Gr-1 compared to LSCs alone (
A colony-forming unit assay starting with an equal number of LSCs was also performed. LSCs cultured with IFN-γ−/− ILC1s formed similar numbers of colonies as LSCs cultured without ILC1s, whereas LSCs cultured with WT or TNF-α−/− ILC1s formed significantly fewer colonies (
The process of LSC differentiation into AML blasts includes transitions from LSCs to LS−K+ leukemia progenitor cells, and from LS−K+ leukemia progenitor cells to AML blasts. To investigate which part of the process was affected by ILC1 and IFN-γ, LS−K+ leukemia progenitor cells were sorted from MllPTD/WT/Flt3ITD/ITD AML mice, then the LS−K+ leukemia progenitor cells were treated with WT ILC1, ILC1s, or recombinant IFN-γ for 5 days. There was no statistical difference in the percentage of Mac-1+ and Gr-1+ cells among any of the groups (
LSCs are hierarchical cells that can give rise to the terminal myeloid blasts that sustain AML28-30. To determine whether ILC1s affect the differentiation of LSCs into terminal myeloid blasts, we co-cultured LSCs with normal ILC1s for 1, 2, 3, or 4 days. On days 3 and 4, the ILC1s had significantly inhibited LSC differentiation into terminal myeloid blasts (compared to no ILCs), as indicated by reduced populations of cells expressing macrophage-1 antigen (Mac-1) and the myeloid differentiation antigen Gr-1 (
LSCs transition into LS−K+ leukemia progenitor cells before becoming AML blasts. To investigate which step in this sequence is affected by ILC1s and IFN-γ, we sorted LS−K+ leukemia progenitor cells from MllPTD/WT: Flt3ITD/ITD mice with AML, and then treated them with WT or IFN-γ−/− ILC1s or recombinant IFN-γ for 5 days. The percentages of cells expressing Mac-1 and Gr-1 remained constant among the groups (
The data indicate that ILC1s suppress LSC differentiation into AML blasts via a process mediated by IFN-γ. This suppression occurs during the first transition from LSCs into LS−K+ leukemia progenitor cells—rather than during the subsequent step that converts progenitor cells into AML blasts (
As shown in
Experiments were designed to test whether ILC1s could suppress leukemia development and growth in vivo. When we initiated the in vivo efficacy experiment, we did not know whether ILC could survive well in vivo after their adoptive transfer. Since IL-15 is a critical cytokine that supports the survival of ILC1s35-36,63, we first tested whether adoptively transferred WT ILC1s can suppress the development of leukemia derived from LSCs when bone marrow cells from IL-15 transgenic (IL-15tg) mice34 were co-injected as support cells into recipient mice pre-integrated with LSCs (
However, the above model could not distinguish WBCs derived from LSCs and those derived from transplanted normal donor bone marrow. Therefore, we utilized CD45.1 and CD45.2 congenic mice to further test our hypothesis. In this congenic mouse model, we sorted CD45.2 LSCs from MllPTD/WT: Flt3ITD/ITD mice with AML, and co-injected them along with CD45.1+ bone marrow cells as support cells into lethally irradiated CD45.1 recipient mice. The next day, we injected WT ILC or IFN-γ−/− ILC i.v. or recombinant IFN-γ cytokine intraperitoneally (i.p.) (
To investigate the mechanisms by which ILC1 and ILC1-secreted IFN-γ regulate LSCs, Ribozero RNA-seq analysis was performed on LSCs co-cultured with or without ILC1s isolated or treated with recombinant murine IFN-65 . Following the ILC1—LSC co-culture, the LSCs from separated from the ILC1s using FACS prior to RNA-seq analysis. RNA-seq revealed that 445 and 93 LSC genes were significantly up- and downregulated, respectively, following co-culture with ILC1s as compared to LSC alone (
Gene set enrichment analysis (GSEA) was used to identify the top 10 pathways in which those upregulated and downregulated genes were enriched (
We conducted Ribozero RNA-seq analysis of LSCs co-cultured with or without ILC1s isolated from normal mice or mice treated with or without recombinant murine IFN-γ. Of note, after ILC1-LSC co-culture, we separated the LSCs from the ILC1s using FACS (
Collectively, these data suggest that IFN-γ derived from ILC1s regulates the differentiation of LSCs through JAK-STAT and PI3K-AKT signaling pathways.
In
In
Ex vivo expanded ILC1 cells isolated using methods described herein exhibit rapid, reproducible expansion and show good persistence (
Both ILC1s and NK cells express IFN-γ, and we assessed each for their ability to produce IFN-γ in the presence or absence of AML or LSCs. We sorted those two cell types from the liver of normal mice and MllPTD/WT: Flt3ITD/ITD mice with AML and co-cultured each preparation separately with LSCs. The ILC1s isolated from mice with AML produced significantly less IFN-γ than those from normal mice. This difference was not observed with the NK cells (
Our data showed that ILC1s likely utilize cell—cell contact with LSCs to produce IFN-γ (
The above results do not suggest that liver NK cells are impotent against LSCs, as they did enhance apoptosis to some extent when the two cell types were co-cultured. However, IFN-γ neutralizing antibody did not affect their action, suggesting that, unlike ILC1s, the induction of LSC apoptosis by liver NK cells is not occurring primarily through IFN-γ (
To evaluate whether NK cells would slow the progression of AML in vivo, we i.p. injected anti-NK1.1 antibody (resulting in depletion of both NK cells and ILC1s) alone)63 into immunocompetent recipient CD45.1 mice (
The data showed that ILC isolated from the liver of mice with AML produce less IFN-γ and TNF-α than ILC1s isolated from normal mice (
ILC1s play critical roles in inflammation and the early anti-viral response40,41,62. However, their role in preventing and/or promoting cancer, including AML, has not been explored42. In particular, it is largely unknown whether ILC1s suppress or promote cancer development. Using in vitro studies in mouse and human as well as in vivo mouse models, we showed that the progression of AML can be controlled by normal ILC1s interacting with LSCs. We discovered that ILC1s have dual roles in regulating LSCs in AML: 1) ILC1s induce apoptosis of LSCs at high effector to target ratios; 2) At a lower dose of effector cells, ILC1s suppress the differentiation of LSCs into leukemia progenitor cells and then to myeloid blasts while facilitating the differentiation of LSCs into non-leukemic cells. Importantly, ILC1s do not affect the apoptosis and differentiation of normal stem cells. Without being bound by theory, although both IFN-γ and TNF-α are secreted by ILC1s, our work demonstrates that IFN-γ mediates ILC1-induced effects on LSCs via both the JAK-STAT and PI3K-AKT signaling pathways in mice. In addition, ILC1s produce higher levels of IFN-γ to control LSCs than do NK cells; DNAM-1 and IL-7Rα expressed on ILC1s interact with their cognate ligands expressed on LSCs. Thus, ILC1s may normally perform critical surveillance by spotting and destroying LSCs as well as other cancer stem cells; consequently, a dysfunction in this innate immune cell population can facilitate tumorigenesis and administering these cells can suppress tumorigenesis.
In AML patients who relapse, a small population of leukemia-initiating cells or LSCs is resistant to standard chemotherapy4,43. Therefore, elucidating the mechanism(s) of LSC resistance is a critical unmet challenge, and developing novel approaches to targeting LSCs offers a potential strategy for prolonging relapse-free survival of patients with AML. Chemotherapy and targeted therapy (e.g., tyrosine kinase inhibitors including FDA-approved midostaurin and gilteritinib) can kill leukemic blasts but may also enrich LSCs44,45. We show that normal ILC act directly on LSCs to control the progression of AML in vivo. Therefore, given the special biologic function of ILC1s, expanding autologous or normal allogeneic ILC ex vivo during times of remission or combining expanded ILC1s with an FDA-approved drug or cytokine may have a positive impact on prolonging relapse-free survival of patients with AML.
IFN-γ plays important roles in anti-viral and anti-tumor immunity, and has been used clinically to treat several diseases46. However, IFN-γ-based therapies have at least two limitations that preclude routine clinical use for cancer patients. The first is that IFN-γ cannot be delivered into local tumor sites to subsequently achieve effective concentrations in the tumor microenvironment (TME) without significant toxicity49-51. The second is that IFN-γ is rapidly cleared from the blood after intravenous administration, further limiting the ability to achieve effective local concentrations. These clinical disadvantages necessitate the development of alternative methods to ensure the effectiveness of IFN-γ in the local milieu of the marrow and/or other organs while limiting toxicity.
This application is the first to provide a promising new approach to treating AML: using a cell-based source of IFN-γ to target LSCs. Although ILC1 s are a minute cell population, they express abundant IFN-γ, especially when they interact with tumor cells in the TME. ILC1s also express high levels of chemokine receptors, including CXCR3 and CXCR6, the respective receptors for CXCL9-1 1 and CXCL16 that are expressed by AML cells41,50. These receptor-ligand interactions may help recruit ILC1 s to the bone marrow or tumor sites, where most LSCs reside51.
Furthermore, ILC 1 s rapidly and persistently produce IFN-γ locally after contacting LSCs or more mature tumor cells, yielding sufficient cytokine to locally target AML blasts53. Our data suggest that ILC1 s can also induce apoptosis and differentiation of LSCs within the TME. Moreover, ILC1 s are associated with reducing severe progression of graft-versus-host disease (GVHD) after allogeneic HSCT treatment for AML65. This suggests that ILC1 s can control AML through their multifaceted roles.
Like ILC1s, NK cells also belong to group 1 ILCs6. Although more than a dozen studies have assessed the efficacy of infusing NK cells into patients in remission following AML treatment, some of which showed promising result54, none have yet explored therapeutic ex vivo expansion and infusion of ILC1s during AML remission. Our data, especially our in vivo data, provide a strong rationale for developing methodologies to expand normal ILC1 populations rapidly and reproducibly for application as a cellular therapy to prolong relapse-free survival in patients with AML who achieve complete remission but may carry quiescent LSCs. This would be especially valuable for patients who are ineligible for HSCT.
The IFN-γ signaling pathway is associated with several biological responses and plays an important role in innate and adaptive immunity. It not only induces apoptosis of tumor cells51, but it also activates immune cells, two processes that are crucial for combatting cancer46,55. IFN-γ induces PD-L1 expression in tumor cells, including AML blast cells56 and immune cells57-58; it regulates PD-L1 expression mainly through the JAK1/2-STAT1/3-IRF1 axis in melanoma cells59. Our data demonstrates that both ILC1 s and recombinant IFN-γ block the differentiation of LSCs into leukemia progenitor cells through the JAK-STAT signaling pathway. This suggests that IFN-γ has a broad reach, covering both tumor cells and immune cells, as well as both mature tumor cells and cancer-stem-like cells among which it can induce different outcomes. The action of IFN-γ on tumors, tumor stem cells, and immune cells can induce PD-L1 expression, which can block T cell responses to tumor cells and their stem cell60 , differentiation of cancer stem cells, and activation of immune cells61. Although these roles are complex and clinical use of IFN-γ should consider all of these effects, the ability of an anti-PD-L1 antibody to block the adverse effects of IFN-γ-upregulated PD-L1 provides a good rationale for combining IFN-γ or if too toxic, combining cells that produce this cytokine, such as ILC1s, with anti-PD-L1 antibody to treat cancers, including AML. Such an anti-leukemic approach may bring new hope to patients with AML, especially relapsed older patients who have a dismal prognosis.
In summary, this study identified novel functions of ILC1s: they can closely regulate AML LSCs by inducing apoptosis; they prevent LSCs from differentiating into leukemia progenitors and then myeloid blasts; and they promote the differentiation of LSCs into a non-leukemic lineage. All of these actions are mediated by IFN-γ that ILC1s secrete when they form cell—cell contact with LSCs. We therefore believe that, by uncovering the mechanisms underlying these processes, our study could unveil a new immunotherapeutic approach—administration of ILC1s that have been expanded ex vivo—to prolong relapse-free survival of patients diagnosed with AML.
1 Nair, R., Salinas-Illarena, A. & Baldauf, H. M. New strategies to treat AML: novel insights into AML survival pathways and combination therapies. Leukemia, doi:10.1038/s41375-020-01069-1 (2020).
2 Ballester, G., Tirona, M. T. & Ballester, O. Hematopoietic stem cell transplantation in the elderly. Oncology (Williston Park, N. Y.) 21, 1576-1583; discussion 1587, 1590-1571, 1606 (2007).
3 Siveen, K. S., Uddin, S. & Mohammad, R. M. Targeting acute myeloid leukemia stem cell signaling by natural products. Molecular cancer 16, 13, doi:10.1186/s12943-016-0571-x (2017).
4 Yamashita, M., Dellorusso, P. V., Olson, O. C. & Passegué, E. Dysregulated haematopoietic stem cell behaviour in myeloid leukaemogenesis. Nature reviews. Cancer 20, 365-382, doi:10.1038/s41568-020-0260-3 (2020).
5 Shlush, L. I. et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 506, 328-333, doi:10.1038/nature13038 (2014).
6 Klose, C. S. N. & Artis, D. Innate lymphoid cells control signaling circuits to regulate tissue-specific immunity. Cell research 30, 475-491, doi:10.1038/s41422-020-0323-8 (2020).
7 Colonna, M. Innate Lymphoid Cells: Diversity, Plasticity, and Unique Functions in Immunity. Immunity 48, 1104-1117, doi:10.1016/j.immuni.2018.05.013 (2018).
8 Diefenbach, A., Colonna, M. & Koyasu, S. Development, differentiation, and diversity of innate lymphoid cells. Immunity 41, 354-365, doi:10.1016/j.immuni.2014.09.005 (2014).
9 Moral, J. A. et al. ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature 579, 130-135, doi:10.1038/s41586-020-2015-4 (2020).
Chevalier, M. F. et al. ILC2-modulated T cell-to-MDSC balance is associated with bladder cancer recurrence. The Journal of clinical investigation 127, 2916-2929, doi:10.1172/jci89717 (2017).
11 Koh, J. et al. IL23-Producing Human Lung Cancer Cells Promote Tumor Growth via Conversion of Innate Lymphoid Cell 1 (ILC1) into ILC3. Clinical cancer research: an official journal of the American Association for Cancer Research 25, 4026-4037, doi:10.1158/1078-0432.Ccr-18-3458 (2019).
12 Carrega, P. et al. NCR(+)ILC3 concentrate in human lung cancer and associate with intratumoral lymphoid structures. Nature communications 6, 8280, doi:10.1038/ncomms9280 (2015).
13 Zhang, Y. et al. IL-22 promotes tumor growth of breast cancer cells in mice. Aging 12, 13354-13364, doi:10.18632/aging.103439 (2020).
14 Eisenring, M., vom Berg, J., Kristiansen, G., Saller, E. & Becher, B. IL-12 initiates tumor rejection via lymphoid tissue-inducer cells bearing the natural cytotoxicity receptor NKp46. Nature immunology 11, 1030-1038, doi:10.1038/ni.1947 (2010).
15 Jovanovic, I. P. et al. Interleukin-33/ST2 axis promotes breast cancer growth and metastases by facilitating intratumoral accumulation of immunosuppressive and innate lymphoid cells. International journal of cancer 134, 1669-1682, doi:10.1002/ijc.28481 (2014).
16 Seillet, C., Belz, G. T. & Huntington, N. D. Development, Homeostasis, and Heterogeneity of NK Cells and ILC1. Current topics in microbiology and immunology 395, 37-61, doi:10.1007/82_2015_474 (2016).
17 Wang, Y., Dong, W., Zhang, Y., Caligiuri, M. A. & Yu, J. Dependence of innate lymphoid cell 1 development on NKp46. PLoS biology 16, e2004867, doi:10.1371/journal.pbio.2004867 (2018).
18 Mopin, A., Driss, V. & Brinster, C. A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model. Journal of visualized experiments: JoVE, doi:10.3791/54270 (2016).
19 Almishri, W. et al. TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2. Journal of innate immunity 8, 617-629, doi:10.1159/000448077 (2016).
20 Zhang, B. et al. Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer cell 21, 577-592, doi:10.1016/j.ccr.2012.02.018 (2012).
21 Zhang, B. et al. Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia. Nature medicine 24, 450-462, doi:10.1038/nm.4499 (2018).
22 Zorko, N. A. et al. Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias. Blood 120, 1130-1136, doi:10.1182/blood-2012-03-415067 (2012).
23 Artis, D. & Spits, H. The biology of innate lymphoid cells. Nature 517, 293-301, doi:10.1038/nature14189 (2015).
24 Appelbaum, F. R., Rowe, J. M., Radich, J. & Dick, J. E. Acute myeloid leukemia. Hematology. American Society of Hematology. Education Program, 62-86, doi:10.1182/asheducation-2001.1.62 (2001).
25 Hoshii, T. et al. mTORC1 is essential for leukemia propagation but not stem cell self-renewal. The Journal of clinical investigation 122, 2114-2129, doi:10.1172/jci62279 (2012).
26 Kumar, R., Fossati, V., Israel, M. & Snoeck, H. W. Lin-Scal+kit- bone marrow cells contain early lymphoid-committed precursors that are distinct from common lymphoid progenitors. Journal of immunology (Baltimore, Md.: 1950) 181, 7507-7513, doi:10.4049/jimmunol.181.11.7507 (2008).
27 Peng, C. et al. LSK derived LSK− cells have a high apoptotic rate related to survival regulation of hematopoietic and leukemic stem cells. PLoS One 7, e38614, doi:10.1371/journal.pone.0038614 (2012).
28 Thomas, D. & Majeti, R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 129, 1577-1585, doi:10.1182/blood-2016-10-696054 (2017).
29 Joshi, K., Zhang, L., Breslin, S. J. P. & Zhang, J. Leukemia Stem Cells in the Pathogenesis, Progression, and Treatment of Acute Myeloid Leukemia. Advances in experimental medicine and biology 1143, 95-128, doi:10.1007/978-981-13-7342-8 5 (2019).
30 Graf, M. et al. Expression of MAC-1 (CD11b) in acute myeloid leukemia (AML) is associated with an unfavorable prognosis. American journal of hematology 81, 227-235, doi:10.1002/ajh.20526 (2006).
31 Adane, B. et al. The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells. Cell reports 27, 238-254.e236, doi:10.1016/j.celrep.2019.03.009 (2019).
32 Park, S. M. et al. IKZF2 Drives Leukemia Stem Cell Self-Renewal and Inhibits Myeloid Differentiation. Cell stem cell 24, 153-165.e157, doi:10.1016/j.stem.2018.10.016 (2019).
33 Sharma, A. et al. Constitutive IRF8 expression inhibits AML by activation of repressed immune response signaling. Leukemia 29, 157-168, doi:10.1038/1eu.2014.162 (2015).
34 Fehniger, T. A. et al. Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells. The Journal of experimental medicine 193, 219-231, doi:10.1084/jem.193.2.219 (2001).
35 Bank, U. et al. c-FLIP is crucial for IL-7/IL-15-dependent NKp46(+) ILC development and protection from intestinal inflammation in mice. Nature communications 11, 1056, doi:10.1038/s41467-020-14782-3 (2020).
36 Klose, C. S. & Diefenbach, A. Transcription factors controlling innate lymphoid cell fate decisions. Current topics in microbiology and immunology 381, 215-255, doi:10.1007/82_2014_381 (2014).
37 Pandey, R. et al. SHP2 inhibition reduces leukemogenesis in models of combined genetic and epigenetic mutations. The Journal of clinical investigation 129, 5468-5473, doi:10.1172/jci130520 (2019).
38 Villarino, A. V., Kanno, Y. & O'Shea, J. J. Mechanisms and consequences of Jak-STAT signaling in the immune system. Nature immunology 18, 374-384, doi:10.1038/ni.3691 (2017).
39 Shuai, K. & Liu, B. Regulation of JAK—STAT signalling in the immune system. Nature Reviews Immunology 3, 900-911, doi:10.1038/nri1226 (2003).
40 Wang, S. et al. Transdifferentiation of tumor infiltrating innate lymphoid cells during progression of colorectal cancer. Cell research 30, 610-622, doi:10.1038/s41422-020-0312-γ (2020).
41 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.ce11.2017.09.052 (2017).
42 Crinier, A. et al. Multidimensional molecular controls defining NK/ILC1 identity in cancers. Seminars in immunology, 101424, doi:10.1016/j.smim.2020.101424 (2020).
43 Taussig, D. C. et al. Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(−) fraction. Blood 115, 1976-1984, doi:10.1182/blood-2009-02-206565 (2010).
44 Trabanelli, S. et al. CD127+ innate lymphoid cells are dysregulated in treatment naïve acute myeloid leukemia patients at diagnosis. Haematologica 100, e257-260, doi:10.3324/haematol.2014.119602 (2015).
45 Levis, M. & Perl, A. E. Gilteritinib: potent targeting of FLT3 mutations in AML. Blood advances 4, 1178-1191, doi:10.1182/bloodadvances.2019000174 (2020).
46 Castro, F., Cardoso, A. P., Gonçalves, R. M., Serre, K. & Oliveira, M. J. Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Frontiers in immunology 9, 847, doi:10.3389/fimmu.2018.00847 (2018).
47 Sharma, A. & Jusko, W. J. Characteristics of indirect pharmacodynamic models and applications to clinical drug responses. British journal of clinical pharmacology 45, 229-239, doi:10.1046/j.1365-2125.1998.00676.x (1998).
48 Razaghi, A., Owens, L. & Heimann, K. Review of the recombinant human interferon gamma as an immunotherapeutic: Impacts of production platforms and glycosylation. Journal of biotechnology 240, 48-60, doi:10.1016/j.jbiotec.2016.10.022 (2016).
49 Kurzrock, R. et al. Pharmacokinetics, single-dose tolerance, and biological activity of recombinant gamma-interferon in cancer patients. Cancer research 45, 2866-2872 (1985).
50 Maloy, K. J. & Uhlig, H. H. ILC1 populations join the border patrol. Immunity 38, 630-632, doi:10.1016/j.immuni.2013.03.005 (2013).
51 Houshmand, M. et al. Bone marrow microenvironment: The guardian of leukemia stem cells. World journal of stem cells 11, 476-490, doi:10.4252/wjsc.v11.i8.476 (2019).
52 Dunn, G. P., Koebel, C. M. & Schreiber, R. D. Interferons, immunity and cancer immunoediting. Nature reviews. Immunology 6, 836-848, doi:10.1038/nri1961 (2006).
53 Munneke, J. M. et al. Activated innate lymphoid cells are associated with a reduced susceptibility to graft-versus-host disease. Blood 124, 812-821, doi:10.1182/blood-2013-11-536888 (2014).
54 Berrien-Elliott, M. M. et al. Multidimensional Analyses of Donor Memory-Like NK Cells Reveal New Associations with Response after Adoptive Immunotherapy for Leukemia. Cancer discovery 10, 1854-1871, doi:10.1158/2159-8290.Cd-20-0312 (2020).
55 Gresser, I. Biologic effects of interferons. The Journal of investigative dermatology 95, 66s-71s, doi:10.1111/1523-1747.ep12874776 (1990).
56 Krönig, H. et al. Interferon-induced programmed death-ligand 1 (PD-L1/B7-H1) expression increases on human acute myeloid leukemia blast cells during treatment. European journal of haematology 92, 195-203, doi:10.1111/ejh.12228 (2014).
57 Hartley, G. et al. Immune regulation of canine tumour and macrophage PD-L1 expression. Veterinary and comparative oncology 15, 534-549, doi:10.1111/vco.12197 (2017).
58 Munir, S. et al. Inflammation induced PD-L1-specific T cells. Cell stress 3, 319-327, doi:10.15698/cst2019.10.201 (2019).
59 Garcia-Diaz, A. et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell reports 19, 1189-1201, doi:10.1016/j.celrep.2017.04.031 (2017).
60 Srivastava, S. et al. Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade. Cancer cell, doi.org/10.1016/j.ccell.2020.11.005 (2020).
61 Song, M. et al. Low-Dose IFNγ Induces Tumor Cell Stemness in Tumor Microenvironment of Non-Small Cell Lung Cancer. Cancer research 79, 3737-3748, doi:10.1158/0008-5472.Can-19-0596 (2019).
62. Shannon, J. P. et al. Group 1 innate lymphoid-cell-derived interferon-γ maintains anti-viral vigilance in the mucosal epithelium. Immunity (2021).
63. 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 Bc1-xL Expression in Hepatocytes. Immunity 52, 96-108.e109 (2020).
64. Gao, Y. et al. Tumor immunoevasion by the conversion of effector NK cells into type 1 innate lymphoid cells. Nature immunology 18, 1004-1015 (2017).
65. Robinette, M. L. et al. Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets. Nat Immunol 16, 306-317 (2015).
66. Sojka, D. K. et al. Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells. Elife 3, e01659 (2014).
67. Spits, H., Bernink, J. H. & Lanier, L. NK cells and type 1 innate lymphoid cells: partners in host defense. Nat Immunol 17, 758-764 (2016).
68. Klose, C. S. N. et al. Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 157, 340-356 (2014).
69. Smith, M. et al. Adult acute myeloid leukaemia. Critical reviews in oncology/hematology 50, 197-222 (2004).
70. Filén, J. J. et al. Quantitative proteomics reveals GIMAP family proteins 1 and 4 to be differentially regulated during human T helper cell differentiation. Molecular & cellular proteomics : MCP 8, 32-44 (2009).
71. Laouedj, M. et al. S100A9 induces differentiation of acute myeloid leukemia cells through TLR4. Blood 129, 1980-1990 (2017).
72. Shipounova Nifontova, I. N., Bigil'diev, A. E., Svinareva, D. A. & Drize, N. I. Characteristics of leukemia stem cells of murine myeloproliferative disease involving the liver. Bulletin of experimental biology and medicine 149, 293-297 (2010).
74. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics (Oxford, England) 30, 2114-2120 (2014).
15 75. Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics (Oxford, England) 34, i884-i890 (2018).
76. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics (Oxford, England) 29, 15-21 (2013).
77. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome biology 11, R106 (2010).
78. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics (Oxford, England) 26, 139-140 (2010).
79. Mootha, V. K. et al. PGC-lalpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature genetics 34, 267-273 (2003).
80. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America 102, 15545-15550 (2005).
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/138,376, filed on Jan. 15, 2021. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/012731 | 1/18/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63138376 | Jan 2021 | US |
