Human type-1 innate lymphoid cells control leukemia stem cell differentiation and limit acute myeloid leukemia development

Domain: neuroscience
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
Innate lymphoid cells (ILCs) are crucial for cancer immunosurveillance. While mouse type 1 ILCs (ILC1s) control acute myeloid leukemia (AML) by targeting leukemia stem cells (LSCs), the role of human ILC1s in AML remains largely undefined. Here, we find that ILC1s in AML patients are impaired, with reduced total ILC1 numbers and diminished function. In contrast, healthy donor (HD) ILC1s-derived TNFα inhibits the leukemic transition from CD34+CD38+ to CD34−CD38+ cells and blocks the differentiation of LSCs (CD34+CD38−) into immunosuppressive, macrophage-like leukemia-supporting cells. HD ILC1-derived IFNγ partially suppresses the differentiation of CD34−CD38+ to CD34−CD38− cells. These combined effects limit human leukemogenesis in vivo. We also identify a human ILC1 subset as Lin−CD127+CD161−CRTH2−CD117− (CD161− ILC1s) that can be generated from umbilical cord blood CD34+ hematopoietic stem cells. This method could provide a reliable source of ILC1s for potential adoptive transfer therapies in AML, offering a therapeutic approach to prolong disease-free survival in AML.

Introduction
Acute myeloid leukemia (AML) is a devastating disease, with a median 5-year survival is 30–35% for patients younger than age 65 treated with standard chemotherapy1. Although allogeneic stem cell transplantation has been 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, defined by a CD34+CD38− phenotype) remain relatively high2,3. LSCs possess self-renewal capacity and are believed to be the driving force behind leukemia initiation, maintenance, and relapse2,4. They have the potential to differentiate into more mature leukemic progenitor cells (CD34+CD38+) and terminally differentiated blasts (myeloid blasts or leukemic cells; CD34−CD38+), which contribute to disease progression5,6. CD34+CD38+ leukemic progenitor cells have progressed beyond the undifferentiated LSC stage7. These cells exhibit increased differentiation compared to LSCs but are still considered immature with self-renewal capacity and the ability to propagate the disease. CD34−CD38+ cells represent a more terminally differentiated leukemic population, having lost some of the stem cell-like properties compared to LSCs and CD34+CD38+ leukemic progenitor cells5,8. While they are more differentiated, CD34−CD38+ cells still possess some self-renewal capacity and the ability to give rise to more mature leukemic cells, perpetuating the disease9. LSCs can also differentiate into CD34⁻CD38⁻ cells, a subset of leukemic cells that are highly resistant to standard therapies. Higher levels of these cells are often associated with a poorer prognosis and a higher likelihood of disease relapse after treatment5,10. Due to the complexity and heterogeneity of LSC differentiation and their relatively lower sensitivity to treatment compared to later stages of AML, understanding the differentiation pathways and properties of these cell populations, as well as developing safer and more effective therapeutic approaches, is crucial for improving the clinical outcome of patients with AML.

Innate lymphoid cells (ILCs) play a critical role in mediating immune responses and regulating tissue homeostasis and inflammation11–22. Unlike adaptive immune cells, ILCs lack antigen-specific receptors and instead are activated by cytokine signals23. ILCs are classified into three main subsets based on their cytokine production profiles and transcription factor (TF) requirements: Group 1 ILCs, including ILC1s and natural killer (NK) cells that produce interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα), and are primarily involved in immune responses against intracellular pathogens such as viruses and certain bacteria as well as tumors24–26. These cells express the TF T-BET. Group 2 ILCs, known as ILC2s, secrete type 2 cytokines such as IL-4, IL-5, IL-9, and IL-13, which play a pivotal role in defense against extracellular parasites like helminths, in allergic inflammation, and defense against tumors22,27,28. These cells are characterized by the expression of the TF GATA3. Group 3 ILCs, or ILC3s, produce IL-17 and IL-22, contributing significantly to mucosal immunity, tumor immunity, and the maintenance of barrier integrity, particularly within the gastrointestinal tract29–32. These cells express the TF RORγt as a defining feature of their phenotype. We recently reported that ILC1s from healthy mice contribute to the control of murine AML by eliminating LSCs and inhibiting their differentiation into myeloid blasts33. However, the potent anti-cancer properties and mechanisms of human ILC1s in AML remain to be further explored.

In this study, we demonstrate that human ILC1s are quantitatively and qualitatively impaired in AML patients, thereby playing a pivotal role in influencing the fate of LSCs. Specifically, we demonstrate that ILC1s isolated from healthy donors (HD) can hinder the differentiation of LSCs into leukemic progenitor cells or a more differentiated population of leukemic cells via their secretion of IFNγ or TNFα. Additionally, these ILC1s can limit the differentiation of LSCs into immunosuppressive, macrophage-like leukemia-supporting cells via their secretion of TNFα, while ILC1s from AML patients cannot. These regulatory functions of HD ILC1s converge to limit human leukemogenesis in vivo. Our study also reveals the inherent heterogeneity within the human ILC1 population, comprising both CD161+ and CD161− subsets. Despite this diversity, both subsets demonstrate similar expression of crucial TFs and effector molecules. We also demonstrate how umbilical cord blood (UCB) CD34+ cells can efficiently generate ILC1s with promising anti-AML activity. Our study shows that human ILC1s are defective in AML patients, yet human HD ILC1s or those generated from UCB CD34+ cells can halt the progression of AML and have the potential to be manufactured ex vivo for improved treatment of AML in vivo.

Results
ILC1 population and their functional roles are disrupted in the setting of AML
We have reported that DNAM-1(+) and IL-7 receptor (R)(+)-ILC1s isolated from healthy mice can recognize CD155/CD112 and IL-7, respectively, on mouse LSCs and induce IFNγ that in turn promotes apoptosis and blocks their differentiation into myeloid blasts33. We have also reported that human ILC1s (defined as Lin−CD127+CD161+/−CRTH2−CD117−, hereafter referred to as ILC1s) promote apoptosis of human LSCs (defined by CD34+CD38−) at a high ratio of ILC1s to LSCs33. Whether human ILC1s control LSC fate by affecting the differentiation of human LSCs remains largely undefined. Human ILC1s can be defined as Lin−CD127+, with “Lin−” defined as depletion of CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FceRI, and CD56 positive cells. Following analysis of ILC1s in the peripheral blood (PB) of healthy donors (HDs) and AML patients, we observed a highly significant increase in the percentage of ILC1s among total ILCs (CD127+) in AML patients relative to HDs (Fig. 1a and Supplementary Fig. 1a). However, within total lineage-negative cells from the same patient population, there was a notable decrease in both the percentage (Fig. 1b) and absolute number of the ILC1 subset compared to HDs (Fig. 1c). This finding is further supported by our assessment of T-BET expression (encoded by TBX21), a critical TF of human ILC1s. Data showed that the number of T-BET+ ILC1s was decreased in AML, while T-BET expression on a per cell basis is equivalent between HDs and AML patients (Fig. 1d and Supplementary Fig. 1b).

We also analyzed 10× Genomics high-throughput droplet-based single-cell RNA sequencing (scRNA-seq) that we previously performed22. In our scRNA-seq, we initially employed the EasySep™ Human Pan-ILC Enrichment Kit to isolate total ILCs. Following this, we performed cell sorting using a comprehensive set of lineage markers34–36 (CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FcεRI, and CD56), in addition to CD127. This strategy of utilizing specific surface markers ensured the exclusion of T cells (including CD4+ T cells, CD8+ T cells, and other T cell lineage cells), B cells, NK cells, myeloid cells, and CD34+ hematopoietic precursors, as well as non hematopoietic cells from the sorted population. We also excluded cells that expressed CD4 and CD8 during the analysis. This exclusion of CD4 and CD8 differs from an earlier report37. By selecting Lin−CD127+ cells, we were able to focus on ILCs, ensuring that no other known immune cell types were present in our analysis. The examination of transcriptomic profiles in sorted total ILCs identified the ILC1 population (cluster 8; Supplementary Fig. 1c) from HDs and AML patients using previously reported ILC1 markers34 (Fig. 1e, f and Supplementary Fig. 1c, d). This population expressed ILC1-specific markers, IL7R, GZMK, IKZF3, IL2RB, LEF1, and GPR183, without the expression of NK cell markers such as FGFBP2, FCGR3A, KIR2DL1, KIR2DL3, and GZMH and without expression of T cell markers, CD4 and CD8 (Supplementary Fig. 1e). Compared to AML ILC1s, HD ILC1s exhibited higher expression of cytotoxicity-associated genes, including NKG7, PRF1, CST7, and CTSW38,39 (Fig. 1g). Gene ontology (GO) pathway analysis for ILC1s from AML patients demonstrated a decrease in pathways associated with cell killing, cell activation, and cytokine production when compared to HD ILC1s (Supplementary Fig. 1f). Additionally, compared to HD ILC1s, AML ILC1s showed a lower functional score of signaling pathways for type 2 IFN production, TNF superfamily cytokine production, cell killing, and cytolysis (Fig. 1h). These findings collectively indicate a dysfunctional state of ILC1s in AML patients. The results were validated by flow cytometry, revealing a significant reduction in the production of IFNγ and TNFα in ILC1s from AML patients compared to HD ILC1s (Fig. 1i, j). Further, ILC1s from AML patients exhibited decreased surface expression of activation receptors DNAM-1 and NKG2D relative to HD ILC1s (Fig. 1k, l). Finally, a negative correlation was observed between the percentage of ILC1s and the percentage of leukemic blasts in AML patients (R = −0.9024, P = 0.0004) (Supplementary Fig. 2a, b). Collectively, these data suggest that the functional role(s) of human ILC1s become dysregulated in the context of AML. A high level of human ILC1s appear to be associated with a lower AML blast count, suggesting that ILC1s could have a role in controlling AML.

HD ILC1 TNFα reduces the differentiation of CD34+CD38+ cells into CD34−CD38+ cells, while HD ILC1 IFNγ reduces the differentiation of CD34−CD38+ cells into CD34−CD38− cells
As noted above, HD human ILC1s induce cell death of human LSCs at a high ILC1:LSC ratio of 4:133. We also explored the impact of HD ILC1s on LSCs at a low effector-to-target ratio (E:T) of one ILC1 to four LSCs (1:4), as well as a culture of LSCs alone with recombinant human (rh) IFNγ (50ng/ml) or rhTNFα (20ng/ml) for 3 days. Our results show that compared to LSC alone, (1) coculturing LSCs with HD ILC1s or treatment with rhIFNγ or rhTNF did not significantly change the absolute number and percentage of LSCs during coculture (Fig. 2a, b and Supplementary Fig. 3a); (2) coculturing LSCs with HD ILC1s or rhTNFα, but not rhIFNγ, significantly increased the number of LSC-derived CD34+CD38+ cells (Fig. 2a, c), while the percentage of LSC-derived CD34+CD38+ cells was elevated only in the rhTNFα-treated group (Supplementary Fig. 3b); (3) coculturing LSCs with HD ILC1s or rhIFNγ or rhTNFα significantly reduced the absolute number of LSC-derived CD34−CD38+ cells (Fig. 2a, d). The percentage of LSC-derived CD34−CD38+ cells was higher in the ILC1 and rhIFNγ-treated groups but reduced in the rhTNFα-treated group (Supplementary Fig. 3c); and (4) coculturing LSCs with HD ILC1s or rhIFNγ or rhTNFα significantly decreased the number of LSC-derived CD34−CD38− cells (Fig. 2a, e). The percentage of LSC-derived CD34−CD38− cells was decreased only in the HD ILC1 and rhIFNγ-treated groups but not in the rhTNFα-treated group (Supplementary Fig. 3d).

However, compared to HD ILC1s cocultured with LSCs, HD ILC1s cocultured with LSCs and neutralizing mAb to TNFα or IFNγ did not significantly change the absolute number or percentage of CD34+CD38− LSCs during coculture (Fig. 2f, g and Supplementary Fig. 3e). The addition of a neutralizing mAb to TNFα but not to IFNγ, suppressed the increased number of CD34+CD38+ cells from LSCs induced by HD ILC1s; however, the percentage of CD34+CD38+ cells was unaffected (Fig. 2f, h and Supplementary Fig. 3f). In contrast, HD ILC1s cocultured with LSCs and neutralizing mAb to IFNγ but not TNFα reversed the decrease in number and the increase in percentage of CD34−CD38+ cells that were originally differentiated from LSCs cocultured with HD ILC1s (Fig. 2f, i and Supplementary Fig. 3g). These data highlight the distinct roles of HD ILC1-derived IFNγ and TNFα in the regulation of LSC differentiation. However, the absence of any notable impact via the neutralization of either cytokine on the differentiation of CD34−CD38− cells from LSCs in the presence of ILC1s suggests that other factors may regulate this specific cell population (Fig. 2f, j and Supplementary Fig. 3h).

HD ILC1-derived TNFα rather than IFNγ reduces the differentiation of immunosuppresive, macrophage-like leukemia-supporting cells from LSCs
Several reports have described that CD11b+CD206+ immunosuppressive, macrophage-like leukemia-supporting cells inhibit effective immune responses and contribute to a favorable environment for LSCs by secreting factors that promote leukemia progression40–42. However, it remains unclear whether LSCs can directly differentiate into CD11b+CD206+ immunosuppressive, macrophage-like leukemia-supporting cells. To investigate this, we cocultured HD ILC1s with LSCs at the E:T of 1:4 for 3 days. By Wright-Giemsa staining and flow cytometry, we observed that ILC1s blocked the differentiation of LSCs into CD11b+CD206+ macrophage-like leukemia-supporting cells. The addition of a neutralizing mAb to TNFα but not to IFNγ reversed this effect (Fig. 3a–c). This was further supported by the observation that rhTNFα, but not rhIFNγ, reduces the differentiation of LSCs into these supportive cells (Fig. 3d, e). Importantly, the inhibitory effect of ILC1s on LSC differentiation into immunosuppressive, macrophage-like leukemia-supporting cells was significantly diminished when cocultured with ILC1s from AML patients (Supplementary Fig. 4a, b), consistent with the lower TNFα production by ILC1s from AML patients (Fig. 1j). These findings underscore the dysfunction of ILC1s in AML patients and highlight the importance of TNFα signaling along with IFNγ in regulating LSC differentiation. The colony-forming unit (CFU) assay further confirmed that HD ILC1s impede the differentiation of human LSCs into munosuppresive, macrophage-like leukemia-supporting cells, as indicated by decreased numbers of total colonies and granulocyte-macrophage progenitor (CFU-GM) colonies after coculture with HD ILC1s compared to culture without HD ILC1s, while there was no significant difference in the number of granulocytes (CFU-G) colonies (Fig. 3f, g and Supplementary Fig. 4c).

To provide definitive evidence that these munsuppresive, macrophage-like leukemia-supporting cells are, in fact, derived from LSCs, we sorted LSCs from two AML patients with the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation and induced their differentiation into CD11b+CD206+ immunosuppressive, macrophage-like leukemia-supporting cells. The CD11b+CD206+ cells were then sorted, and the presence of the FLT3-ITD was detected by polymerase chain reaction (PCR) using primers specific for the mutation, thus demonstrating their derivation from the LSC43,44 (Fig. 3h, Lane 1 and Lane 2).

Collectively, these data indicate that produced by HD ILC1 TNFα contributes to reducing the differentiation of LSCs into CD11b+CD206+ immunosupressibe, macrophage-like leukemia-supporting cells in vitro, whereas this same effect is impaired in ILC1s from AML patients.

HD ILC1s prolong the survival of mice bearing human LSCs in vivo
We also performed the in vivo transplantation experiment, in which human CD34+CD38− LSCs and human HD ILC1s were administrated intravenously (i.v.) into NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ (NSG-SGM3) mice. On each of days 1–7, the mice received an intraperitoneal (i.p.) injection of human IL-15 (hIL-15) (Fig. 4a). The mice were monitored for over 600 days. We found that the injection of human HD ILC1s reduced the LSC engraftment in NSG-SGM3 mice and suppressed the progression of AML, as evidenced by a significantly decreased number of CD45+CD33+ blast cells, a significantly decreased number of CD34+CD38− LSCs, and significantly prolonged survival of NSG-SGM3 mice, all compared to mice that did not receive an injection of HD ILC1s but received daily i.p. injections of hIL-15 from days 1 to 7 (Fig. 4b–d). These data indicate that human HD ILC1s can suppress the differentiation of human LSCs and the development of AML in vivo. Taken together, these findings strongly suggest that human HD ILC1s have a role in protecting against AML and provide a rationale for using HD ILC1s as a cellular-based therapy to extend disease-free survival in AML.

CD161− ILC1s exhibit a phenotype similar to CD161+ ILC1s
ILC1s have conventionally been characterized as CD161-expressing cells. However, isolation and ex vivo expansion of Lin−CD161+ ILC1s from either HDs or AML patients does not result in a homogeneous population of ILC1s. Through extensive flow cytometric analyses, we found that ILC1s are heterogeneous in human PB using ILC1-specific surface markers (Lin, CD127, CRTH2, and CD117) that were reported previously35,45 along with an anti-CD161 mAb (Supplementary Fig. 5). The heterogeneous ILC1s in the blood of HDs included the conventional CD161+ ILC1s (hereafter referred to as 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 HD PB (Fig. 5a). Moreover, both CD161+ and CD161− ILC1s exhibited comparable expression levels of T-BET and EOMES (Note: human EOMES+ ILC1s were also previously reported in human ILC1s34) (Fig. 5b), comparable production of IFNγ and TNFα (Fig. 5c), and comparable expression of the cytolytic molecules GZMA and GZMB (Note: human GZMA- and GZMB-expressing ILC1s were also previously reported11 (Fig. 5d, e).

We further expanded the total ILCs on either 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 (Supplementary Fig. 6a–d). The CD161− ILC1s were further identified by the expression of IFNγ, T-BET, and EOMES (Supplementary Fig. 6e, f), indicative of an ILC1 lineage.

Efficient generation of ILC1s from umbilical cord blood CD34+ cells confers anti-AML activity
Using the same method recently reported for ILCs differentiated from umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs)46, we obtained ILC1s that were almost entirely CD161−, regardless of co-culture on either DL1-expressing or DL4-expressing OP9 stromal cells, as demonstrated by the phenotype and the expression of IFNγ, EOMES, and T-BET (Fig. 6a–c). These cells were referred to as UCB CD34+ cell-derived CD161− ILC1s. We also observed that both UCB freshly isolated CD161+/− ILC1s and UCB CD34+ cell-derived CD161+/− ILC1s expressed NKG2D and CD200R1 (Fig. 6d). However, unlike NK cells, they do not express KIR (including KIR2DL4, KIR2DL5, and KIR2DL1/S1/S3/S5). Additionally, unlike UCB CD34 cell-derived CD161+/− ILC1s, UCB-freshly isolated CD161+/− ILC1s do not express NKG2A (Fig. 6d). Furthermore, both UCB freshly isolated CD161− ILC1s and UCB CD34+ cell-derived CD161− ILC1s expressed GZMB and perforin (Supplementary Fig. 7). An early 700-fold increase from UCB CD34+ cells to CD161− ILC1s (>97% purity) was observed on day 28 (Fig. 6e). The cytotoxicity against LSCs was comparable between UCB CD34+ cell-derived CD161− ILC1s and the CD161+ ILC1s isolated from UCB mononuclear cells (UBMCs; Fig. 6f, g). Neither UCB CD34+ cell-derived CD161− ILC1s nor UBMC-derived CD161+ ILC1s displayed high cytotoxicity against HSCs isolated from HD PB (Fig. 6f, g). Injection of UCB CD34+ cell-derived CD161− ILC1s significantly extended the survival of mice bearing human LSCs when compared to the group injected with LSCs alone (Fig. 6h, i). Importantly, this effect was abolished upon administration of anti-IFNγ (Fig. 6j, k). Furthermore, we also compared the anti-tumor effect of ILC1s and another innate effector cell type (NK cells). Our results demonstrated that injections of both UCB CD34+ cell-derived CD161− ILC1s and UCB CD34+ cell-derived NK cells delayed the progression of AML compared to the control group without these injections. However, the injection of UCB CD34+ cell-derived CD161− ILC1s showed a trend towards better prolongation of survival than the injection of UCB CD34+ cell-derived NK cells in our LSC model (Fig. 6j, k). Taken together, the results suggest that ILC1s derived from UCB CD34+ cells exhibit anti-AML efficacy both in vitro and in vivo.

Methods
Human samples
Peripheral blood (PB) samples were obtained from healthy donors (HDs) and acute myeloid leukemia (AML) patients at the City of Hope National Medical Center (COHNMC). All HDs and AML patients signed an informed consent form approved by the City of Hope Institutional Review Board. Human specimens were collected from individuals with AML registered at the COHNMC who consented to an Institutional Review Board (IRB)-approved protocol (IRB 18067); healthy donor specimens were collected from individuals who consented to IRB 06229. Recruitment in this cohort was independent of sex, as any patient with newly diagnosed, relapsed, or refractory AML was eligible. All participants in this study were patients with relapsed or refractory acute myeloid leukemia. Eligibility criteria included being at least 18 years old and providing written informed consent in accordance with national legal and regulatory requirements before any project-specific procedures. Information on race, ethnicity, or other socio-economical parameters was not collected for this cohort. Healthy donors and patients with AML were not compensated for their blood cell or bone marrow donations. Detailed characteristics of the study population can be found in Supplementary Tables 1, 2. Given the limited size of the cohort, we did not conduct any analyses stratified by sex. Patients who met the inclusion criteria outlined above were recruited for this study, so we do not anticipate any self-selection or other biases. Mononuclear cells were isolated using Ficoll separation. LSCs were sorted using a BD FACSAriaTM Fusion (BD Biosciences). ILCs were isolated using EasySep™ Human Pan-ILC Enrichment Kit (STEMCELL) or were sorted using a BD FACSAriaTM Fusion. UCB was obtained from StemCyte, Inc. (Baldwin Park, California). CD34+ cells were isolated using the CD34 MicroBead Kit (Miltenyi Biotec) and then sorted using a BD FACSAriaTM.

Cells and cell culture
OP9 cells were purchased from the American Type Culture Collection (ATCC; Cat# CRL-2749). Using this base cell line, human DLL (delta-like canonical Notch ligand) 1 and DLL4 genes were overexpressed to create OP9-DL1 and OP9-DL4 cell lines, respectively22. OP9-DL1 and OP9-DL4 were maintained in MEMα GlutaMAX media supplemented with 20% FBS, 100 U/ml penicillin, 0.1mg/ml streptomycin, and 50μM β-Mercaptoethanol. Human LSCs were cultured in StemSpan™ Serum-Free Expansion Medium II with penicillin (100U/mL), streptomycin (100mg/mL), stem cell factor (SCF, 20ng/mL), thrombopoietin (TPO, 20ng/mL), Flt3-L (20ng/mL), IL-3 (10ng/mL), and IL-6 (10ng/mL). Human ILC1s were cultured in MEMα GlutaMAX media. The media was supplemented with 10% human AB serum, IL-2 (500IU/mL), IL-7 (20ng/mL), and IL-15 (20ng/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, Cat# 561802, Clone: HIT3a; anti-CD4, Cat# 555346, Clone: RPA-T4; anti-CD8, Cat# 555634, Clone: HIT8a; anti-CD14, Cat# 555397, Clone: M5E2; anti-CD15, Cat# 555401, Clone: HI98; anti-CD16, Cat# 555406, Clone: 3G8; anti-CD19, Cat# 555412, Clone: HIB19; anti-CD20, Cat# 555622, Clone: 2H7; anti-CD33, Cat# 555626, Clone: HIM3-4; anti-CD34, Cat# 555821, Clone: 581; anti-CD203c, Cat# MA5-28586, Clone: NP4D6; anti-FceRI Cat# 334608, Clone: AER-37 (CRA-1)), anti-CD56 (FITC, Cat# 562794, Clone: B159; AF700, Cat# 557919, Clone: B159; BV421, Cat# 562751, Clone: NCAM16.2, or BUV395, Cat# 563555, Clone: NCAM16.2), anti-CD127 (APC, Cat# 558598, Clone: HIL-7R-M21), anti-CRTH2 (PE-Cy7, Cat# 350118, Clone: BM16), and anti-c-Kit (PE, Cat# 555714, Clone: YB5.B8 or BV711, Cat# 313230, Clone: 104D2). Human LSCs were identified by lineage (FITC-anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD19, anti-CD20, anti-CD11b, anti-CD56, and anti-CD235a, Cat# 349104, Clone: HI264), anti-CD45 (BV510, Cat# 368526, Clone: 2D1; or BUV395, Cat# 563791, Clone: HI30), anti-CD34 (PE, Cat# 555822, Clone: 581; FITC, at# 555821, Clone: 581), and anti-CD38 (BV605, Cat# 303532, Clone: HIT2; PE-Cy7, Cat# 560677, Clone: HIT2). To examine intracellular cytokine production, intracellular staining for IFN-γ, TNFα, GZMA, GZMB, perforin, EOMES, and T-BET was performed using a Fix/Perm kit (eBiosciences), followed by staining with an anti-IFN-γ antibody (BV786, Cat# 5563731, Clone: 4S.B3; BV421, Cat# 564791, Clone: 4S.B3), an anti-TNFα antibody (APC, Cat# 502912, Clone: MAb11; BV650, Cat# 563418, clone: MAb11), an anti-GZMA antibody (Pacific Blue, Cat# 507207, Clone: CB9 or APC, Cat# IC29051A, Clone: 356412), an anti-GZMB antibody (PE, Cat# 561142, Clone: GB11), perforin (APC, Cat# 30811, Clone: dG9), anti-EOMES antibody (BUV395, Cat# 567171, Clone: X4-83 or PE, Cat# 566749, Clone: X4-83), or an anti-T-BET antibody (APC, Cat# 644814, Clone: 4B10), respectively. All mouse antibodies were used at 1:200. All human antibodies were used at 1:50, except for CD34, CD38, CRTH2, and CD117, which were used at 1:100. All analyses were performed on a Fortessa X-20 flow cytometer (BD Biosciences), and sorting was performed using a BD FACSAriaTM Fusion.

Single-cell RNA-seq data analysis
The single-cell RNA-seq data were analyzed following the methodology outlined in our previous report, utilizing the same analysis approach as reported22. Briefly, single-cell RNA sequencing data were processed and analyzed using the Scanpy framework. Cells with fewer than 300 detected genes or with a mitochondrial UMI fraction >15% were removed, and genes detected in fewer than three cells were excluded. Potential doublets were identified and filtered out using Scrublet. The remaining count matrices were normalized using scanpy.pp.normalize_total with ‘target_sum=1e4’. To enrich for innate lymphoid cells (ILCs) and minimize T-cell contamination, we retained cells exhibiting positive expression of IL7R (CD127) and negative expression of CD4 and CD8A. Highly variable genes (HVGs) were identified using scanpy.pp.highly_variable_genes. To reduce technical variation, the effects of total UMI counts and mitochondrial transcript percentages were regressed out using scanpy.pp.regress_out on the HVG matrix. Principal component analysis was then performed using scanpy.tl.pca, and the top 30 components were retained. Batch effects were corrected using BBKNN, followed by Leiden clustering (scanpy.tl.leiden, resolution = 1). Uniform Manifold Approximation and Projection (UMAP) embeddings were generated using scanpy.tl.umap with default parameters.

Gene-set activity scores were computed using scanpy.tl.score_genes in Scanpy. Gene signatures were obtained from the MSigDB database. Statistical significance of scored differences between groups was assessed using the Mann–Whitney U test.

Differential expression analysis was carried out using the Wilcoxon rank-sum test implemented in the scanpy.tl.rank_genes_groups. Genes with an adjusted p-value < 0.05 and an absolute log fold change > 0.25 were considered significantly differentially expressed. Differentially expressed gene lists were subsequently subjected to Gene Ontology (GO) enrichment analysis to identify overrepresented biological processes.

In vitro LSCs and ILC1s co-culture assays
Human LSCs from AML patients were labeled with 5mM CellTrace™ Violet dye (CTV) and co-cultured with or without ILC1s (Lin−CD127+CRTH2−CD117−) isolated from the PB of HD in the presence of human IL-12 (10ng/ml) and IL-15 (100ng/ml). For coculture assays with cytokines and antibodies, human LSCs were cocultured with or without IFNγ (50ng/ml), TNFα (20ng/ml), anti-IFNγ antibody (20µg/ml), or anti-TNFα (20µg/ml) antibody. For all coculture assays, after three days of coculture, 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 manufacturer’s instructions.

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

Analysis of FLT3-ITD mutation
Detection of the FLT3-ITD mutation was performed using a conventional PCR technique as outlined by Kiyoi et al. in 199755. Specific primers were designed to amplify both the FLT3-ITD mutant and wild-type FLT3 at exons 14 and 15. The forward primer 11F (5′-GCAATTTAGGTATGAAAGCCAGC-3′) and the reverse primer 12R (5′-CTTTCAGCATTTTGACGGCAACC-3′) were used to produce a 329bp wild-type product. The presence of an additional PCR product longer than 329bp indicated the FLT3-ITD mutant type56. The PCR products were analyzed using 3% agarose gel electrophoresis.

In vivo LSC transplantation assay
Mice were housed in the City of Hope Animal Facility with a 12-h light/12-h dark cycle and temperatures of ~18–23°C with 40–60% air humidity. 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 under protocol numbers 18008, 20003, and 24049. Tumor-bearing mice were monitored twice per week and at more frequent intervals depending on the status of the mice. Mice exhibiting evidence of distress, discomfort, pain, lethargy, inability to properly groom, or inability to obtain food and/or water were killed immediately via CO2 inhalation. Tumor-bearing mice with 20% weight loss from the age-matched controls without receiving tumor cell inoculation were killed.

For the human LSC engraftment experiments related to Fig. 4, 0.5× 104 human LSCs were isolated from the peripheral blood of AML patients and then transplanted via intravenously (i.v.) injection into sublethally irradiated (200cGy) 6–8-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ (NSG-SGM3) mice purchased from the Jackson laboratory. One day later, 5× 104 human ILC1s isolated from the PBMCs of healthy donors were injected via the tail vein into these mice. A control group had no ILC1 injection. Recombinant human IL-15 (2µg/mouse) was injected intraperitoneally (i.p.) into recipient mice daily for 7 days. Engraftment of human CD45+CD33+ and CD34+CD38− cells in the peripheral blood of mice was monitored at three weeks by flow cytometry.

For the human LSC engraftment experiments related to Fig. 6h, i, 0.5× 104 LSCs were injected i.v. into sublethally irradiated (200cGy) 6–8-week-old female NSG-SGM3 mice on day 0. One day later, 5× 104 UCB CD34+ cell-derived CD161− ILC1s were i.v. injected into these mice. A control group had no CD161− ILC1 injection. Recombinant human IL-15 (2µg/mouse) was injected i.p. into recipient mice daily for 7 days.

For the human LSC engraftment experiments related to Fig. 6j, k, 5× 104 LSCs were injected i.v. into sublethally irradiated (200cGy) 6–8-week-old female NSG-SGM3 mice on day 0. One day later, mice were randomized to receive (1) no ILC1 or NK cell infusion; (2) 1.5× 105 UCB CD34+ cell-derived CD161− ILC1s; (3) 1.5× 105 UCB CD34+ cell-derived NK cells; or (4) 1.5× 105 UCB CD34+ cell-derived CD161− ILC1s plus anti-IFNγ (500μg given intraperitoneally every 2 days for 8 days). Recombinant human IL-15 (2µg/mouse) was injected i.p. into recipient mice daily for 7 days.

In vitro ILC1 induction from UCB CD34+ HSCs
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 (20ng/ml), Flt3-L (20ng/ml), IL-7 (20ng/ml), IL-15 (20ng/ml), and IL-3 (5ng/ml) in the first week. One week later, IL-3 was removed from the media. Two weeks later, Flt3-L was reduced to 5ng/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 in six-well plates.

In vitro cytotoxicity assay
NK cells were first enriched from PBMCs using the RosetteSep Human NK Cell Enrichment Kit to remove non-NK cells and red blood cells. Next, total ILCs were enriched to remove non-ILCs using the EasySep™ Human Pan-ILC Enrichment Kit. The CD161+ ILC1s and CD161− ILC1s were sorted from the enriched pan-ILCs to be cocultured with HSCs sorted from HD blood and LSCs from the blood of AML patients at various ratios.

Statistics & reproducibility
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. Paired t test or one-way ANOVA models with repeated measures were used to compare two or more donor-matched groups. 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. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications33. No data were excluded from the analyses. Experimenters were blinded to observe the survival of mice. Otherwise, blinding was not performed, such as during in vitro experiments, where experimenters were required to know the conditions of each well. Data distribution was assumed to be normal, but this was not formally tested. Data are presented as mean ± s.d. Prism software v.10 (GraphPad, CA, USA), and SAS v.9.4 (SAS Institute, NC, USA) were used to perform statistical analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Li et al represents a follow up work of a previous paper recently published in Nat Immunol by the same team, reporting on the tumor-supporting action of ILC1s in AML.\nIn the current work, the authors focus on human ILCs, while the previous work was centered, but not exclusively, on assessing the role of murine ILCs in the interaction with LCSs and in leukemogenesis. While new data are provided, in particular on the in vitro generation of CD161-ILC1 with anti-tumoral functions, several observations have been previously reported by others or by the same group, and no in vivo validation of the antitumor potential of this novel cell-based product has been shown.\nFirst part of the results reporting on the relative ILC1 expansion in AML, with concomitant impaired Type 1 cytokine secretion has already been published by others (E.g., Trabanelli et al, Haematologica, 2015).\nAlthough the authors claim that the mechanisms of action of human ILC1 on LSCs were not reported in the previous work, the pro-apoptotic role of human ILC1 on human LSCs or HSC was already shown by the same group in Ref 8 published in 2022 (Fig 1g-k, n; ext Fig 2e-f; Fig 6b).\nFig2A-F. Although difficult due to low cell numbers, at least for some patients, the authors should perform the same experiments with ILC1s isolated from AML patients, +/- rhIFNg, +/- rhTNFa depending on the experimental setting (i.e., apoptosis vs M2 differentiation vs CFU assay).\nFig2G-J. It would be very informative to have a comparison of the anti-tumor effect of conventional NK cells and ILC1s in vivo. This would support the validity of the claim that producing ILC1s for in vivo adoptive cell transfer is superior to the easier expansion of cNK cell products.\nExtended Figure 1A and 1H-J. Which markers define CD161- ILC1s, and allow to claim that they are ILC1s? The reviewer has this obvious question since the authors use T-bet, Eomes and IFNg to describe these cells (known markers to be expressed by cNKs). Eomes should not be expressed by helper ILC1s. Do these cells express CD200R1, CD16, CD56, KIR, NKG2D, NKG2A, Granzymes, Perforin…before/after expansion?\nIf the in vitro expanded CD161- ILC1s from CB should be used for cellular-based therapies, the authors need to show tumor protective data in vivo in AML bearing animals, as done for the primary human ILC1s.\nHow do the authors define the “ILC1 signature”, and are confident to be specific for ILC1, a cell type that has no specific marker expression? Is the definition based on the use of IFNG, TNFA, TRAIL, CD49A, NKP46 and RORA only, as reported in the methods?\nSide-by-side experiments using LSC and HSC should be performed to determine the effect of human ILC1s and CD161- ILC1s generated by the CB (Ext Fig 1L) on the healthy stem cell counterpart.\nMinor:\nFig1D. Although the p value gives a statistical number, the biological significance of such a correlation “cloud” remains questionable to the reviewer.\nFig 2J. How many mice were used per cohort? Was the experiment performed several times?", "reviewer_2": "Zhenlong LI and Rui Ma et al. showed type I innate lymphoid cells (ILC1s) suppressed leukemic stem cells (LSCs) by production of IFNγ and by blocking differentiation of LSCs toward M2 macrophage-like, leukemia-supporting leukemic blasts through TNF. They further identified human ILC1 subsets as Lin-CD127+CD161-CRTH2-CD117- and tested the possibility of clinical usage of expanded human ILC1s from human umbilical cord blood.\nAuthors showed interesting findings about the interaction of LSCs and recently identified immune cell subsets through in vitro and in vivo experiments. I have several suggestions as below.\nMajor comments>\n1. Z Li et al. used CD34+CD38- cell fractions as human LSCs for co-culture with human ILC1s. CD34+CD38- cell fractions have been widely used for LSC studies using primary AML samples. However, it is also revealed that many true LSCs which are engrafted in immunocompromised mice do not always reside in CD34+CD38- fractions but in CD34+CD38+ or even in CD34- fractions [Stanley N K Ng et al. Nature 2016, 17 gene stemness score in AML]. Have you done experiments using 5 different patients’ samples with AML and every sample was engrafted into mice OR using a divided samples from the same patient for 5 mice groups?\nSince AML is a remarkably heterogenous disease entity, it is very difficult to generalize the cross-talk between LSCs and immune cells using small number of patients’ samples. It would be also good to see the relationships between LSCs and immune cells in view of different mutational subtypes of AML OR only in a specific molecular subtype of AML. (ex. FLT3-mutated AML).\nAt least, detailed description about baseline characteristics, molecular and cytogenetic profile of AML patients used in this experiment is needed.\n2. CD34, CD33, CD133, CD7 and CD13 can be AML markers with aberrant expressions, but inappropriate as LSC markers. Have they seen relationships between ILC signature and AML-LSC signatures previously proposed (ex. MPP-like, LMPP-like, and GMP-like LSCs [Goardon N et al. Coexitence of LMPP-and GMP-like..... Cancer Cell 2011] or LSC-17 gene signature [Stanley N K Ng et al. Nature 2016] for the analyses using public databases?\nMinor comments>\n1. In figure 1B, cell distributions in dot plots are very different between healthy donors and patient with AML. What’s the cause of this difference? Please check single staining control, compensation plot, and positive/negative control in each sample.\n2. Presentation of absolute cell numbers of ILCs and IFN+ ILCs might enhance understanding of readers.\n3. Authors insisted that IFNγ production of ILC1s have an impact on LSCs. Did they measure the IFNγ level by ELISA? Main AML cell niche is bone marrow rather than peripheral blood in many cases. How was the percentage and ILC subsets in AML samples from patients’ BM? Is information from TCGA LAML database the results from PB samples or BM samples?\n4. Please provide representative images of G- and GM- colonies that authors counted.", "reviewer_3": "In this manuscript the author investigated the functional role of human ILC1s in targeting/controlling AML stem cells and development. Although the study contains interesting findings some comments should be addressed. Major points include the following. First, authors claim that human ILC1s are reduced in blood of AML patients compared to healthy donors. But a very limited number of patients have been analyzed, and as AML is very heterogeneous disease and mostly found in elderly, a larger group of patients vs age-matched controls should be analyzed. Further only percentage ILC1s are depicted of the small group of AML patients, but also absolute numbers should be analyzed before concluding that ILC1 numbers are reduced. Secondly, using TCGA database they observed significant positive correlation between ILC1s and leukemia blast/LSC signatures, and prolonged overall survival of AML patients. But ILC1 signature is based on IFNG, TNFA, TRAIL, CD49A, NKp46, RORA, but most of these genes are also expressed by CD56+ conventional NK cells. So it is not clear whether confounding factors with cNK signatures are involved in their analysis? Thirdly, though in figure 2A they show effect on CD34+CD38- AML cells by co-culture with ILC1s or IFNgama, they also show it on percentage (while gating on this subsets is tricky), and not depict CD34+CD38- abs number while they do in Fig 2B? Further, to get the complete picture also the effect on the other CD34+CD38+ and CD34- AML populations should be shown. With regard of the ILC1 dependent blockade and only partial TNFalpha dependent differentiation effect of CD34+CD38- cells into M2-like macrophage-like cells it would be good to poof that these differentiating cells have the AML genetic aberration of the malignant clones. Finally, as the anti-TNFalpha effect is only partially, are others factors involved from ILC1s in this differentiation effects?", "reviewer_4": "In this work by Li et al., the authors investigated the role of human ILC1 in the killing of leukemia stem cells (LSCs). This work is based on a previous report by the same team showing similar findings in the mouse. However, the current study presents several flaws and conclusions are not supported by the data.\nAcute myeloid leukemia (AML) is a very heterogeneous disease. The authors analyzed only 4 patients and did not provide information about the patients.\nThe authors excluded the analysis of CD56+ cells. However human ILC1s occasionally express CD56 (doi: 10.1016/j.bj.2020.10.001).\nFigure 1:\n-Because of the disease, absolute counts have to be shown and not % of cells. Experiments have to be performed with true count beads or similar system.\n-ILC1 are defined by the absence of CD117 expression and CRTH2 expression. There is no marker defined so far to gate on ILC1. In Figure 1B, there is a decrease in the expression of CRTH2 and CD117 but it is not clear if it is a real increased in ILC1 population. Thus, it may explain a poor % of IFNg+ cells. Analysis of the transcription factors could help.\nThe gene signature of ILC1 in Figure 1D is not common (NKp46 is associated to NK cell signature and not ILC1). Please check Bjorklund et al. Nat Imm 2006; Lopes et al., Cell Rep Med 2022.\nFigure 1E: What is the cut off? This representation can be misleading and not statistically relevant with other cut off. Please provide a forrest plot. Also, in the text it is indicated that 53 patients were analyzed but the double is presented in the figure.\nFigure 2:\n-In 2A, the real decrease is observed for the CD38+CD34+ double positive cells. CD34+ cells are not a well-defined population on the graph and the difference between conditions are not convincing.\n-In 2B the number of cells analyzed is too small and not relevant. Statistical analysis on 3 samples is not appropriate.\n-2D please check compensations, the staining is weird. Statistical analysis on 3 samples is not appropriate.\n-2J: experiment was performed once? There is no control with the injection of another cellular cell type. There is no transfer of ILC1 from ILC1 patients or in vivo IFNg blockade\nExtended data A\n-extremely concerning, it is the same dot plot in A and in E and it should not be.\nAgain ,the identification of ILC1 relies on the absence of expression of CD117 and CRTH2. It is not clear if the cells generated are ILC1 or NK cells as in the Hernandes et al., Immunity 2021.\nIt is not clear how the authors have succeeded to purify ILC1 CD161+ from PBMC as there is virtually no cells shown in A.\nIt would have been of much interest to compare the in vivo potential of UCB CD34+ derived CD161- and PBMC ILC1 CD161 in experiment such as in Figure 2H."}, "author_response": "Li et al. NCOMMS-22-50954-T\nPoint-by-Point Responses to Reviewers' Comments:\nWe appreciate the reviewers’ careful evaluation of our data and analyses, which contributes to\nimproving the quality and rigor of our research. Please see the point-by-point responses to the\nreviewers’ comments below. Reviewer #1 (Remarks to the Author): with expertise in ILCs\nReviewer #1 General Comment: The manuscript by Li et al represents a follow up work of a previous\npaper recently published in Nat Immunol by the same team, reporting on the tumor-supporting action\nof ILC1s in AML.\nIn the current work, the authors focus on human ILCs, while the previous work was centered, but not\nexclusively, on assessing the role of murine ILCs in the interaction with LCSs and in leukemogenesis.\nWhile new data are provided, in particular on the in vitro generation of CD161-ILC1 with anti-tumoral\nfunctions, several observations have been previously reported by others or by the same group, and no\nin vivo validation of the antitumor potential of this novel cell-based product has been shown.\nResponse: Thank you for the reviewer's comments. In the human part of our previously published\nwork, our focus was solely on the examination of human ILC1-induced LSC cell death in vitro. In the\nprevious publication, we did not delve into whether human ILC1s could influence human LSC\ndifferentiation, nor did we investigate whether human ILC1s possess an anti-AML function in vivo.\nIn the current manuscript, we present novel findings that reveal the mechanism by which the in vitro\nobservations occur, and we combine this mechanistic work with our first in vivo studies in the human\nsystem. We reveal that the production of IFNγ by ILC1s contributes to the reduction of CD34−CD38+\ncells that were differentiated from LSCs, while TNFα production by ILC1s suppresses CD34+CD38+\ncells that were derived from LSCs differentiation into CD34−CD38+ cells. Interestingly, the decrease in\nCD34−CD38− cell numbers induced by ILC1s was not suppressed by either IFNγ or TNFα, indicating\nthat additional factors or pathways, independent of these cytokines, may play a role in regulating\nCD34−CD38− cell numbers in the context of ILC1-LSC interactions (Fig. 2, in the revised manuscript,\nrecapitulated below for the reviewer’s convenience). Additionally, we demonstrate that human ILC1\nproduction of TNFα impedes LSC differentiation into M2 macrophage-like leukemia-supporting cells,\nwhich have been shown to support leukemia growth (Fig. 3a-3h, in the revised manuscript,\nrecapitulated below for the reviewer’s convenience). Finally, we demonstrated that these combined\neffects play a crucial role in limiting human leukemogenesis in vivo (Fig. 4, in the revised manuscript,\nrecapitulated below for the reviewer’s convenience). [...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the additional work performed by the authors to substantiate their findings. However, in my opinion, the study and the presented data remain problematic in 3 aspects: novelty of the findings, accuracy of ILC1 definition, and potential advantage of the use of ILC1 in translational settings in AML patients.\nSpecific comments:\n1) The authors outline the characterization of novel findings as compared to previous work by them and by others. The novelty mainly resides in the capacity of human ILC1s to alter M2 polarization of AML cells in vitro, an observation that was not shown previously and that is interesting. However, the in vivo relevance of this observation has not been tested and the TNF blockade in vitro only has partial effect, suggesting that other factors might have even stronger effects.\nThroughout the manuscript, the definition of human ILC1s (as also pointed out by Reviewer 4) remains problematic, to adequately distinguish ILC1s from NKs and T cells.\nThe new scRNAseq performed after EasySep™ Human Pan-ILC Enrichment Kit purification gives very different results as compared to a very similar scRNAseq recently published by Jaeger et al, Nat Immunol, 2024. In that work, bona fide human ILC1s are not identified in the peripheral blood, but only in tissues. The authors don’t refer to this work, while they should verify the identity of their « ILC1s » with respect to both NK cells and T cells.\nAs mentioned previously (also by Reviewer 4), the signature used to define ILC1s for survival analyses includes NKp46, which is not an ILC1 marker, since it is expressed by a large proportion of NK cells. This analysis does not allow correlation of ILC1 with survival, since it includes NK cells. On top of this, the results are not statistically significant (p=0.1, p=0.6). Hence, conclusions on the impact of increased ILC1 (high ILC1) on survival are not supported by the data.\nI believe that the population studied in this work is comprised in “group 1 ILC1s”, sharing attributes from both NK cells and helper ILC1s.\n2) I thank the authors for the new in vivo experiment.\nThe new experiment presented in Fig 6x does not show a significant advantage of using UCB-expanded ILC1 as compared to conventional NK cells. Therefore, the overall claim that : “ILC1 generated from UCB CD34+ cells can be manufactured ex vivo for improved treatment of AML in vivo”; “provide a rationale for using HD ILC1s as a cellular-based therapy to extend disease-free survival in AML”, even if biologically true, would be too time-consuming and costly in the clinics, as compared to NK cell manufacturing. This aspect is not at all discussed.", "reviewer_3": "The authors adequately addressed all my comments and performed additional experiments to verify their claims. The work is original and extends the knowledge of ILC1 cells in limiting human leukemogenesis.", "reviewer_4": "Additional experiments and comments provided by the authors are truly appreciated and extremely relevant to confirm their findings. The revised manuscript is greatly improved and of high quality."}, "author_response": "Point-by-Point Responses to Reviewers' Comments:\nWe appreciate the reviewers’ careful evaluation of our data and analyses, which contributes to\nimproving the quality and rigor of our research. Please see the point-by-point responses to the\nreviewers’ comments below. We are happy to see Reviewers #3 and #4 found our revised manuscript\nacceptable. Below we provide some final comments to the thoughtful review provided by Reviewer #1.\nReviewer #1 (Remarks to the Author)\nReviewer #1 General comment: The manuscript by Li et al represents a follow up work of a previous\npaper recently published in Nat Immunol by the same team, reporting on the tumor-supporting action\nof ILC1s in AML.\nIn the current work, the authors focus on human ILCs, while the previous work was centered, but not\nexclusively, on assessing the role of murine ILCs in the interaction with LCSs and in leukemogenesis.\nWhile new data are provided, in particular on the in vitro generation of CD161-ILC1 with anti-tumoral\nfunctions, several observations have been previously reported by others or by the same group, and no\nin vivo validation of the antitumor potential of this novel cell-based product has been shown.\nResponse: We appreciate the Reviewer’s comments. Below, we provide a detailed response to each\npoint. [...]"}, {"round": 3, "reviewer_comments": {"reviewer_1": "I thank the reviewer for the consideration of my comments and for the re-analysis of the Jaeger dataset.\nGiven that the authors have now removed the clinical correlations analyses (that was though one of the claimed novelty of this work) given the lack of statistical power, they should remove the method section that was correlated to it (Page 16, lines 434-439).\nRegarding the in vivo experiment in NSG animals, providing evidence of the leukemia control by ILC1, I remain confused. In the method section, it is stated that a group of mice received purified NK cells. Are these the control animals? Or if the control animals received only vehicle (what I assume), can the authors please provide the data of the animals injected with the NK cells? I did not find these very relevant results."}, "author_response": "Point-by-point responses to the reviewer’s comments:\nREVIEWER #1\nReviewer #1 comment #1: Given that the authors have now removed the clinical correlations\nanalyses (that was though one of the claimed novelty of this work) given the lack of statistical\npower, they should remove the method section that was correlated to it (Page 16, lines 434-439).\nResponse: We appreciate the reviewer’s comment and apologize for our oversight. We have now\nremoved the method section related to the clinical correlation analyses from the revised\nmanuscript.\nReviewer #1 comment #2: Regarding the in vivo experiment in NSG animals, providing evidence\nof the leukemia control by ILC1, I remain confused. In the method section, it is stated that a group\nof mice received purified NK cells. Are these the control animals? Or if the control animals\nreceived only vehicle (what I assume), can the authors please provide the data of the animals\ninjected with the NK cells? I did not find these very relevant result.\nResponse: We apologize for the lack of clarity in the methods section regarding our in vivo\nexperiments. The confusion may have arisen because we summarized the methods for three\ndifferent in vivo models together. One of the three models included NK cell control, while the other\ntwo did not. To prevent this issue, we have now described the methods for each of the three\nmodels separately (Pages 17 and 18), which we have copied and pasted below for the reviewer’s\nconvenience. We hope these changes improve the clarity. [...]"}]

Decision letter