Human ILC1s Target Leukemia Stem Cells and Control Development of AML

Abstract

Acute myeloid leukemia (AML) is a devastating disease with a median 5-year survival of only 40-45% for patients younger than age 65 who are treated with standard chemotherapy. Although in some cases allogeneic stem cell transplantation has proven to be curative, the treatment-related mortality and the risk for disease relapse due to persistence of leukemia stem cells (LSCs) remains relatively high. Therefore, safer and more effective novel therapeutic approaches are needed to improve the clinical outcomes of patients with AML. Innate lymphoid cell (ILC) is critical in mediating immune responses and regulating tissue homeostasis and inflammation. We recently reported that mouse ILC1s contribute to the control of AML by eliminating LSCs and inhibiting their differentiation into myeloid blasts, and functional impairment of mouse ILC1s in AML leads to the outgrowth of LSCs and disease relapse (Li et al., Nature Immunology. 2022). However, the full role and mechanistic characterization of human ILC1s in anti-tumor responses to AML remains to be fully explored. Upon analysis of ILC1s in the blood of patients with AML at the onset of disease, we observed a highly significant reduction in the total ILC1s count among lineage-negative cells (Lin −, defined asdepletion of CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FceRI, and CD56 positive cells) relative to healthy donors ( p = 0.031, n = 6 healthy donors; n = 4 patients with AML). Further, functional ILC1s positive for IFNγ and DNAM-1 were significantly reduced in the patients with AML compared to healthy donors. Analysis of 106 AML cases from the Cancer Genome Atlas (TCGA) showed that AML patients with a high ILC1 gene signature had a significantly prolonged overall survival compared to AML patients with a low ILC1 gene signature. By directly interacting with LSCs, human ILC1s can eliminate LSCs via the production of IFNγ. Through Wright-Giemsa staining, we observed that ILC1s blocked the differentiation of CD34 +CD38 − cells into macrophage-like leukemia-supporting cells, which were previously reported to support the growth of leukemic cells rather than inhibit them. Flow cytometry of these differentiated cells showed that some exhibited the tumor-promoting phenotype with expression of CD11b and CD206. These macrophage-like leukemia-supporting cells significantly decreased when co-cultured with ILC1s. This differentiation is at least partially dependent on TNF secreted by ILC1s. We also performed an in vivo transplantation experiment, in which human CD34 +CD38 − cells and human ILC1s were co-injected intravenously (i.v.) into NOD.Cg- Prkdc scid Il2rg tm1WjlTg (NSG-SGM3) mice that express human IL3, GM-CSF, and SCF to support the stable engraftment of myeloid lineages. Injection of human ILC1s from healthy individuals reduced the LSC engraftment into these mice and suppressed the progression of AML. This was evidenced by a significant decrease in the number of CD45 +CD33 + blast cells, CD34 +CD38 − LSCs, and significantly prolonged survival of the mice ( p = 0.0118, n = 5/group). These results were all in comparison to mice that did not receive an injection of ILC1s. Although ILC1s target LSCs, they have no toxicity on normal HSCs. For a therapeutic purpose, we were able to derive ILC1s from umbilical cord blood (UCB) CD34 + hematopoietic stem cells (HSCs) with over 700-fold expansion and the expanded ILC1s demonstrate authentic ILC1 phenotypes and functions. Moreover, arming the expanded ILC1s with a chimeric antigen receptor (CAR) targeting FLT3, which we previously constructed (Chen et al., Leukemia. 2017), enhanced their effector function. In conclusion, ILC1s in patients with AML are impaired, while a high expression of the ILC1 gene signature is associated with better overall survival. Human ILC1s can both eliminate LSCs via production of IFNγ and block LSC differentiation into tumor-promoting macrophage-like cells through TNF. These effects converge to limit leukemogenesis in vivo. The use of UCB CD34 + HSCs to generate ILC1s, especially after being engineered with a CAR, could allow for a readily available supply of ILC1s to be produced for human adoptive transfer studies. Our findings provide evidence that targeting human ILC1s may be a promising therapeutic approach for extending disease-free survival in patients with AML.