Functions and mechanisms of ILC2s in trained immunity

Trained immunity is a process that is activated in hematopoietic stem and progenitor cells (HSPCs) in response to inflammatory stimuli such as severe infection and cancer. Evidence has now shown that following the resolution of inflammation, HSPCs maintain an altered epigenetic program over time. This process, termed “trained immunity” is a type of epigenetic memory that can result in robust immune responses to subsequent inflammatory challenges. Despite growing evidence of its importance in regulating responses to inflammation, the cellular players and molecular pathways involved in controlling the epigenetic responses that lead to trained immunity are poorly understood. In preliminary studies, we performed scRNA-seq in sorted bone marrow immune cells following challenge with β-glucan, a stimulus which induces trained immunity and robust secondary protection against tumor challenge. This analysis identified a population of bone marrow ILC2s that upregulated cytokine expression following β-glucan challenge, suggesting these cells may play a key role in trained immunity. Strikingly, depletion of ILC2s abolished the protective phenotype of β-glucan in tumor challenge, confirming that ILC2s play an important role in this system. Based on this data, we propose that ILC2s play a critical role in the establishment of trained immunity. While ILC2s were not found within the tumor, they regulated neutrophil differentiation in the tumor microenvironment, blocking acquisition of a pro-tumor phenotype, and maintaining an anti-tumor phenotype. However, how neutrophils are functionally altered by ILC2s in β-glucan training and which of these functions culminates in protection from tumor challenge is not known. Further, whether ILC2s modulate responses in monocytes and macrophages or respond to additional proinflammatory microbial ligands to initiate trained immunity has not been studied. Here we interrogate these questions by (i) defining the functional alterations dependent on ILC2s in tumor-infiltrating myeloid cells in trained immunity; (ii) determining the epigenetic and transcriptomic mechanisms in bone marrow progenitors and mature neutrophils in trained immunity; and (iii) interrogation of the gene expression pathways regulated in ILC2s by inflammatory challenges. Altogether, completion of these studies will fundamentally advance our understanding of the regulation of trained immunity. Furthermore, as trained immunity is actively investigated as an intervention in the clinic, modulation of the ILC2-neutrophil pathway could be a novel therapeutic target for the treatment of inflammatory diseases and cancer. Completion of these studies will establish the groundwork for future efforts to identify key cellular and molecular pathways involved in regulating trained immunity in vivo. Finally, while we considered alternatives to animal models for this work, trained immunity involves integrated multiorgan processes, such as activation of ILC2s, reprogramming of HSPCs in the bone marrow, and skewing of neutrophil responses in the tumor microenvironment, all of which depend on intact systemic physiology and cellular crosstalk that cannot be recapitulated in vitro, necessitating the use of animal models in our studies. Project Number: 1R01CA311426-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Richard Flavell | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $684,252 | Activity Code: R01 | Study Section: Basic Cancer Immunobiology Study Section[BCIB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11340914