Efficient In Vitro Generation of IL-22-Secreting ILC3 From CD34+ Hematopoietic Progenitors in a Human Mesenchymal Stem Cell Niche

Abstract

Innate lymphoid cells (ILCs) and in particular ILC3s have been described to be vital for mucosal barrier functions and homeostasis within the gastrointestinal (GI) tract. Importantly, IL-22-secreting ILC3 have been implicated in the control of inflammatory bowel disease (IBD) and were shown to reduce the incidence of graft-versus-host disease (GvHD) as well as the risk of transplant rejection. Unfortunately, IL-22-secreting ILC3 are primarily located in mucosal tissues and are not found within the circulation, making access to them in humans challenging. On this account, there is a growing desire for clinically applicable protocols for in vitro generation of effector ILC3. Here, we present an approach for faithful generation of functionally competent human ILC3s from cord blood-derived CD34+ hematopoietic progenitors on layers of human mesenchymal stem cells (MSCs) generated in good manufacturing practice (GMP) quality. The in vitro-generated ILC3s phenotypically, functionally, and transcriptionally resemble bona fide tissue ILC3 with high expression of the transcription factors (TF) RorγT, AHR, and ID2, as well as the surface receptors CD117, CD56, and NKp44. Importantly, the majority of ILC3 belonged to the desired effector subtype with high IL-22 and low IL-17 production. The protocol thus combines the advantages of avoiding xenogeneic components, which were necessary in previous protocols, with a high propensity for generation of IL-22-producing ILC3. The present approach is suitable for the generation of large amounts of ILC3 in an all-human system, which could facilitate development of clinical strategies for ILC3-based therapy in inflammatory diseases and cancer.

ILC3: a case of conflicted identity

“…In-vitro culture of enriched human ILC3 is a prolonged process that requires several growth factors (SCF, IL-7, Flt-3L, IL-2, IL-15). The success of ILC3 propagation in vitro can depend upon a layer of cells to support ILC3 proliferation (such as mesenchymal stem cells) ( 79 ). Mouse-enriched ILC3s are usually cultured with SCF and IL-7 and on occasion with the addition of IL-1β, IL-12, or IL-23.…”

“…Having in mind the required precision in ILC3 phenotyping, sorting, and culture conditions and the paucity of tissue ILC3, as well as the limited availability of organs/tissues for their isolation, the use of ILC3 for human cell therapy seems to be complex and unrealistic. Still, alternative strategies, such as propagating tissue-like ILC3 from CD34 + hematopoietic progenitors ( 79 ), could overcome the above-stated obstacles on the road toward ILC3-based therapy.…”

Editorial: Global excellence in cellular immunology: Europe 2021

“…Umbilical cord blood (CB) is another home of immune and hematopoietic cells of basic and applied interest. It not only is a source for allogeneic stem cells used for transplantation, but also holds great promise as a source for cell-based tumor therapy and the in vitro generation of immune cells with therapeutic potential that are rare in peripheral blood such as innate lymphoid cells (ILCs) and plasmacytoid dendritic cells (8)(9)(10)(11). Here, Greppi et al have identified a novel subpopulation of NK cells in CB that express the checkpoint inhibitor programmed death receptor 1 (PD1).…”