Delineating Hierarchical And Spatiotemporal Development Of Human Fetal Innate Lymphoid Cells

The discovery of innate lymphoid cells(ILCs)was recognized as one of the top 20 advances in immunology in the last 20 years by Nature Reviews Immunology[1].They lack specific antigen receptors expressed on T cells and B cells.A common nomenclature was firstly proposed by Hergen Spits and his colleagues in 2013 and have been updated in 2018[2,3].Shortly,they could be divided into 3 groups or 5 subsets.NK(nature killer,NK)and ILC1 belong to Group 1 ILC.ILC2 represents Group 2 ILC.ILC3 and LTi(lymphoid tissue inducer,LTi)cell belong to Group 3 ILC[4,5].They are known as innate counterpart of T cells considering their similarities[6,7].Firstly,both of them are derived from hematopoietic stem cells via lymphoid-primed multipotent progenitors and common lymphoid progenitors in bone marrow in adult[8].Secondly,the transcriptional factors guiding their development and maturation and cytokines released by each subset correspond to different types of T cells[6,9].It’s reported that the cytokine-producing ILCs play an important role in maintaining mucosal tissue homeostasis,rapid anti infection and promoting tissue repair[10,11],indicating their potential clinical use in mucosal related disease such as colorectal cancer and non-small cell lung cancer.Whereas the critical roles of ILCs in adult are increasingly appreciated,their developmental hierarchy in early human fetus remains largely elusive,which has instructive significance on regeneration of functional ILCs.In this study,we designed an enrichment flow sorting strategy to enrich innate lymphocytes and their possible precursors and progenitors.Then we sorted cells from the fetal hematopoietic(liver),lymphoid(thymus and spleen)and non-lymphoid(intestine,lung and skin)tissues,span 8 to 12 post-conception weeks,for single-cell RNA-sequencing.After quality control and doublets exclusion,a total of high-quality31,233 cells were captured,followed by computational analysis and functional validation at bulk and single-cell levels.We delineated the early phase of ILC lineage commitment from hematopoietic stem progenitor cells,which mainly occurred in fetal liver and intestine,where represent the main hematopoietic site at this stage and reported ILC-rich mucosal organ,respectively.We further unveiled that interleukin-3 receptor could be a surface marker for the lymphoid progenitors in fetal liver with T,B,ILC and myeloid potentials.Our functional data further indicated that IL-3RA+ lymphoid progenitors(Lin-CD45+CD127+CD34+)had multiple lineage potentials while IL-3RA-lymphoid progenitors were predominantly B-lineage committed.We further resolved the specification of three groups of ILC from ILC-committed progenitors.The data suggest that all three groups contain a subset with high expression of proliferation signature genes(TUBA1B,TUBB,MKI67,HMGB2).Besides,the proportion of them were highest at week 8,then progressively reduced in week 10 and week 12,suggesting that they may occur earlier than other subsets.So,we named these proliferating subsets as precursors of each ILC.Heterogeneity of each ILC was also investigated.Each group of ILC has subpopulations with obvious tissue distribution preference,such as ILC1_d in Group 1ILC,which is in a relatively specific stage of development and is mainly distributed in the skin.In Group 3 ILC,ILC3_b and ILC_c were mainly distributed in the lymphoid organs including thymus and spleen,and showed lymphoid inducer cell like features.In Group 2 ILC,an unconventional subset mainly present in thymus attract our attention.Classical ILC2 marker CRTH2 encoded by PTGDR2 showed very low expression in them.Moreover,they were featured by high expression of BCL11 B,which is essential for T cell-fate commitment.And CCR9 which is necessary for seeding of T progenitors showed highest expression in them.Flow cytometric analysis determined that this subset(Lin-CD45+CD127+CRTH2-CD117-CCR9+GATA3+)truly exists and enriched in the fetal thymus.The discovery of this subset further suggests that ILC2,especially the non-classical ILC2 subpopulation,may be essential for thymic organogenesis,thymus seeding progenitors’ colonization and T-cell development and maturation.Taken together,our study constructed a single-cell transcriptomic profiling covering the ILC lineage commitment,specification and maturation in human embryos.We unveiled interleukin-3 receptor as the surface marker for the lymphoid progenitors in fetal liver with T,B,ILC and myeloid potentials.Notably,we determined the heterogeneity and tissue distribution of each ILC subpopulation,revealing the proliferating characteristics shared by the precursors of each ILC subtype.Additionally,a novel unconventional ILC2 subpopulation(CRTH2-CCR9+ ILC2)was identified in fetal thymus.All these findings provide important theoretical support and database resources for the study of the development rules and regulatory mechanism of human early ILCs,as well as the in vitro regeneration strategy of ILCs,T and B lymphocytes.