Research On Liver ILC1s

It is extensively conceived that group 1 ILCs include circulating conventional NK(cNK)cells and type 1 innate lymphoid cells(ILC1s)locating in different tissues,and the latter in liver was firstly identified as liver-resident NK cells by our group.Moreover,liver ILC1s account for a relatively large fraction among liver ILCs,making the liver as an important window to study ILC1s.Previous studies on liver ILC1s have shown their functional diversity and specificity.For instance,liver ILC1s can mediate antigenspecific recall responses in murine models of hapten-induced contact hypersensitivity(CHS);they secrete interferon-y(IFN-y)more rapidly than cNK cells upon viral infection.Moreover,in contrast to poor cytotoxic mucosal ILC1s,liver ILC1s have strong cytotoxic potential.Whether such functional diversity and specificity reflect the heterogeneity within the ILC1 population remains unclear.Furthermore,in early life,adaptive immune responses are limited,and the innate immune system may have greater importance than in adulthood.Interestingly,ILC1s are predominant over cNK cells during the embryonic and neonatal periods.However,the roles of neonatal liver ILC1s during early life remain unexplored.In this study,we begin to reveal the heterogeneity of liver ILC1s and to further investigate the functions of each subset.1.Dissecting liver group 1 ILC heterogeneity by scRNA-seqCombining single-cell RNA sequencing and flow cytometric analysis,we found that hepatic group 1 ILCs were grouped into five main clusters(C1-C5),in which C1-C3 clusters were representing three developmental stages of cNK cells;C4-C5 clusters were representing two subpopulations of liver ILC1s.2.Two distinct subsets exist in liver ILClsWe further confirmed the heterogeneity of liver ILC1s at protein level with flow cytometric analysis.We found that Ly49E expression could dissect liver ILC 1 s into two distinct subsets.Furthermore,adoptive transfer experiments and transcriptional analysis both indicated that Ly49E+and Ly49E-cells are likely two independent and distinct subpopulations in liver ILC1s.3.Ontogeny dynamics of two liver ILC1 subsetsThe ontogeny dynamic study revealed that these two populations developed in different waves with distinct molecular signatures.Meanwhile Ly49E+ILC1s were independent of postnatal bone marrow hematopoiesis.Combining with the transcriptional analysis,we found that neonatal Ly49E+ILC1s exhibited the potential of early immune responding to stimuli.4.Ly49E marks a highly cytotoxic subset of liver ILClsTo wonder whether Ly49E+and Ly49E-liver ILC1s had distinct immunologic functions with each other,we stimulated liver lymphocytes with different stimulus and found that Ly49E+ILC1s expressed higher levels of granzyme family molecules than Ly49E-ILC1s and performed more cytotoxic.5.Ly49E+ILCls confer protection against MCMV infection in neonatal miceIn order to explore the immune function of neonatal liver Ly49E+ILC1s,we built murine cytomegalovirus(MCMV)infection model in newborn mice.We found that Ly49E+ILC1s deficient mice were more susceptible to infection,however adoptive transfer of liver Ly49E+ILC1s effectively reduced the viral loads.6.Ly49E-liver ILCls exhibit immune memory potentialConsidering that adaptive immunity is acquired gradually during development but not inherited,and Ly49E-ILC1s were gradually increased with age,we speculated that Ly49E-ILC1s might participate in adaptive immunity.We found that Ly49E-ILC1s expressed immune memory-associated molecules including IL-7Rα,and functionally exerted memory potential in response to haptens in CHS models of adults.Altogether,we found that Ly49E expression could separate liver ILC1s into two subsets.Specifically,Ly49E+ ILC1s with strongest cytotoxicity are predominantly present in neonatal mouse liver and play an essential role in host defense against MCMV infection during early life.In contrast,Ly49E-ILC1s,which are gradually increased with age,occupy most of the adult liver ILC1s and primarily derived from postnatal hematopoiesis,preferentially express memory-associated molecules and have memory potential in CHS models.Thus,these findings reveal a dynamic change of ILC1 composition during ontogeny that corresponds to their functional diversity in order to adapt to age-related immune demands during different periods of life.