Mechanisms Of Memory ILC1 Formation And Long-term Maintenance

Innate lymphoid cells(ILCs)are a heterogeneous populations of innate immune cells that are important for host defense and homeostasis.The ILC family consists of NK cells,ILCls,ILC2s,ILC3s,ILCregs,which mirror the development and functions of CD8+ T cells,Thl,Th2,Th17 cells,and Treg cells.Although ILCs belong to the innate immune system,accumulating evidence indicates that they have adaptive immune features.Evidence has emerged that group 1 ILCs,consisting of conventional NK(cNK)cells and ILC1s,are able to generate a long-term memory response against haptens,mouse cytomegalovirus(MCMV),and cytokine stimulation.CD49a+ liver-resident NK cells,recently referred to as ILC1s,were defined as a subset that confers hapten-specific memory responses;however,little is known about memory ILC1 formation and maintenance.In this study,we describe the phenotype of memory ILC Is and reveal the mechanisms of their memory formation and long-term maintenance in detail.1.Liver IL-7R?~+ILC1s mediate hapten-specific memory responsesPrevious studies have indicated that memory group 1 ILCs are restricted to the liver in CHS models.Liver group 1 ILCs can be divided into three subsets by the expression of CD49a and IL-7R?:CD49a-IL-7R?-cNK cells,CD49a+IL-7R?-LrNK cells and CD49a+IL-7R?~+ ILC1s.We found that the percentage and absolute number of ILC1s,but not cNK cells or LrNK cells,increased 2-fold increase 60 days after hapten sensitization.Compared to cNK cells,both ILC Is and LrNK cells constitutively expressed higher levels of CXCR6 and Thy-1,which are associated with liver group 1 ILC-mediated CHS,suggesting the possibility that IL-7Ra+ILC1s may participate in hapten-induced memory responses.Adoptive transfer of hapten-primed liver IL-7Ra+ILC1s elicited significant CHS responses of recipient mice in a hapten-specific manner.Furthermore,these IL-7R?~+ILC1s can accumulate at the effector site,with an approximately 20-fold increase.Thus,liver IL-7R?~+ILC1s are the cells responsible for the hapten-specific memory responses in CHS model.2.IL-7R?~+ILCls acquire memory potential in skin-draining lymph nodesAfter hapten sensitization,IL-7R?~+ ILC1s in skin-draining LNs initially increased both in frequency and in cell numbers,peaking at 48 h.LN IL-7R?~+ ILC1s from hapten-sensitized(48 h)mice produce significantly higher amounts of TNF-?and IFN-?,suggesting they are primed by haptens.However,expression of the Ki67,in LN IL-7R?~+ILC1s was slightly decreased at 48 h,suggesting the increase of LN IL-7R?~+ILC1s is not due to cell proliferation,but is the result of cell migration.We observed that IL-7R?~+ILC1s expressed high levels of CXCR3 and that the mRNA levels of Cxcl9 and Cxcl10 increased at 48 h in skin-draining LNs after hapten sensitization.Moreover,increased recruitment of IL-7R?~+ ILCls to the ILN and ALN was abolished in Cxcr3-/-mice,suggesting that migration of IL-7R?~+ ILC1s to skin-draining LNs is CXCR3 dependent.Phenotypic analysis revealed that LN IL-7R?~+ ILC1 expressed high levels of surface markers associated with group 1 ILC1-mediated CHS responses,including CXCR6,Thy-1,CD62L,CD 18,NKG2D and CD49a.Interestingly,LN IL-7R?~+ILC1s,rather than IL-7R?-cNK cells,can transfer long-term CHS responses into naive recipient mice.These results show that IL-7R?~+ILC1s acquire their memory characteristics in skin-draining LNs.3.LN-derived memory ILCls selectively reside in the liver for long-term maintenanceDuring the contraction phase,LN IL-7R?~+ ILC1s did not exhibit increased apoptosis(7AAD+),suggesting that the contraction could be due to ILC1 egress from skin-draining LNs.Mice treated with FTY-720 exhibit increased IL-7R?~+ ILC1s at 72 h after hapten sensitization,suggesting that IL-7R?~+ ILC1 egress from LNs relies on S1PR1,similar to T cells.To find out the homing site of LN-derived memory ILC1s,we adoptively transfer the sensitized LN IL-7R?~+ ILC1s into recipient mice,and found that the donor cells reside only in recipient liver two months later.By comparing the migration of Cxcr6-/-LN group 1 ILCs with their WT counterparts,we found that the migration of Cxcr6'-/-LN ILC1s to the recipient liver was inhibited significantly.Furthermore,liver IL-7R?~+ILC1s from OXA-sensitized(96 h)ILN-deficient mice could not transfer CHS responses in naive mice.Collectively,these results indicate that the liver could serves as a unique niche for for the residency and long-term homeostasis of LN-derived memory ILC1s.4.IL-7R signaling is necessary for memory ILC1 longevityMemory ILC1s underwent three steps,including memory formation phase,long-term maintenance phase and recall response phase.To investigate the contribution of IL-7R? to memory ILC1s,we treated hapten sensitized Rag1-/-mice at different time point and we found that long-term blockade of IL-7Ra impaired the skin inflammation.While blocking IL-7R? in OXA-sensitized Rag1-/-mice at memory formation phase and recall response phase did not affect CHS responses.As liver sinusoidal endothelial cells(LSEC)constitutively secrete IL-7,IL-7R signaling has a critical role for the long-term maintenance of memory ILC1s in the liver.Conclusion:We show that memory ILCls,distinguished by IL-7Ra expression,are generated in lymph nodes(LNs)and maintain long-term survival in the liver through IL-7R signaling.Hapten sensitization initiates recruitment of IL-7R?~+ ILC1s into skin-draining LNs,in a CXCR3-dependent fashion,where they are primed and acquire hapten-specific memory potential.Memory IL-7R?~+ ILC1s then exit draining LNs and preferentially reside in the liver via CXCR6.Moreover,IL-7R signaling is critical in maintaining longevity of memory ILC1s in the liver.Thus,our results identify a memory IL-7R?~+ ILC1 population and reveal that a LN-liver axis is essential for memory formation and long-term maintenance of ILC1s.