Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.
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The RNA-seq and ATAC-seq data are deposited in Gene Expression Omnibus database under the accession number GSE128906. The data that support the findings of this study are available from the corresponding author upon request.
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We thank members of the labs of T.E.O., C.S.L., J.R.C., A. Iwasaki and J.C.S. for helpful comments and discussions. We thank J. Reichel and S. Jonjic for reagents. E.S. was supported by the National Institutes of Health (NIH) Medical Scientist Training Program training grant (no. T32GM007205). N.M.A. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH (no. T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD/PhD Program) and an F30 Predoctoral Fellowship from the National Institute of Allergy and Infectious Diseases of the NIH (no. F30 AI136239). J.C.S. was supported by the Ludwig Center for Cancer Immunotherapy, the Burroughs Wellcome Fund, the American Cancer Society and grants from the NIH (nos. AI100874, AI130043 and P30CA008748). T.E.O. was supported by the NIH (nos. P30DK063491 and AI145997).
The authors declare no competing interests.
Journal Peer Review Information: Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Integrated supplementary information
Supplementary Figure 1 Related to Figure 2. Phenotypic and functional stability of Liver ILC1 during MCMV challenge.
(a) Flow cytometry gating strategy for identifying Liver ILC1. (b) 4×104 liver ILC1 (Lin-NK1.1+ CD49b-CD200r1+CD11b-Ly49H-) were sort purified from CD45.1+ mice, adoptively transferred i.v. into Ly49H-deficient CD45.2+ WT hosts, and subsequently infected with MCMV. Histograms show indicated cell surface markers on liver ILC1 and mNK cells from uninfected WT mice and adoptively transferred liver ILC1 recovered 7 days PI. Data are representative of 3 independent experiments with n=3 mice per group.
Supplementary Figure 2 Related to Figure 3. Memory T cell populations increase the expression of cytokine receptors following the resolution of infection.
(a,b) Graph shows average mRNA expression of indicated cytokine receptors in splenic OT-I CD8+ T Cell sorted from (a) Listeria-OVA and (b) VSV-OVA infected mice at indicated time-points PI, as assessed by microarray (Data provided by Immunological Genome Consortium. (c-d) Graph shows mRNA expression of indicated cytokine receptors by (c) naïve and LCMV experienced primary and secondary memory GP66-CD4+ T cells post LCMV infection and by (d) resting and inflammation experienced memory Tregs, assed by RNA-sequencing as was previously reported23. Data are representative of 2 independent experiments with (c) n=3 and (d) n=5 mice per group. Data are presented as the mean ± SEM.
Supplementary Figure 3 Related to Figure 4. Il-18rα+ ILC1 display enhanced IFN-γ production following stimulation of activating receptors.
WT mice were infected with MCMV (i.p.) and liver was harvested and analyzed 30 days PI. Graph shows percentage of IFN-γ+ cells within indicated liver ILC1 populations following plate-bound stimulation with either media alone, αNKp46, or αNK1.1 plate-bound antibodies compared to uninfected mice. Data are representative of 3 independent experiments with n=3 mice per group. Samples were compared using a two-tailed Student′s t test, and data are presented as the mean ± SEM (*p<0.05).
Supplementary Figure 4 Related to Supplementary Figure 5. Memory ILC1 have distinct transcriptome and epigenomes compared to naïve ILC1.
(a) Gene set enrichment analysis (GSEA) of genes upregulated in D35 IL-18rα+ ILC1 over D0 ILC1 from genes upregulated in effector memory T cells (TEM) (left) or resident memory T cells (TRM) (right) compared to naïve T cells after LCMV infection, assessed by RNAsequencing as was previously reported32. (NES, normalized enrichment score; FDR, false discovery rate; NES, normalized enrichment score assessed by an empirical phenotype based permutation test assuming a null distribution). (b) Absolute numbers and proportion of all peaks (23016 total) and differentially accessible (DA) peaks in peak atlas (373 total, p value less than 0.2). (c) MA plots of differentially accessible regions (red dots) of all peak types comparing D0 vs D35 IL-18rα+ ILC1. (d) Representative ATAC-sequencing tracks show accessible regions for Rora, Itgb3, and Il7r in naïve and memory ILC1. Y axis depicts normalized counts, while X axis displays genomic axis with scale bar. Data are representative of 2 replicate experiments with n=2 samples of n=20 mice per condition. All adjusted p values were indeed determined using DESeq2 and were two-sided.
Supplementary Figure 5 Related to Figure 6. Induction of ILC1 memory is MCMV specific and cannot be formed by other viruses.
WT mice were injected initially with either PBS or Influenza-PR8 intranasally (i.n.). 28 days PI, mice were subsequently challenged with either MCMV (i.n.) or Sendai virus (Sev) and analyzed 48 hours PI. (a) Quantification of intracellular IFN-y staining by percentage and MFI of ILC1 at 48 hours following primary and secondary MCMV challenge in i.v. CD45 unlabeled fraction of the lung. (b) Quantification of intracellular IFN-y staining by percentage and MFI of ILC1 at 48 hours following primary and secondary SeV challenge in i.v. CD45 unlabeled fraction of the lung. Data are representative of 3 independent experiments with (a) n=4 mice and (b) n=3 per group. Data are presented as the mean ± SEM.
Supplementary Figure 6 Related to Figure 6. The MCMV-encoded protein m12 does not drive memory formation of IL-18rα- ILC1.
(a) Graph shows mRNA expression of indicated Group 1 ILC specific activating and inhibitory receptors in resting liver ILC1, liver mNK, and splenic mNK, obtained by RNA-sequencing as was previously reported12. (b-c) WT mice were infected with indicated MCMV strains i.p. and liver ILC1 were analyzed 30 days PI. Quantification of IFN-γ+ cells within liver for (b) IL18rα- ILC1 and (c) mNK following platebound stimulation with either media alone or aNK1.1 antibody. Data are representative of 3 independent experiments with (a) n=3 mice and (b) n=4 per group. Data are presented as the mean ± SEM.