In Situ Maturation and Tissue Adaptation of Type 2 Innate Lymphoid Cell Progenitors

Abstract

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1⁺ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1⁺ ST2^- ILCPs to Il18r^- ST2⁺ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

Keywords: ILC2; ILCP; Innate lymphoid cells (ILC); Nippostrongylus Brasiliensis; bone marrow; immune system development; progenitors; single cell atlas; single-cell RNA-seq; tissue immunity.

Figure 1. Heterogeneity of ILC2s in healthy adult lung tissue and immature Il18r1⁺ ILCs.

(A) Workflow for lung ILC scRNA-seq library preparation. (B) Representative sorting strategy for Lin^- NK1.1^-Il7ra⁺Thy1.2^hi/im ‘pan’-ILCs from lung. (C) t-SNE map of single-cell lung ILC transcriptomes clustered with RaceID3. (D) t-SNE maps indicating log2 normalized expression of candidate genes. (E) Candidate gene expression for clusters with at least 20 cells. Color represents z-score mean expression across clusters and dot size represents fraction of cells in the cluster expressing the gene. (F) Lineage inference using StemID2. Node color depicts transcriptome entropy and link color indicates p-value of StemID2 links (P < 0.05, Methods). (G) Pseudo-temporal gene expression profiles (local regression) of representative ILC2 genes (top) and ILC progenitor genes (bottom) along the predicted trajectory. Color bars indicate cluster identity. (H,I), Representative FACS analyses of Lin^- Il7ra⁺ ILCs in adult lung at steady state. Gating (H) and percentage (I) of Il18r1⁺ST2^-RORγt⁺Klrg1^- ILCs. (J-K) Fraction of Ki67⁺ (J) and Il5⁺ (K) cells within the indicated lung ILC subsets (ICS after incubation with monensin,RORγt⁺ ILCs are not excluded). (L) FACS analysis of Lin^-Il7ra⁺ lung ILCs subdivided into Il18r1⁺ST2^-RORγt⁺Klrg1^-, Il18r1⁺ST2⁺ and Il18r1^-ST2⁺ ILCs. Depicted is the mean fluorescence intensities (MFI) for Tcf7, Il18r1, Il17rb, Gata3, ST2 and CD25. Data in (H-L) are representative of 2-3 independent experiments (n=4-12 mice). Graphs in (I), (J) and (K) depict data as mean ± SD, (one-way ANOVA Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant).

Figure 2. IL18rl⁺ST2^- ILCs in neonatal and adult lung share similarities with IL18rl⁺ BM ILCPs.

(A-C), t-SNE maps of neonatal lung ILC single-cell transcriptomes highlighting RaceID3 clusters (A) or sorted ST2^- and ST2⁺ ILCs (B), and log2 normalized expression of representative genes marking putative ILC progenitors (C). (D) Expression of candidate genes within neonatal lung clusters with at least 10 cells. Color represents z-score mean expression across clusters and dot size represents fraction of cells in the cluster expressing the gene. (E) FACS analysis of neonatal (P4) and adult (6-8w) Il18r1⁺ST2^- lung ILCs, represented as t-SNE maps, indicating expression levels for Gata3, RORγt, PLZF, Tcf7, C-kit, CD103 and Ki67. (F) Histograms comparing ILC subsets (PLZF⁺Gata3⁺, RORγt⁺CD103^-, RORγt⁺CD103⁺, Il18r1^-ST2^- ILC2s and RORγt⁺ ILC3) in adult lung for Gata3, Icos, a4p7, PD1, CD25 and Il17rb expression levels. Data are representative of 2-3 independent experiments (n=6-12 mice). (G) t-SNE map of Il18r1⁺Icos⁺ BM ILCs highlighting RaceID3 clusters. (H) Expression of candidate genes for Il18r1⁺Icos⁺ BM clusters with at least 10 cells. Color represents z-score mean expression across clusters and dot size represents fraction of cells in the cluster expressing the gene. (I-J) FACS analysis of human lung and PB ILCs pre-gated on CD45⁺Lin^- CD127⁺EOMES^-T-bet^-CRTH2^-NKp44^-c-KIT^+/- (I) or c-KIT ILCs (J). Figures depict representative staining (I) and t-SNE maps (J) indicating the expression levels of selected markers. Data are representative of 3 independent experiments (n=2-4 samples each).

Figure 3. Il18r1⁺ ILCs are immature cells that give rise to Gata3^hi ILC2s in the lung.

(A-B) FACS analysis of Ki67 expression and intravenous CD45 label (ivCD45) in Il18r1⁺ST2^-RORγt⁺Klrg1^- and Il18r1^-ST2⁺ adult lung ILC2s. Representative gating (A) and percentage of ivCD45⁺ cells within indicated ILC subsets (B). (C) Number of Lin^-Il7ra⁺Il18r1⁺ cells per ml blood in PBMCs (Il18r1⁺ST2^- ILCs) and lung (ivCD45⁺Il18r1⁺ST2^-RORγt⁺Klrg1^- ILCs). Graphs in (B) and (C) depict data as mean ± SD (unpaired t-test, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant). (D-E) Representative gating (D) and percentage of Ki67⁺ and ivCD45⁺ cells (E) for indicated Il18r1⁺ST2^- ILC subsets in adult lung. Data are pooled from 3-4 independent experiments (n=4-16 mice). (F) Percentage of Il5^Cre fate-mapped (Il5FM⁺) Il18r1⁺ST2^-RORγt^-CD103^- ILCs in lung and BM. RORγt staining was used to exclude ILC3. Data are pooled from 2-3 independent experiments (n=4-9 mice). Graphs in (E) and (F) depict data as mean ± SD (one-way ANOVA Tukey’s multiple comparisons test (E) or unpaired t-test (b, c, f), ***P < 0.001; ****P < 0.0001). Data are pooled from 3-4 independent experiments with a total of n = 8-15 mice). (G) t-SNE representation of single-cell transcriptomes of lung Il18r1⁺Icos⁺ ILCs before (“input”) and after culture (“output”) highlighting RaceID3 clusters. (H) Cluster composition of samples (left) and sample composition of clusters (right, for legend see (G)). (I) Expression of candidate genes for input and output populations. Color represents log2 mean expression in the respective cluster and dot size indicates fraction of cells expressing the gene in the cluster. (J) Total number of Gata3⁺ ILC2s after culture of indicated lung ILCs for 15 days in the presence of Il2, Il7, SCF, Il25 and Il33 on OP9-DL1 for 15 days. (K) FACS analysis of Lin^-Il7ra⁺ lung ILCs on d21 post transfer into sublethally irradiated CD45.2⁺RAG^-/-γc^-/- mice. Congenically marked Il18r1⁺ST2^- and Il18r1^-ST2⁺ ILCs sorted from lungs of d2 Nb infected mice were cotransferred (2,000 cells each). Data are representative of 2 individual experiments (n=3 mice). (L) Total number of Gata3⁺ ILC2 after culture of indicated lung ILCs as in (J). Data in (J) and (L), are pooled from 2 independent experiments with n=3-4 repeat wells each. (M) Indicated lung and BM ILC subsets were sorted as single cells onto OP9-DL1 monolayers and cultured for 19 days. Clonal progeny was analyzed by FACS. Pie charts indicate distribution of culture output across positive single cell cultures.

Figure 4. Emerging heterogeneity of lung ILC2s during Nippostrongylus brasiliensis infection.

(A) FACS analysis of the fraction of Lin^-Gata3⁺ ILC2s among live CD45⁺ cells (left) and of Ki67⁺ cells among Lin^-Gata3⁺ ILC2s (right) in Nb infected lungs. Data are pooled from 3 independent experiments with a total of n=12-16 mice per time point. (B) Sequenced samples at the respective time-points during Nb infection. (C) t-SNE map of combined Nb infection time-course and uninfected lung data (cf. Figure 1) highlighting RaceID3 clusters. (D) Overlay of t-SNE map from (C) with pie charts indicating normalized sample contribution to clusters. (E) Expression of candidate genes for ILC subsets for inferred time-course clusters with at least 20 cells (left) or the respective sample (right). Color represents z-score of mean expression across clusters/samples and dot size represents fraction of cells positive for the gene in the cluster/sample. (F) Time-course FACS analysis of the fraction of ivCD45⁺ ILC2s in Nb infected lungs. FACS data are pooled from 2-3 independent experiments with n=6-12 mice per time point. (G) Time-course FACS analysis of Il7ra⁺ ILCs in Nb infected lungs (black) compared with fractions of RNA expressing cells of time-course dataset (red). FACS data are pooled from 2-3 independent experiments with n=6-9 mice per time point. (H) Time-course FACS analysis of Nb infected lungs. Expression of indicated markers represented as t-SNE maps for Il5⁺ ILCs. ICS after incubation with PMA, ionomycin and monensin.Data are pooled from 5 mice for each time point and merged from equal numbers of Lin^-Il7ra⁺ ILCs. Graphs in (A) and (F) depict data as mean ± SD (one-way ANOVA Tukey’s multiple compari sons test, ***P < 0.001; ****P < 0.0001).

Figure 5. Differentiation trajectories of lung ILC2s during Nippostrongylus brasiliensis infection.

(A) t-SNE maps showing the distribution of uninfected lung immature clusters (cf. Figure 1) within the combined data set (top) and pie charts of the distributions (bottom) (cf. Figure 4C). (B) StemID2 lineage inference and normalized sample contribution to clusters depicted as pie-charts. Link color indicates p-value of StemID2 links (P < 0.05, Methods). (C) Pseudo-temporal gene expression profiles (local regression) along depicted trajectories. Color bars indicate cluster and sample identity, respectively, of cells ordered along the depicted trajectory by StemID2. (D) Time course analysis for the percentage of Il18r1⁺ST2^-RORγt⁺Klrg1^- ILCs (left) and the comparison of ivCD45 labelled and unlabelled fractions (right). (E) Time course analysis for the percentage of Il18r1⁺ST2⁺ (left) and Il18r1^-ST2⁺ (right) ILC2s. Data in (D) and (E) are representative of 2 independent experiments with n=4-6 mice per timepoint. Graphs depict data as mean ± SD (one-way ANOVA Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001). (F) Time-course FACS analysis of Nb infected lungs. Expression of indicated markers represented as t-SNE maps for Il18r1⁺ ILCs. ICS after incubation with PMA, ionomycin and monensin. Data are pooled from 5 mice for each time point and merged from equal numbers of Lin^-Il7ra⁺ ILCs.

Figure 6. Recruited cells contribute to the entire phenotypic spectrum of lung ILC2s during Nippostrongylus brasiliensis infection.

(A) t-SNE map of cells derived from parabiotic mice at d15 p.i. highlighting RaceID3 clusters. (B) StemID2 lineage inference and normalized donor and host contribution to clusters depicted as pie-charts. Link color indicates p-value of StemID2 links (P < 0.05, Methods). (C) Differentially expressed genes (P < 0.05, Methods) between donor and host cells within the depicted clusters. (D) t-SNE maps indicating log2 normalized expression of candidate genes in the parabiosis data. (E) Expression of representative candidate genes for ILC subsets in parabiosis clusters with at least 20 cells. Color represents z-score of mean expression across clusters and dot size represents fraction of cells expressing the gene in the cluster. (F) t-SNE map of parabiosis data showing normalized weights of indicated parabiosis cluster medoids for cells within the Nb timecourse data (cf. Figure 4) as computed by cluster mapping (Methods).

Figure 7. Bone marrow-derived ILCs give rise to the full phenotypic spectrum of ILC2s in the adult lung.

(A) Nb infection of the shield chimera model. (B) Frequency of donor-derived cells from transferred bone marrow (BM). (C-D) t-SNE map of ILCs from shield chimeric mice isolated at d15 p.i. highlighting RaceID3 clusters (C) and donor or host origin for each cell (D). (E) StemID2 lineage inference and normalized donor and host contribution to clusters depicted as pie-charts. Link color indicates p-value of StemID2 links (P < 0.05, Methods). (F) Expression of candidate genes for ILC subsets in the shield chimera clusters with at least 10 cells. Color represents z-score of mean expression in a cluster and dot size represents fraction of cells expressing the gene in the cluster. (G) Differentially expressed genes (P < 0.05, Methods) between donor and host cells within the depicted clusters.