Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity

Abstract

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-β (TGF-β) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

Figure 1.. S1pr5 and granzyme C mark subsets of circulating and tissue-resident group 1 innate lymphocytes, respectively.

A. Log fold change (LFC) of gene expression from microarray of liver (Liv) ILC1s versus Liv NK cells (y-axis) versus mean accessibility LFC for Liv ILC1s versus Liv NK cells. Genes with significant differentially accessible peaks are included; genes with significant differential expression are shown in black, red (enriched in ILC1) or green (enriched in NK cell), and genes without significant differential expression are shown in gray. B. Gene accessibility tracks for S1pr5 and Gzmc, displaying average peaks for splenic (Spl) NK cell, Liv NK cell, Liv ILC1, and salivary gland (SG) ILC1, which had differential accessibility between overall ILC1 and NK cell as well as differential gene expression between Liv ILC1 and Liv NK cell. Differentially accessible peaks, as listed in Table S1, are highlighted in the red box. C. Representative histograms (left) and quantification (right) of CD49b, CD11b, KLRG1, CD49a, CD103, CXCR6, CD127, CD200R1, CD69, Eomes, Tbet, and IFN-γ expression in Spl, Liv, and SG S1pr5^eGFP-positive or granzyme C (GzmC)-positive CD3⁻NK1.1⁺NKp46⁺ cells of S1pr5^eGFP-iCre mice, or fluorescence minus one (FMO) staining. Each dot represents one mouse, 3–12 mice per group. All data are combined from three or more independent experiments and shown as mean +/− SEM (one-way ANOVA with Tukey’s multiple comparisons test, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 2.. Granzyme C-expressing innate lymphocytes differentiate from ILCps, but not from NK cells, ILC2s, or ILC3s.

A. Experimental design for S1pr5 fate-mapper (S1pr5^FM) chimeric mice. Expression of S1pr5^FM and granzyme C (GzmC) in splenic (Spl), liver (Liv), and salivary gland (SG) CD3⁻NK1.1⁺ cells. B. Expression of S1pr5^FM in Liv and SG CXCR6⁺CD49a⁺, SG Eomes⁺CD49a⁺, and Liv CD49a⁻CD49b⁺ NK cells from S1pr5^FM chimeric mice. C. Experimental design for ILCp (Lin⁻CD127⁺α4β7⁺Flt3⁻CD25⁻PD-1⁺) transfer. Expression of CD49a and GzmC in Liv ILCp-derived CD3⁻NK1.1⁺NKp46⁺ cells. D. Experimental design for IL-5 fate-mapper (Il5^FM) mice. Expression of Il5^FM and GzmC in Liv, SG, and small intestine lamina propria (SI LP) Lin⁻Thy1.2⁺ and/or NK1.1⁺ innate lymphocytes. E. Experimental design for IL-17A fate-mapper (Il17a^FM) mice. Expression of Il17a^FM and GzmC in Liv, SG, and SI LP Lin⁻Thy1.2⁺ and/or NK1.1⁺ innate lymphocytes. F. Experimental design for IL-22 fate-mapper (Il22^FM) mice. Expression of Il22^FM and GzmC in Liv, SG, and SI LP Lin⁻Thy1.2⁺ and/or NK1.1⁺ innate lymphocytes. Each dot represents one mouse, n = 3–6 mice per group. All data are combined from three or more independent experiments and shown as mean +/− SEM (one-way ANOVA with Tukey’s multiple comparisons test, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 3.. Granzyme C-expressing cells are not precursors for NK cells, ILC2s, or ILC3s.

A. Representative flow cytometric analysis (left) and quantification (right) of granzyme C (GzmC) and S1pr5^eGFP expression in NK1.1⁺NKp46⁺ innate lymphocytes in livers of 0.5 day-, 7 day-, 14 day-old and adult (8–12 weeks of age) mice. B. Representative plots of Gzmc^td-Tomato and CD49a expression in splenic (Spl) and liver (Liv) NK phenotype and Liv and salivary gland (SG) ILC1 phenotype cells of Gzmc^tdT-T2A-iCre mice. C. Experimental design of granzyme C fate-mapper (Gzmc^FM) mice. D. Quantification of Gzmc^FM expression in NK and ILC1-phenotype cells in the Spl, Liv and SG. E. Expression of Gzmc^FM and IL-5 in Liv, SG, and small intestine lamina propria (SI LP) Lin⁻Thy1.2⁺ and/or NK1.1⁺ innate lymphocytes after four-hour PMA/Ionomycin/Golgi stop treatment. F. Expression of Gzmc^FM and IL-17A in Liv, SG, and SI LP Lin⁻Thy1.2⁺ and/or NK1.1⁺ innate lymphocytes after four-hour PMA/Ionomycin/Golgi stop treatment. G. Expression of Gzmc^FM and IL-22 in Liv, SG, and SI LP Lin⁻Thy1.2⁺ and/or NK1.1⁺ innate lymphocytes after four-hour PMA/Ionomycin/Golgi stop treatment. Each dot represents one mouse, n = 3–10 mice per group. All data are combined from three or more independent experiments and shown as mean +/− SEM (one-way ANOVA with Tukey’s multiple comparisons test, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 4.. Differential regulation of ILC1 populations across tissues by T-bet, Eomes and TGF-β.

A. Representative (left) flow cytometric analysis of granzyme C (GzmC) and CD49a expression among NK1.1⁺CD3⁻ cells in the liver (upper) and salivary gland (lower) of wild-type (WT), GzmC^iCreTbx21^fl/fl (Gzmc^ΔTbx21) (yellow), GzmC^iCreEomes^fl/fl (Gzmc^ΔEomes) (green), and GzmC^iCreTbx21^fl/flEomes^fl/fl (Gzmc^{ΔTbx21Δeomes}) (blue) mice. (Right) abundance of GzmC⁺NK1.1⁺CD3⁻ (GzmC⁺) and CD49a⁺NK1.1⁺CD3⁻ (ILC1) cells out of total CD45⁺ cells in liver and salivary gland. B. Representative (left) and quantification (right) of flow cytometric analysis of group 1 innate lymphocytes in liver (upper) and salivary gland (lower) of WT (white bar) and GzmC^iCreTgfbr2^fl/fl (Gzmc^ΔTgfbr2) (gray bar) mice, quantifying CD49a⁺ cells among NK1.1⁺CD3⁻ cells and CD103⁺ and GzmC⁺ cells among CD49a⁺NK1.1⁺CD3⁻ cells in each organ. Each dot represents one mouse (n = 4–10 mice per group). All data are combined from three or more independent experiments and shown as mean +/− SEM (unpaired student’s t-test for two groups, one-way ANOVA with Tukey’s multiple comparisons test with more than two groups, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 5.. Granzyme C-expressing ILC1s expand and mediate cancer immunosurveillance in the PyMT model of breast cancer

A. Abundance of CXCR6⁺CD49a⁺NK1.1⁺CD3⁻ (CXCR6⁺CD49a⁺), Eomes⁺CD49a⁺NK1.1⁺CD3⁻ (Eomes⁺CD49a⁺) and CD49a⁻CD49b⁺NK1.1⁺CD3⁻ (NK) cells out of total CD45⁺ cells in PyMT tumors. Expression of S1pr5^eGFP (B), granzyme C (GzmC) (C), S1pr5^FM (D), and Gzmc^FM (E) in CXCR6⁺CD49a⁺ ILC1 (red), Eomes⁺CD49a⁺ ILC1 (blue), and CD49a⁻CD49b⁺ NK cell (green) populations in PyMT tumors of S1pr5^eGFPPyMT (B), PyMT (C), S1pr5^FMPyMT chimera (D), and Gzmc^FMPyMT (E) mice. F. Abundance of ILC1s in tumors of PyMT (white bar) and GzmC^iCreTgfbr2^fl/flPyMT (Gzmc^ΔTgfbr2PyMT) (gray bar) mice, quantifying CD49a⁺ cells among NK1.1⁺CD3⁻ cells and CD103⁺ and GzmC⁺ cells among CD49a⁺NK1.1⁺CD3⁻ cells in the tumor. G. Tumor volume at 12 and 16 weeks of age from PyMT (white bar) and Gzmc^ΔTgfbr2PyMT (gray bar) mice. H. Abundance of ILC1s in tumors of PyMT (white bar) and GzmC^iCreRosa26^LSL-DTA/+ PyMT (Gzmc^DTAPyMT) (black bar) mice, quantifying CD49a⁺ cells among NK1.1⁺CD3⁻ cells and CD103⁺ and GzmC⁺ cells among CD49a⁺NK1.1⁺CD3⁻ cells in the tumor. I. Tumor volume at 12 and 16 weeks of age from PyMT (white bar) and Gzmc^DTAPyMT (black bar) mice. Each dot represents one mouse (n = 5–9 mice per group). All data are combined from three or more independent experiments and shown as mean +/− SEM (unpaired student’s t-test for two groups, one-way ANOVA with Tukey’s multiple comparisons test with more than two groups, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 6.. ILC1s broadly express cytotoxic molecules and granzyme C expression marks a mature effector state.

A. Representative (upper) and quantification (lower) of granzyme B (GzmB) and perforin expression among splenic (Spl) and liver (Liv) NK cells as well as Liv and salivary gland (SG) ILC1s. B. Representative and quantification of GzmB and perforin protein expression among subsets of ILC1s in Liv (upper) and SG (lower) based on current and history of granzyme C expression (GzmC⁻Gzmc^FM−, double-negative [DN], blue; GzmC⁻Gzmc^FM+, fate-mapped single positive [FMSP], purple; GzmC⁺Gzmc^FM+, double positive [DP], red). C. The number of differentially expressed genes in each of six pairwise comparisons conducted between the four sequenced populations. The color indicates direction of higher expression. D. 62 of 74 genes significantly differentially expressed between DP and DN ILC1s with higher expression in DP, grouped by function and localization. 12 genes could not be grouped due to unknown cellular localization of the proteins they encode and are listed in Table S3. Each dot represents one mouse (n = 4–5 mice per group). Flow cytometry data are combined from three or more independent experiments and shown as mean +/− SEM (one-way ANOVA with Tukey’s multiple comparisons test, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 7.. CXCR6⁺ ILC1s from liver can mediate perforin-dependent cytotoxicity regardless of initial granzyme C expression.

A. Expression of granzyme C (GzmC) in group 1 innate lymphocyte subsets sorted based on expression of GzmC protein and/or Gzmc^FM and cultured in 100 ng/mL IL-15/IL-15Rα for 24 hours (h). Sorted populations included DP (CD49a⁺CXCR6⁺Gzmc^tdT+Gzmc^FM+) (red), FMSP (CD49a⁺CXCR6⁺Gzmc^tdT−Gzmc^FM+) (purple), DN (CD49a⁺CXCR6⁺Gzmc^tdT−Gzmc^FM−) (blue), and NK (CD49a⁻CD49b⁺CD11b⁺) (green) cell subsets. Data are combined from four independent experiments. B. Killing assay, displaying death rate of RMA-S target cells after coincubation with subsets of CXCR6⁺ ILC1s from the liver. Effector cells were either Gzmc^tdT+ or Gzmc^tdT− ILC1s (CXCR6⁺CD49a⁺NK1.1⁺CD3⁻) and were sorted from the livers of Gzmc^{tdT-T2A-iCre/+}Prf1^+/+ or Gzmc^{tdT-T2A-iCre/+}Prf1^−/− mice. Effectors were expanded in 100 ng/mL IL-15/IL-15Rα and cocultured with CTV-labeled RMA-S target cells for 16h at a 10:1 effector:target ratio in media supplemented with 100 ng/mL IL-15/IL-15Rα. Data are representative from one of three independent experiments. Each dot represents one mouse (n = 3–6 mice per group). All data are shown as mean +/− SEM (one-way ANOVA with Tukey’s multiple comparisons test for more than two groups, “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).

Figure 8.. Constitutively active IL-15 signaling in granzyme C-expressing ILC1s causes perforin-dependent lethal autoimmunity.

A. Granzyme C (GzmC) expression in liver (Liv) and salivary gland (SG) CD3⁻NK1.1⁺NKp46⁺ cells of wild-type (WT) and Il15^−/− mice. B. Experimental design of Gzmc^Stat5b-CA mice. C. Kaplan-Meier survival curves for Gzmc^Stat5b-CA/+, Gzmc^Stat5b-CA/+Rag1^−/−, and littermate control mice. D. GzmC⁺NK1.1⁺NKp46⁺ cells per gram of Liv tissue from seven-day-old WT and Gzmc^Stat5b-CA/+ mice. E. Representative images of hematoxylin and eosin staining (H&E, first row) and NKp46 (second row) and cleaved caspase 3 (CC3, third row) immunoreactivities (indicated by arrows) of Liv sections from 14-day-old WT, Gzmc^Stat5b-CA/+, and Gzmc^Stat5b-CA/+Prf1^−/− mice. Images and inserts are 200X and 400X, respectively. NKp46 and CC3 immunoreactivity was also quantified on serial sections as percent of total tissue using digital pathology software (Halo). Scale bar indicates 100 μm. F. Kaplan-Meier survival curves for Gzmc^Stat5b-CA/+ and Gzmc^Stat5b-CA/+Prf1^−/− littermate control mice (n = 15–18 per group). Each dot represents one mouse; A, D, E: n = 3–4 mice per group, C: n = 7–18 per group, F: n = 15–18 mice per group. All data are combined from three or more independent experiments and shown as mean +/− SEM (A, D, E: one-way ANOVA with Tukey’s multiple comparisons test, C, F: Log-rank [Mantel Cox] test; “ns” = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001)