In order to investigate the importance of the IL-33 receptor for ILC2 regulation in vivo, we aimed to target the ST2 chain of the IL-33 receptor (encoded by the Il1rl1 gene) in ILC2 using the Cre-loxP system. To this end, we crossed the recently developed BAC-transgenic Nmur1 ^iCre-eGFP mouse, which allows gene targeting in ILC2 (20, 21), to Il1rl1 ^flox/flox mice (22). Indeed, Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox lacked ST2 mRNA and protein expression in ILC2s of the small intestine, lung, adipose tissue, mesenteric lymph node, and skin similar to Il1rl1 ^-/- mice but not in CD4⁺ T cells, CD8⁺ T cells, eosinophils, basophils or mast cells ( Figures 1A–G ; Supplementary Figures 1A–E ).

To validate whether ST2-deficient ILC2s lost the capacity to sense IL-33, we sort-purified ILC2s from the small intestine, which have low ST2 expression, and from the bone marrow, which have high ST2 expression ( Figure 1D ), and cultured the cells with IL-7 alone to provide a survival signal, or IL-7 + IL-33 to induce activation. While control ILC2s massively expanded, blasted and secreted type 2 effector cytokines upon IL-33 stimulation, ST2-deficient ILC2s failed to respond to IL-33, indicating efficient deletion of ST2 and unresponsiveness to react to the ligand ( Figures 2A–C ; Supplementary Figure 2A). Next, we aimed to test if ILC2 still properly responded to stimulation with other alarmins despite the conditional deletion of ST2. To this end, we performed a similar in vitro assay with sort-purified ILC2s from the small intestine but now exposed the cells to IL-7 or IL-7 + IL-25. ST2-deficient ILC2s expanded and blasted to a comparable degree to ST2-sufficient ILC2s upon IL-25 stimulation ( Figures 2A, B ). We also exposed ILC2s to combinations of TSLP together with IL-7 or IL-25 and IL-33 and assessed blasting and cytokine production of ILC2s ( Supplementary Figures 2B–D ). However, ST2-deficient ILC2s could still respond to TSLP stimulation. Taken together, these data suggest that ILC2 were selectively unresponsive to IL-33 but could still respond to other alarmins, such as IL-25 and TSLP.

Consistent with previous reports investigating mice with germline mutations of ST2, ILC2 were not reduced in frequencies or absolute cell numbers in Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice, suggesting that IL-33-ST2 signals are not essential for proper expansion and survival of ILC2s (23, 24) ( Figure 3A ). Similar results were obtained for bone marrow ILC2 progenitors, indicating that lack of ST2 signals did not lead to overt developmental defects ( Figure 3B ) (25).

To test whether deletion of ST2 altered the heterogeneity of ILC2s in tissues, we performed multicolor phenotyping of ILC2s by flow cytometry. Overall, the heterogeneity of ILC2s in tissue was not perturbed and most surface markers used for ILC2 phenotyping, in particular IL-25R or TSLPR were expressed equally in ILC2s from Il1rl1 ^flox/flox and Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice ( Figure 3C ). In contrast, the marker PD-1 was consistently downregulated in ST2-deficient ILC2s ( Figures 3C–E ; Supplementary Figure 3 ). Since PD-1 was described as an activation marker on ILC2s (26), these data suggest that ILC2 fail to terminally mature without ST2 signals. In summary, Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice are a suitable model to investigate the role of ST2 for ILC2 function in vivo.

Several publications have highlighted the essential role of ILC2 and ILC2-derived IL-5 for eosinophil homeostasis (21, 27). Additional evidence suggests that IL-5 secretion is under the control of alarmin signals (23, 28) and that PD-1⁺ ILC2 are potent cytokine producers (26). Therefore, we asked if ST2 signals are required for type 2 cytokine production by ILC2. Indeed, IL-5 and IL-13 mRNA levels were significantly reduced in sort-purified ILC2s from Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox compared to control ILC2s ( Figure 4A ). Next, we assessed the number of eosinophils in Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice at steady state due to the IL-5 cytokine being crucial for eosinophil homeostasis (29, 30). Consistent with reduced IL-5 levels in ILC2s, we detected a significant reduction of eosinophils in the lung, adipose tissue, mesenteric lymph nodes and skin, suggesting that ILC2-intrinsic sensing of IL-33 via ST2 is necessary for type 2 cytokine production regulating proper eosinophil homeostasis ( Figures 4B, C ).

Next, we tested how the lack of ST2 on ILC2s influences the type 2 immune responses in the context of allergic lung inflammation, which was provoked by the administration of the protease allergen papain. Papain administration resulted in expansion of ILC2s, which was significantly reduced in Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice ( Figures 5A, B ). We also found a strong increase in eosinophil counts in lung and BAL fluid upon papain treatment ( Figures 5C–E ). This increase in eosinophil counts was drastically reduced in papain-treated Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice ( Figures 5C–E ). A more detailed analysis revealed that both CD11b⁺ SiglecF^int tissue-resident and CD11b⁺ SiglecF^high inflammatory eosinophils (31) were reduced in Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice, suggesting that ST2 signals in ILC2s are required for recruitment and local proliferation of eosinophils ( Figures 5C, F ).

To test whether ST2 is required in a cell-intrinsic manner on ILC2s to mediate resistance against helminth infections, we infected Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox and littermate control mice with the worm N. brasiliensis. Littermate control mice mounted a potent type 2 immune response upon worm infection, which was diminished in Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice ( Figure 6 ). The massive expansion of ILC2s upon infection was weakened in Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice compared to littermate controls ( Figures 6A–C ). Furthermore, eosinophil frequencies and tuft cell hyperplasia were reduced in conditional knockout mice, underlining the importance of ST2 signals for a proper type 2 immune response ( Figures 6D–I ). As a consequence, Nmur1 ^iCre-eGFP Il1rl1 ^flox/flox mice had higher worm counts in the intestine ( Figure 6J ). Therefore, our data expose cell-intrinsic ST2 signals in ILC2s as a non-redundant stimulatory pathway for triggering protective type 2 immunity.

C57BL/6 mice were purchased from Janvier. Il1rl1 ^flox/flox mice (22) (provided by the UC Davis/MMRRC repository), Il1rl1 ^-/- mice (28) and Nmur1 ^iCre-eGFP mice (20, 21) bred locally at Charité. Sex and age-matched animals were used for experiments if not otherwise indicated. We did not use randomization to assign animals to experimental groups. All animal experiments were approved and are in accordance with the local animal care committees (Lageso Berlin).

Small intestine was removed, cleaned from remaining fat tissue and washed in ice-cold PBS. Peyer’s patches were eliminated, small intestine was opened longitudinally and washed in ice-cold PBS. Dissociation of epithelial cells was performed by incubation on a shaker at 37°C in HBSS (Sigma-Aldrich) containing 10 mM Hepes (Gibco) and 5 mM EDTA (Roboklon) two times for 15 min. After each step, samples were vortexed and the epithelial fraction discarded. Afterwards, remaining tissue was chopped into small pieces and enzymatic digestion was performed using dispase (0,5 U/ml; Corning), collagenase D (0,5 mg/ml; Roche) and DNaseI (100 μg/ml; Sigma-Aldrich). Leukocytes were further enriched by Percoll gradient centrifugation (GE Healthcare). Lungs were chopped and incubated in the enzyme cocktail described above for 40 min on a shaker at 37°C. The remaining tissues were mashed with a syringe plunger and single cell suspensions were filtered through a 70 μm cell strainer. Leukocytes were then further enriched by Percoll gradient centrifugation. Mesenteric lymph nodes were chopped and incubated in RPMI 1640 medium (Gibco) supplemented with 1% BSA (Sigma-Aldrich), collagenase II (1 mg/ml; Sigma-Aldrich) and DNaseI (100 μg/ml) for 20 min on a shaker at 37°C. Afterwards, cells were dissociated using a pasteur pipette, and filtered through a 70 μm cell strainer. Epididymal white adipose tissue was removed and incubated in the same digestion buffer for 45 min on a shaker at 37°C. After incubation, cells were dissociated using a Pasteur pipette, filtered through a 70 μm cell strainer, spun down and the adipocyte layer was aspirated. For skin preparation, ears were removed, split and put dermal side down in DMEM supplemented with Liberase TL (0,5 mg/ml; Roche) at 37°C for 1,5 h. The tissue was then mashed through a 70 μm cell strainer and the leukocytes further enriched by Percoll gradient centrifugation. For isolation of bone marrow cells, femur and tibia bone were crushed with a pestle, rinsed and cells were filtered through a 70 μm cell strainer. Red cell lysis was performed in ACK lysis buffer for 3 min.

Dead cells were routinely excluded with Fixable Aqua Dead Cell Stain or SYTOX Blue Dead Cell Stain (Thermo Fisher Scientific). Single cell suspensions were incubated on ice with anti-CD16/CD32 antibody and the following conjugated antibodies in PBS (Ca2+ and Mg²⁺-free, Sigma-Aldrich). If indicated, lineage-positive cells were excluded by staining for CD3e (145-2C11 or 500A2), CD5 (53-7.3), CD19 (1D3 or 6D5), FcϵRIα (Mar-1) and Ly6G (1A8). For surface staining the following antibodies were used: c-Kit (2B8), CCR9 (9B1), CD11b (M1/70), CD11c (N418), CD127 (A7R34), CD25 (PCG1.5), CD4 (GK1.5 and RM4-5), CD44 (IM7), CD45.2 (104), CD49b (DX5), CD64 (X54-5/7.1), CD8a (53-6.7), CD90.2 (53-2.1), F4/80 (BM8), ICOS(C398.4A), IL-25R (Munc33), KLRG1 (2F1 or MAFA), Ly6G (1A8), NK1.1 (PK136), PD-1 (29F.1A12), Sca-1 (D7), SiglecF (E50-2440), ST2 (RMST2-33), TSLPR (22H9), Rat IgG2b kappa Isotype (eB149/10H5). All antibodies used in flow cytometry were purchased from eBioscience, Biolegend or BD Biosciences if not otherwise indicated. All flow cytometry experiments were acquired using a custom configuration Fortessa flow cytometer and the FACS Diva software (BD Biosciences) and were analyzed with FlowJo V9.9.3 or V10.6.2 software (TreeStar) and for the t-SNE plots analyzed with the Cytobank Software using custom configurations (Backman Coulter) or sort-purified by using a custom configuration FACSAria cell sorter (BD Biosciences).

Sort-purified cells were homogenized in Trizol (Thermo Fisher Scientific) and stored at -80°C. RNA was extracted with chloroform and RNA concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription of total RNA was performed using the High Capacity cDNA Reverse Transcription kit according to the protocol provided by the manufacturer (Thermo Fisher Scientific). Reaction was detected on a QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) using Taqman Gene Expression Assay (Applied Biosystems) with Il5 (Mm00439646_m1), Il13 (Mm00434204_m1) or SYBR Green Master Mix with Il1rl1 (forward: 5’- GGGCACACAGGTCCTACTTG-3’, reverse: 5’- ATGTAGTTGGTTCCATTCTCCG-3’). Gene expression was normalized to the housekeeping gene Hprt1 (Mm00446968_m1) for Taqman and Hprt1 (forward: 5’-GATACAGGCCAGACTTTGTTGG-3’, reverse: 5’-CAACAGGACTCCTCGTATTTGC-3’) for SYBR Green.

Third-stage larvae (L3) of N. brasiliensis were purified with a Baermann apparatus. After washing three times in PBS, larvae were counted and 500 purified larvae were injected subcutaneously in PBS. Mice were killed, organs were analyzed and worm burden was determined in the small intestine 7 days post infection.

For allergic asthma induction, 30 μg of Papain (Roche) in PBS were administered intranasally on three consecutive days. Mice were killed on day seven after initial administration, organs were collected and analyzed.

Sort-purified ILC2s (as live CD45⁺, Lin^-, NK1.1^-, CD127⁺ and KLRG1⁺ for small intestine or CD25⁺ for bone marrow) were incubated in DMEM with high glucose supplemented with 10% FCS, 10 mM Hepes, 1 mM sodium pyruvate, non-essential amino acids, 80 μM 2-Mercaptoethanol, 2mM Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin (all from Gibco) in 96-well U-bottom microtiter plates (Nunc) for three days at 37°C and 5% CO₂. As indicated, the culture was supplemented with a cocktail of IL-7 only, IL-7 and IL-33 or IL-7 and IL-25, IL-7 and TSLP or IL-7 and IL-33, IL-25, TSLP (Biolegend, TSLP from R&D, 20 ng/ml each).

Cytokine concentrations in culture supernatants were determined by using the LEGENDplex murine T_H2 Panel V03 multiplex beads-based assay (Biolegend) according to the manufacture’s protocol. Samples were recorded on a custom configuration Fortessa flow cytometer and the FACS Diva software (BD Biosciences) and the flow cytometry data files were analyzed using the Legendplex cloud-based analysis software suite (Biolegend).

For immunofluorescence staining, the tissue was fixed in 4% PFA at 4°C until tissue embedding. Paraffin-embedded sections were de-paraffinized and rehydrated. Sections were permeabilized with 0.5% Triton-X in PBS and blocked with PBS 0,5% Triton X-100 and 10% serum and stained with rabbit anti-DCLK1 (Abcam) followed by donkey anti-rabbit antibody coupled to Alexa Fluor 555 (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Images were captured on a Zeiss Axio Observer 7 microscope and analysed with Zen software (Zeiss). For tuft cell numbers, five to ten representative villi were counted on four independent images per mouse using ImageJ.

Data is plotted showing the mean +/- standard deviation. P values of data sets were determined by unpaired two-tailed Student’s t-test with 95% confidence interval. If equal variances could not be assumed, Welch test was performed. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to analyze several groups. Normal distribution was assumed. Before mentioned statistical tests were performed with Graph Pad Prism V9 software (GraphPad Software, Inc.). (*p <0.05; **p <0.01; ***p <0.001; ****p <0.0001 and ns, not significant).