Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct immune signatures for predicting the immunotherapy efficacy of esophageal squamous cell carcinoma or adenocarcinoma

doi: 10.1007/s00262-024-03904-1

Figure Lengend Snippet: Detecting unique cytokines linked to clinical outcomes in esophageal squamous cell carcinoma and adenocarcinoma separately. a Venn diagram shows genes in the cytokine-cytokine receptor pathway for ESCC and EAC from TCGA, annotated with gene counts in distinct and overlapping categories. b Overall survival of patients with TCGA-ESCC and different expression levels of TNFSF10 and CXCL14. c TNFSF10 and CXCL14 expression comparison between early and advanced ESCC tissues (TCGA). d Survival probabilities of patients with EAC classified by CXCL8 and IL17RB expression levels (TCGA). e CXCL8 and IL17RB expression during EAC progression from early to advanced stages. f TNFSF10 and CXCL14 expression in early and advanced ESCC tissues of Chinese patients. g, h The left panel presents immunohistochemical images illustrating TNFSF10 or CXCL14 staining in tissue microarrays obtained from patients diagnosed with ESCC, arranged sequentially from left to right based on ascending expression levels quantified by the H-score. On the far left, the lowest expression level is shown, followed by a sample in the lowest 33.33 percentile, then a moderate expression sample in the next 33.33 percentile, and finally, the highest expression level on the far right. The right panel displays survival curves stratified by the expression of TNFSF10 and CXCL14, categorized into low and high expression groups based on their H-scores. i, j The left panel displays immunohistochemical staining for CXCL8 or IL17RB in tissue microarrays from patients with EAC, with samples ordered from left to right by increasing H-scores. The sequence starts with the lowest expression level, followed by a sample in the first 33.33 percentile, then a sample with moderate expression in the next percentile, and ends with the highest expression level on the far right. P values were calculated using the Mann–Whitney (log-rank) test. Early stage: Stage 0-II; Advanced stage: Stage III-IV; ns: no significance; * P < 0.05, Mann–Whitney test

Article Snippet: The tissue sections were incubated with primary antibodies against TNFSF10 (Catalog No. ab2056; Abcam), CXCL14 (catalog no. ab137541; Abcam), CXCL8 (catalog no. ab106350; Abcam), IL17RB (Catalog No. AF1207; R&D Systems), CXCL10 (Catalog No. ab306587; Abcam, Cambridge, UK), HIF1α (catalog no. ab51608; Abcam), CSF2 (catalog no. ab316862; Abcam), and NOS2 (catalog no. ab283655; Abcam) (diluted at 1:500 in phosphate-buffered saline) overnight at 4°C.

Techniques: Expressing, Comparison, Immunohistochemical staining, Staining, Sequencing, MANN-WHITNEY

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Flow diagram of the i.p. HDM sensitization model. Challenge phase: D24, Remission phase: D30. B Airway resistance of mice was determined on day 1 (D1), D8, D15, D24 and D30 under methacholine (0, 6.25, 12.5, 25 and 50 mg/mL in PBS) stimulation. C Serum IgE ( n = 8), D lung IL-4, IL-5 and IL-13 ( n = 8), E eosinophils in peripheral blood ( n = 6), and F cytology of bronchoalveolar lavage fluid (BALF) ( n = 10) were determined at indicated time points. G Gating strategy. In live CD45 + lymphocytes, Th2s are defined as CD4 + GATA3 + , and total ILC2s are defined as lineage – CD90.2 + NK1.1 – NKp46 – Rorγt – GATA3 + . H Kinetics of the frequency and number of ST2 – KLRG1 + IL-17RB + ILC2 subset in the lamina propria of small intestines (siLP) ( n = 10). I , J IL-13 + percentage and MFI I and IL-5 + percentage and MFI J of ST2 – KLRG1 + IL-17RB + ILC2s in siLP after stimulation with PMA, ionomycin, brefeldin A and monensin. K , L Pie charts of IL-13 + K and IL-5 + L cell populations in siLP in remission under stimulation ( n = 10). Mice in control group (Ctrl) were treated with corresponding volume of sterile normal saline. Data points are individual mice pooled from three independent experiments for all panels. Data are shown as the mean ± SD. Statistical comparisons were performed using unpaired one-way ANOVA with Dunnett’s test except in B and F using unpaired two-way ANOVA. p values are shown on the graphs. Source data are provided as a Source Data file.

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Control, Sterility, Saline

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Donor CD45.1 + mice were sensitized and challenged with HDM follow the i.p. HDM sensitization model protocol. On day 30 (remission), lineage-negative cells were isolated from bone marrow (BM), spleen, lungs and siLP, respectively and cultured in vitro for expansion. Subsequently, 5 × 10 5 CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ILC2s were sorted and injected into the irradiated CD45.2 + mice via tail vein. Recipient CD45.2 + mice were then treated with 25 μg HDM intranasally for 3 times once daily. 24 hours later, single cells in lungs were prepared for flow cytometry analysis. B – E Representative plots and statistical results (bar graph) showing the percentages and the numbers of ST2 – KLRG1 + IL-17RB + ILC2s at lungs in mice that received cells derived from B BM, C spleen, D lungs, or E siLP, respectively. F , G IgE levels in serum F and IL-13 and IL-5 protein levels in lungs ( G ) of recipient mice in each group were detected by ELISA. H Histological scores and representative images of lungs of recipient mice by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. Results are representative of three independent experiments. Means ± SD from 8 mice each group. Statistical comparisons were performed using unpaired, two-sided Welch’s t-test except in F – H using unpaired two-way ANOVA. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 2A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Isolation, Cell Culture, In Vitro, Injection, Irradiation, Flow Cytometry, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A CD45.1 + and irradiated CD45.2 + mice were surgically jointed from olecranon to knee joint and recovered for 4 weeks to establish blood chimerism. Subsequently, CD45.1 + mice were treated with HDM as Fig. . B The percentages and the numbers of ST2 – KLRG1 + IL-17RB + ml-ILC2s in siLP of CD45.1 + and CD45.2 + were analyzed on day 30. Cells were gated on CD45.1 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + . For in vivo imaging experiments, mice were treated with HDM to establish the asthma relapse model as portrayed in Fig. . C On day 30 (remission), ST2 – KLRG1 + IL-17RB + ILC2s were sorted from siLP and stained with 5 mM Xenolight DiR, and then injected intravenously (5 × 10 5 cells/each) into various groups of mice. Specifically, mice in DiR- group were injected with unstained cells. For the remission + CCX282-B group, recipient mice resting from asthma symptoms were treated with the CCR9 antagonist CCX282-B at 50 mg/kg (daily injection subcutaneously) from day 24 to day 30. For the remission + Etrolizumab group, recipient mice resting from asthma symptoms were treated with the anti-α4β7 monoclonal antibody Etrolizumab at 5 mg/kg (injection intravenously) on day 24, 26, 28 and 30. For the relapse group, mice in remission were injected with DiR-stained cells, followed with treatment of HDM at 50 μg intranasally for 2 times once per hour. Mice in the relapse + FTY720 group received intravenous injection of FTY720 along with 2-time intranasal administration of HDM. Mice in all groups were imaged 2 hours post cell injection, and organs including lungs, spleen, liver, SI and large intestine were then rapidly harvested and imaged. D S1P expression in plasma collected from mice post asthma relapse (D34, D37 and D40) was measured by ELISA. The S1P signaling antagonist FTY720 was administered intraperitoneally to asthmatic mice on days 31 to 33 for 3 times. E The percentages and the numbers of ST2 – KLRG1 + IL-17RB + ILC2s in lungs were detected on D34. F Histological examination of lungs by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. G Protein levels of IL-13 in lungs was detected by ELISA. Results are representative of three independent experiments. Means ± SD from 6 mice for each group. Statistical comparison was conducted using unpaired, two-sided Welch’s t test for B and unpaired one-way ANOVA with Dunnett’s test for the rest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 3A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Irradiation, In Vivo Imaging, Staining, Injection, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A WT or Rag1 – / – mice were stimulated with HDM, and anti-KLRG-1 mAb or isotype IgG were administered intraperitoneally as indicated. B The percentage and the number of CD45 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in lungs were analyzed on day 24. C Pulmonary IL-13 was detected by ELISA. D Airway resistance was measured under stimulation of methacholine. E Pulmonary histological changes were observed by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. F Rag1 – / – mice were stimulated with HDM, and CD45 + lineage – CD90.2 + ST2 – KLRG1 + IL-17RB + ILC2s were sorted from siLP on D30. In vitro culturing cells were treated with anti-KLRG1 mAb or isotype control, and then adoptively transferred into untreated ILC2 depleted (anti-CD90.2 mAb or isotype control) Rag1 – / – mice via tail vein (5 × 10 5 cells/mouse). Recipient mice were intranasally treated (i.n.) with 25 μg HDM or 500 ng recombinant IL-25 for 3 consecutive days. G The percentage and the number of CD45 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in lungs. H , I IL-13 level in lungs homogenates H and airway resistance I was measured. J Pulmonary histological changes were analyzed. Magnification: ×200, scale bar = 100 μm. Results are representative of three independent experiments. Data was shown as Means ± SD. n = 6 in all panels except for n = 3 in D and I . Statistical comparison was conducted using unpaired, two-sided Welch’s t test, except in ( D ) and ( I ) using unpaired two-way ANOVA with the Bonferroni posttest to analyze dose response to methacholine. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 4F Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Control, Recombinant, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Flow chart of asthma mice model establishment and sample collection. B , C On days 21 to 23, BrdU (0.8 mg/mice) was given intranasally. At indicated time points, single-cell suspension was prepared and subjected to flow cytometry analysis; Dynamic changes of the percentage B and numbers C of CD45 + lineage – CD90.2 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in siLP. D The proportions of BrdU + ST2 – KLRG1 + IL-17RB + ILC2s in siLP of HDM-challenged mice were analyzed by flow cytometry. A control histogram (shaded in blue) was generated using cells from ctrl mice stained with BrdU. E During asthma remission (AR) (day 30) of the i.p. HDM sensitization model, three ILC2 subsets including KLRG1 – , KLRG1 + IL-17RB + and KLRG1 + IL-17RB + (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB + and AR-KLRG1 + IL-17RB + ) were purified from the total CD45 + lineage – CD90.2 + ST2 – cells from siLP. Besides, ST2 – KLRG1 + IL-17RB + ILC2s (marked as naive-KLRG1 + IL-17RB + ILC2s) were sorted from siLP of untreated WT mice. Cells were then cultured (5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins + ) or without (alarmins-) 200 ng/mL IL-25 and 200 ng/mL IL-33 every other day for 14 days. Subsequently, cells were collected for further analysis. (F-H) Ki-67 + percentage F , number ( G ) and fold changes H of expansion of cells were determined. I Representative plots and statistical results (bar graph) showing the percentages of IL-13 + and IL-5 + of these four ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. Data are representative of four independent experiments. Means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD; n = 5 for the naive-KLRG1 + IL-17RB + group, and n = 9 for the other groups. B , C statistical comparisons were performed using unpaired, two-sided Welch’s t test. F – I unpaired two-way ANOVA with the Bonferroni posttest analysis was performed. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 5E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Suspension, Flow Cytometry, Control, Generated, Staining, Purification, Cell Culture

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A In asthma remission, KLRG1 – , KLRG1 + IL-17RB – , KLRG1 + IL-17RB + subsets (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB – and AR-KLRG1 + IL-17RB + ) in total CD45 + lineage – CD90.2 + ST2 – ILC2s in siLP were sorted. KLRG1 + IL-17RB + ILC2 (marked as naive-KLRG1 + IL-17RB + ) were isolated from siLP of naive WT mice (5 ~ 7 × 10 5 for every subset, purity > 98%). Total RNA was isolated, and qRT-PCR was performed. Heat map of mRNA expression values of selected genes (see Fig. ). n = 6 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and AR-KLRG1 + IL-17RB + ILC2, n = 3 for naive-KLRG1 + IL-17RB + ILC2. B – F Sorted CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ml-ILC2s were transfected with siRNAs targeting Tcf7 or Tox (si Tcf7 or si Tox ) or negative control (Negative Ctrl). Together with AR-KLRG1 – , AR-KLRG1 + IL-17RB – and naive-KLRG1 + IL-17RB + ILC2s (5 ± 10 3 per well), a combination of 200 ng/mL IL-25 and 200 ng/mL IL-33 were added every two days with IL-2 and IL-7 for 8 days. Knockdown efficiency of Tcf7 or Tox was confirmed (see Fig. ). B Cell expansion was calculated relative to day 0. C Protein levels of IL-13 in culture medium were detected by ELISA. n = 8 for ml-ILC2 Ctrl, ml-ILC2 Negative Ctrl, ml-ILC2 si Tcf7 and ml-ILC2 si Tox , n = 5 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and naive-KLRG1 + IL-17RB + ILC2. D Heat map of mRNA expression values of selected genes (see Fig. ). n = 3. Expression of E CCR9 and F S1PR1 of all cells in each group were detected with flow cytometry. n = 3. G Flow chart of asthma mice model, BrdU administration, RNA interference and sample collection. On days 7–11, BrdU (0.8 mg/mice) was given intranasally daily. Rag1 – / – mice ( n = 5) were injected with Ambion in vivo si-negative control or indicated siRNAs on days 12–18, and were intranasally treated with 25 μg HDM on days 72-74. The percentage of BrdU + in ST2 – KLRG1 + IL-17RB + ILC2s in H siLP on day 71 and in ( I ) lungs on day 75 were detected with flow cytometry. J Flow chart of asthma mice model, adoptive transfer, RNA interference, and sample collection. Rag1 – / – mice were exposed with HDM and rested till AHR vanished as described in Fig. . Mice were injected with Ambion in vivo si-negative control, si Tcf7 or si Tox on days 16-18, and were intranasally treated with 25 μg HDM on days 19–21. For the groups of in vivo si Tcf7 +ml-ILC2 AT + HDM and in vivo si Tox +ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of si-negative control on day 19 before HDM treatment; For the groups of in vivo si Tcf7 +si Tcf7 -ml-ILC2 AT + HDM and in vivo si Tox +si Tox -ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of siRNAs on day 19 before HDM treatment. Twenty-four hours after the last HDM treatment, K airway resistance ( n = 3) and L IL-13 level in lung homogenates were measured ( n = 6). M Pulmonary histological changes were analyzed ( n = 6). Magnification: ×200, scale bar = 100 μm. Data was shown as Means ± SD. Results are representative of three independent experiments. Statistical comparison was conducted using unpaired one-way ANOVA with Dunnett’s test, except in ( B ) and ( K ) using unpaired two-way ANOVA with the Bonferroni posttest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 6G, J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Transfection, Negative Control, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Injection, In Vivo, Adoptive Transfer Assay, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: Flow cytometry was performed to analyze CD45 + lineage – CRTH2 + CD127 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in PBMCs purified from asthmatic patients during remission phase (all rested for 50 days or longer) and healthy donors. A Gating strategy, B statistical plots, C IL-13 + percentage and MFI and D IL-5 + percentage and MFI of CD45 + lineage – CRTH2 + CD127 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s. E KLRG1 – , KLRG1 + IL-17RB – and KLRG1 + IL-17RB + subsets were purified from the total CD45 + lineage – CRTH2 + CD127 + ST2 – ILC2s in PBMC of 6 asymptomatic asthmatic patients (marked as A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ). KLRG1 + IL-17RB + subsets were sorted from total ILC2s in PBMC of 3 healthy donors (marked as H-KLRG1 + IL-17RB + ). Cells were then cultured (5 × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins+) or without (alarmins−) 200 ng/mL recombinant human IL-17E/IL-25 and 200 ng/mL recombinant human IL-33 every other day for 21 days. Subsequently, cells were collected for further analysis. F Ki-67 + percentage, G absolute number and H fold changes of expansion of cells were determined. Representative plots and statistical results (bar graph) showing the percentages of I IL-13 + and J IL-5 + of three ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. KLRG1 – , KLRG1 + IL-17RB – and KLRG1 + IL-17RB + subsets in total CD45 + lineage – CRTH2 + CD127 + ST2 – ILC2s in PBMC of asymptomatic asthmatic patients (marked as A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ) and KLRG1 + IL-17RB + subsets in total ILC2s in PBMC of healthy donors (marked as H-KLRG1 + IL-17RB + ) were sorted (4.5 ~ 6.8 × 10 5 for every subset, purity > 98%). K , L Total RNA was isolated, and qRT-PCR was performed. K Heat map of mRNA expression values of selected genes. L Bar plots of mRNA expression values (relative to GAPDH ) for genes significantly changed. Data was shown as Means ± SD. n = 8 H and 15 A for ( A – D ). n = 11 ( H ) and 18 A for E – J . n = 6 (A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ) and 3 (H-KLRG1 + IL-17RB + ) for ( K, L ). Statistical comparisons were performed using unpaired, two-sided Welch’s t test for ( B – D ), and unpaired one-way ANOVA with Dunnett’s test for ( L ). p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 7E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Flow Cytometry, Purification, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Expressing