Journal: Cell reports

Article Title: Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

doi: 10.1016/j.celrep.2022.111281

Figure Lengend Snippet: Figure 3. SIOs provide an essentially GF model of gut-specific ILCs (A) Representative flow plots of NKp46 expression in ILCP + SIO co-culture-derived ILCs or SI-LP-derived CD127+ ILCs, with the frequency of NKp46+ ILC3s (co- culture: Live, EpCAM, Lin, CD45+, RORgt+; primary tissue: Live, CD45+, Lin, CD127+, Klrg1, NK1.1+/, RORgt+) additionally quantified for ILCPs cultured without SIOs or with GF SIOs in (B) (N = 2–5 animals, pooled from two experiments). (C) Relative frequency of mature ILC subsets excluding immature or other cells, depicting group 1 (magenta; Live, EpCAM, CD45+, Lin, RORgt-, ST2, Klrg1, NK1.1+, NKp46+), group 2 (green; Live, EpCAM, CD45+, Lin, RORgt, NK1.1, ST2+, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; Live, EpCAM, CD45+, Lin, ST2, Klrg1, RORgt+, NKp46+), and NKp46 group 3 ILCs (blue; Live, EpCAM, CD45+, Lin, ST2, Klrg1, RORgt+, NKp46) in live, unstimulated co-cultures derived from SPF-SIOs or GF-SIOs compared with primary SPF ileum (no Peyer’s patches). (D) Diagram of transwell culture strategy. (E) Relative frequency of group 1, 2, and 3 ILCs derived from PD-1+ ILCP + SIO +/ transwell insert (TW) separation (N = 3, two experiments).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human CRTh2 – PE Miltenyi Biotec Cat# 130-113-600 Human CRTh2– BV711 BioLegend Cat# 350124 Human CRTh2 – BV421 BioLegend Cat# 350112 Human CD161 APC, A700 BioLegend Cat# 302012 Human ST2 – APC R&D Sys Cat# FAB5231A Human ST2 – PE R&D Sys Cat# FAB5231P Human NKp44 – PE Cy7 BioLegend Cat# 325116 Human NKp44 – PerCP Cy5.5 BioLegend Cat# 325114 Human/Mouse Tbet – PE-Cy7 BioLegend Cat# 644824 Human RORgt – APC Thermofisher (eBioscience) Cat# 17-6988-82 Human RORgt – PE BDBiosciences Cat# 563081 Human GATA3 – APC Cy7 Santa Cruz Cat# sc-268 Human IL22 – PerCP Cy5.5 BioLegend Cat# 366709 Human IL17A – PE-Dazzle BioLegend Cat# 512335 Human IL17A – e450 BD Horizon/ bioscience Cat# 560610 Human IL17A – BV786 BD Horizon/ bioscience Cat# 563745 Human IFNg –APCe780 Invitrogen Cat# 47-7319-41 Human IL-5 – APC BioLegend Cat# 504305 Human IL-13 – FITC eBioscience Cat# 11-7139-41 Human IL-13 – bv711 BD Biosciences Cat# 564288 Human CD25 – PerCP-Cy 5.5 BD Biosciences Cat# 560503 Human Klrg1 – APC Thermofisher (eBioscience) Cat# 25-5893-80 Human CCR6 – APC BioLegend Cat# 353416 Human CCR6 – BV605 BioLegend Cat# 353419 Human NKp46 APC BioLegend Cat# 331918 FcR CD16/32 blocking, mouse BioCell Cat# BE0307 FcR blocking, human Miltenyi Biotec Cat# 130-059-901 Anti-rhIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3625 Anti-rmIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3626 E-Cadherin – anti-human (rat) Thermofisher (eBioscience) Cat# 51-3249-82 CDX2 – anti-human (rabbit) abcam Cat# Ab76541 EpCAM – anti-mouse (rabbit) abcam Cat# Ab71916 CD45 anti-human (mouse) BioLegend Cat# 304001 ZO-1 anti-mouse (rabbit) Abcam Cat# Ab96587 Dclk1 anti-mouse (rabbit) Abcam Cat# Ab31704 CD44 anti-mouse/human (rat) Thermofisher (eBioscience) Cat# 14-0551-82 Critical commercial assays CellTrace FarRed ThermoFischer Cat# C34564 Foxp3 / Transcription Factor Staining Buffer Set Invitrogen eBioscience Cat# 00-5523-00 Live Dead fixable blue/UV ThermoFischer Cat# L34961 UltraComp eBeads Invitrogen Cat# 01-2222-42 RNeasy Qiagen Cat# 74106 RevertAid First Strand cDNA Synthesis Kit ThermoFisher Cat# K1622 Fast SYBR green master mix Applied Biosystems Cat# 4385612 (Continued on next page) Cell Reports 40, 111281, August 30, 2022 e2

Techniques: Expressing, Co-Culture Assay, Derivative Assay, Cell Culture

Journal: Cell reports

Article Title: Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

doi: 10.1016/j.celrep.2022.111281

Figure Lengend Snippet: Figure 7. Gut-matured ILC2 upregulates ST2 on transfer to HLO culture (A) Representative image of SD-HIOs and SD-HLOs showing E-cadherin+ (E-CAD) epithelium, CD45+ ILCs, and nuclei (Hoechst) after 14-day co-culture (scale bars: 50 mm). (B) Count of EpCAM, CD45+, LIN ILCs after 14-day co-culture with SD-HIOs or SD-HLOs, with corresponding count of EpCAM, CD45 mesenchyme.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human CRTh2 – PE Miltenyi Biotec Cat# 130-113-600 Human CRTh2– BV711 BioLegend Cat# 350124 Human CRTh2 – BV421 BioLegend Cat# 350112 Human CD161 APC, A700 BioLegend Cat# 302012 Human ST2 – APC R&D Sys Cat# FAB5231A Human ST2 – PE R&D Sys Cat# FAB5231P Human NKp44 – PE Cy7 BioLegend Cat# 325116 Human NKp44 – PerCP Cy5.5 BioLegend Cat# 325114 Human/Mouse Tbet – PE-Cy7 BioLegend Cat# 644824 Human RORgt – APC Thermofisher (eBioscience) Cat# 17-6988-82 Human RORgt – PE BDBiosciences Cat# 563081 Human GATA3 – APC Cy7 Santa Cruz Cat# sc-268 Human IL22 – PerCP Cy5.5 BioLegend Cat# 366709 Human IL17A – PE-Dazzle BioLegend Cat# 512335 Human IL17A – e450 BD Horizon/ bioscience Cat# 560610 Human IL17A – BV786 BD Horizon/ bioscience Cat# 563745 Human IFNg –APCe780 Invitrogen Cat# 47-7319-41 Human IL-5 – APC BioLegend Cat# 504305 Human IL-13 – FITC eBioscience Cat# 11-7139-41 Human IL-13 – bv711 BD Biosciences Cat# 564288 Human CD25 – PerCP-Cy 5.5 BD Biosciences Cat# 560503 Human Klrg1 – APC Thermofisher (eBioscience) Cat# 25-5893-80 Human CCR6 – APC BioLegend Cat# 353416 Human CCR6 – BV605 BioLegend Cat# 353419 Human NKp46 APC BioLegend Cat# 331918 FcR CD16/32 blocking, mouse BioCell Cat# BE0307 FcR blocking, human Miltenyi Biotec Cat# 130-059-901 Anti-rhIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3625 Anti-rmIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3626 E-Cadherin – anti-human (rat) Thermofisher (eBioscience) Cat# 51-3249-82 CDX2 – anti-human (rabbit) abcam Cat# Ab76541 EpCAM – anti-mouse (rabbit) abcam Cat# Ab71916 CD45 anti-human (mouse) BioLegend Cat# 304001 ZO-1 anti-mouse (rabbit) Abcam Cat# Ab96587 Dclk1 anti-mouse (rabbit) Abcam Cat# Ab31704 CD44 anti-mouse/human (rat) Thermofisher (eBioscience) Cat# 14-0551-82 Critical commercial assays CellTrace FarRed ThermoFischer Cat# C34564 Foxp3 / Transcription Factor Staining Buffer Set Invitrogen eBioscience Cat# 00-5523-00 Live Dead fixable blue/UV ThermoFischer Cat# L34961 UltraComp eBeads Invitrogen Cat# 01-2222-42 RNeasy Qiagen Cat# 74106 RevertAid First Strand cDNA Synthesis Kit ThermoFisher Cat# K1622 Fast SYBR green master mix Applied Biosystems Cat# 4385612 (Continued on next page) Cell Reports 40, 111281, August 30, 2022 e2

Techniques: Co-Culture Assay

Journal: Nature Communications

Article Title: GM-CSF drives dysregulated hematopoietic stem cell activity and pathogenic extramedullary myelopoiesis in experimental spondyloarthritis

doi: 10.1038/s41467-019-13853-4

Figure Lengend Snippet: a FACS staining, frequencies and absolute numbers of MCs in the SI LPL of healthy vs. spondyloarthritic (week 5) SKG mice. b Percentage of CD16/32 hi and ST2 hi cells among MCs in SI LPL. c – e SKG mice were treated daily with PBS or cromolyn sodium following SpA induction and culled at week 4. Healthy SKG mice (“uninjected”) were used as controls in ( c , d ). c Absolute numbers of GM-CSF + MCs in the SI LPL. d Absolute numbers of GM-CSF + MCs in the spleen. e Absolute numbers of neutrophils in joints and SI. f Clinical score of arthritis, ankle, and paw size increase. Dots represent individual mice; horizontal bars indicate mean. Data are representative of ( a – e ) or pooled from ( f ) two independent experiments. Groups were compared using Mann–Whitney U tests. Source data are provided as Source Data file.

Article Snippet: Female SKG mice were injected with curdlan, then ST2-Fc fragment (mouse ST2-mouse IgG1-Fc, Medimmune) or with a control mouse IgG1 (NIP228, Medimmune), both at 0.3 mg per IP injection three times weekly for 5 weeks.

Techniques: Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: GM-CSF drives dysregulated hematopoietic stem cell activity and pathogenic extramedullary myelopoiesis in experimental spondyloarthritis

doi: 10.1038/s41467-019-13853-4

Figure Lengend Snippet: a IL-33 protein levels from joints and SI of healthy and spondyloarthritic mice, n = 13 mice per group. b BMMC were stimulated with PBS or IL-33 for 16 h and secreted IL-6 and GM-CSF quantified by ELISA. Error bars = SEM of 3 independent experiments. c – i SpA-triggered male SKG mice were injected IP twice weekly with PBS or 1 µg of rIL-33. c Experimental protocol. d GM-CSF protein levels from joints and SI from PBS or IL-33 treated mice, n = 7 mice per group. e Frequencies and absolute numbers of splenic MC. f Staining of intracellular cytokines (compared to GM-CSF FMO control) and frequencies of GM-CSF + cells among splenic MCs. g Frequencies of LT-HSCs and MPPs in BM (left). Staining of GMPs and MEPs in BM (middle). Frequencies of GMPs and neutrophils in BM (right). h Absolute numbers of GMP and neutrophils in spleens (left) and joints (right). Dots represent individual mice; horizontal bars indicate mean. i Mean arthritis scores ( n = 7 mice in each group) and increase in ankle thickness. j – k SKG females were injected with ST2-Fc or control Fc fragment for 5 weeks after injection of curdlan. j Frequency of GM-CSF + mast cells in SI of Fc control- and ST2-Fc-treated mice after 5 weeks. k Change in paw size (left) and clinical arthritis score (right) in Fc control- and ST2-Fc-treated mice after 2 (top) or 5 (bottom) weeks. l SKG males were injected with curdlan then IL-33, with or without isotype or anti-GM-CSF antibodies. Dots represent individual mice; horizontal bars indicate mean. Clinical arthritis score (left) and change in ankle thickness (right) after 4 weeks. Data are representative of three independent experiments ( a − i ) or pooled from two independent experiments ( k ). Groups were compared using Mann–Whitney U tests. Source data are provided as Source Data file.

Article Snippet: Female SKG mice were injected with curdlan, then ST2-Fc fragment (mouse ST2-mouse IgG1-Fc, Medimmune) or with a control mouse IgG1 (NIP228, Medimmune), both at 0.3 mg per IP injection three times weekly for 5 weeks.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Staining, Control, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.

doi: 10.4049/jimmunol.181.9.5981

Figure Lengend Snippet: FIGURE 1. Real-time quantitative PCR and flow cytometric analysis for ST2. A, cDNAs from highly purified basophils (n 7), eosinophils (n 7), and neutrophils (n 7) were tested. The data are expressed as the following ratio: copy number of ST2 gene/copy number of -actin gene 10,000. , p 0.05 and , p 0.001 vs the ratio of basophils. Data for the positive-control ST2-expressing cells, i.e., cultured mast cells, are reported in our recent article (27) and included in this figure (n 4). B, Highly purified basophils were incubated with and without IL-33 at 100 ng/ml for 4 h before RNA extraction (n 5). The calculated ratios for the same donor are connected with a solid line in the graph. C, Highly purified basophils were stained for surface-expressed ST2. Cells stained with control Ab are shown as a shaded area. D, Purified basophils were cultured with 300 pM IL-3 for 18 h and then stained for surface-expressed ST2. The cells incubated in medium alone are shown as a thin line, and the cells cultured with IL-3 are shown as a thick line. The cells stained with control Ab are shown as a shaded area. E, Intracellular ST2 staining of freshly isolated basophils. The cells stained with control Ab are shown as a shaded area. All the flow cytometry data are representative of three separate experiments using cells from different donors and showing similar results.

Article Snippet: The following Abs were purchased as indicated: mouse anti-IL-4 mAb (IgG1, clone 8D4–8) and biotin-conjugated rat anti-IL-4 mAb (IgG1, clone MP4–25D2) (eBioscience); FITC-conjugated goat anti-human IgE Ab (Biosource International); mouse neutralizing anti-ST2 mAb (IgG1, clone 97203) (R&D Systems); rabbit anti-IL-33 pAb (Adipogen); mouse anti-ST2 mAb (IgG1, clone HB12) (MBL); control mouse IgG1 (MOPC21) and mouse IgG2a (UPC10) (Sigma-Aldrich); mouse IgG2b mAb (MOPC 195) (Cappel); and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Expressing, Cell Culture, Incubation, RNA Extraction, Staining, Control, Isolation, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.

doi: 10.4049/jimmunol.181.9.5981

Figure Lengend Snippet: FIGURE 4. IL-33 enhances basophil CD11b expression through their surface ST2 receptor. A, Percoll-separated basophils were incubated with medium alone (thin line) or IL-33 at 10 ng/ml (thick line) for 30 min. Then the surface CD11b expression level was assessed by flow cytometry. Data are representative of three separate experiments yielding similar results. B, Percoll-separated basophils were incubated with and without cytokines for 30 min, and then the levels of surface CD11b expression were analyzed. The data are expressed as the percentages of MESF values of basophils treated without any stimulus (nil). Error bars represent the SEM (n 3). , p 0.05; , p 0.01 vs nil. C, Basophils were incubated with the indicated concentrations of IL-33 for either 30 min () or 18 h (f). Then the surface CD11b expression level was assessed. Error bars represent the SEM (n 5). , p 0.001 vs nil. D, Basophils were incubated with IL-33 plus either IL-3 (u) or CRA-1 mAb (f) for 30 min, and then the expression level of CD11b was quantified. Error bars represent the SEM (n 3–6). , p 0.01; , p 0.001 vs corresponding values of cells cultured without IL-33. E, Basophils were preincubated with medium alone (u), control IgG (), or neutralizing anti-ST2 Ab (f) at 10 g/ml for 60 min and then treated with IL-33 at indicated concentrations for 30 min. Error bars represent the SEM (n 3). , p 0.05; , p 0.001 vs nil.

Article Snippet: The following Abs were purchased as indicated: mouse anti-IL-4 mAb (IgG1, clone 8D4–8) and biotin-conjugated rat anti-IL-4 mAb (IgG1, clone MP4–25D2) (eBioscience); FITC-conjugated goat anti-human IgE Ab (Biosource International); mouse neutralizing anti-ST2 mAb (IgG1, clone 97203) (R&D Systems); rabbit anti-IL-33 pAb (Adipogen); mouse anti-ST2 mAb (IgG1, clone HB12) (MBL); control mouse IgG1 (MOPC21) and mouse IgG2a (UPC10) (Sigma-Aldrich); mouse IgG2b mAb (MOPC 195) (Cappel); and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Techniques: Expressing, Incubation, Cytometry, Cell Culture, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.

doi: 10.4049/jimmunol.181.9.5981

Figure Lengend Snippet: FIGURE 5. IL-33 enhances human basophil migration toward eotaxin. A, Two 104 Percoll-separated basophils were added to the upper cham- ber. IL-33 at 10 or 100 ng/ml or eotaxin (Eot) at 10 nM was added to the lower chamber. Cells mixed with IL-33 at 10 ng/ml were also tested for migration toward eotaxin. The percentage of migrated cells was calculated by subtracting the spontaneous migration (9.6 0.4% for medium only). Error bars represent the SEM (n 5). , p 0.01 vs spontaneous mi- gration in medium alone. B, Percoll-separated basophils were mixed with the indicated concentrations of IL-33 and then tested for migration toward eotaxin at 10 nM. The percentage of migrated cells was calculated by subtracting the spontaneous migration (16.8 0.5% for medium only). Error bars represent the SEM (n 3). , p 0.01 vs basophil migration toward eotaxin in the absence of IL-33. C, Both Percoll-separated () and MACS-separated (f) basophils were used for the migration assay. Baso- phil preparations with and without IL-33 at 100 ng/ml were placed in the upper chamber, and eotaxin at 50 nM was added to the lower chamber. The percentage of migrated cells was calculated by subtracting the spontaneous migration (16.8 0.8% for Percoll-separated and 12.3 1.2% for MACS- separated preparations). Error bars represent the SEM (n 3). , p 0.01 vs migration of the corresponding basophils without IL-33. D, Effect of neutralizing Ab for ST2 on IL-33 enhancement of basophil migration. Per- coll-separated basophils were mixed without Ab () or with control IgG at 20 g/ml (u) or anti-ST2 Ab at 20 g/ml (f). IL-33 at 10 ng/ml was then added to the cells; eotaxin at 10 nM was added to the lower chamber. Data shown are mean values of an experiment performed in duplicate. Another experiment using basophils from a different donor yielded similar results. E, Effect of IL-33 on CCR3 expression by human basophils. Highly puri- fied basophils were incubated with (bold line) and without (thin line) IL-33 at 10 ng/ml for 1 h at 37°C. The dotted line indicates basophils that were incubated with IL-3 at 300 pM. Cells stained with control Ab are shown as a shaded area. Data are representative of two separate experiments using cells from different donors and showing similar results.

Article Snippet: The following Abs were purchased as indicated: mouse anti-IL-4 mAb (IgG1, clone 8D4–8) and biotin-conjugated rat anti-IL-4 mAb (IgG1, clone MP4–25D2) (eBioscience); FITC-conjugated goat anti-human IgE Ab (Biosource International); mouse neutralizing anti-ST2 mAb (IgG1, clone 97203) (R&D Systems); rabbit anti-IL-33 pAb (Adipogen); mouse anti-ST2 mAb (IgG1, clone HB12) (MBL); control mouse IgG1 (MOPC21) and mouse IgG2a (UPC10) (Sigma-Aldrich); mouse IgG2b mAb (MOPC 195) (Cappel); and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Techniques: Migration, Control, Expressing, Incubation, Staining