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: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) A schematic of experimental design. CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 alone, IL2 and IL-33, or anti-CD3/CD28 under Th2 polarizing conditions for five days. (B) Representative flow plot of CD4 T cells immediately following isolation from secondary lymphoid organs of naïve or Hpoly -infected mice. (C) CD4 T cells from secondary lymphoid organs of Hpoly -infected mice were cultured under indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified. (D) Representative flow plot of CD4 T cells right after isolation from secondary lymphoid organs of Hpoly-infected 4get mice. (E) CD4 T cells from naïve or Hpoly-infected mice were cultured with IL2 and IL-33. (F) CD4 T cells from Hpoly-infected mice were cultured under the indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified after five days. (G) Abundance of amphiregulin in the culture supernatants from (F). (H) Splenocytes from naïve or Hpoly-infected mice cultured with IL2 alone or together with HES. Frequencies and numbers of GATA3+ cells was quantified. The data (B-H) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B-G) or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Representative flow plots and quantification of ST2+GATA3 + in live CD45+TCRβ+CD4+FOXP3-T cells from mesenteric adipose tissue of Hpoly -infected mice or after five days of in vitro culture of CD4 T cells isolated from Hpoly-infected mice. Flow plots and quantification of ST2+IL4+(GFP) (B) or CD44+IL4+(GFP+) (C) cells cultured as above. (D) CD4 T cells from naïve or Hpoly -infected mice were cultured as in (A) and IL-33R+GATA3+ Th2 cells were quantified. (E) Representative flow plots and quantification of AREG+ cells within the IL4+(GFP+) population after 5hr restimulation with PMA/Iono in the presence of Brefeldin. The data (A-E) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (A-C, E) or two-way ANOVA with Sidak post hoc test (D).. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, *** p ≤ 0.0001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Infection, In Vitro, Isolation, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) CD44+IL4+ (GFP)+ early Th2 cells, CD44+IL4-(GPF-) activated cells, and naïve CD44-GFP-cells were FACS-isolated from 4get mice infected with Hpoly and cultured with IL2 and IL-33 for five days. Frequencies of GFP+ and GFP+ST2+ cells (B) as well as ST2 gMFI were quantified (C). The data (A-C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B) or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: RNAseq was performed on CD44+IL4+(GFP+) Th2 cells isolated from spleens and lymph nodes of Hpoly -infected mice or after five-day culture under indicated conditions. (A) Principal component analysis of RNAseq data. (B) Heatmap of hierarchical clustering of differentially expressed genes. (C) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (D) Flow plots and quantification of pS6 expression in CD44+IL4(GFP+) Th2 cells. (E) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 in the presence of rapamycin. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (F) Levels of indicated cytokines after overnight culture in fresh media containing rapamycin. The data (D, E, F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E) or paired T-test (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Comparison, Expressing, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured anti-CD3/CD28 in the presence of rapamycin. The mean fluorescence intensity of GATA3 and cell numbers were quantified. (C) Levels of indicated cytokines after overnight culture of anti-CD3/CD28-stimulated cells in fresh media containing rapamycin. (D) Quantification of results from SCENITH assay in GATA3+ Th2 cells. (E) Representative flow plot showing the expression of TSLPR and ST2 after five days of in vitro culture of CD4 T cells were isolated from Hpoly -infected mice. (F) Representative flow plot and quantification of mitotracker green and mitotracker deep red staining after five days of culture. The data (B-F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, E, F), or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Comparison, Isolation, Infection, Cell Culture, Fluorescence, Expressing, In Vitro, Staining

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Heatmap representing the abundance of the top 100 detected metabolites. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days. The intracellular abundance of indicated amino acids in naïve and CD4+CD44+ activated cells was determined via LC/MS. (C) Heatmap depicting the expression of arginine transporters genes related in RNAseq data from . (D) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days in amino acid drop-out media. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (E) Representative histograms and quantification of CTV dilution of CD4 T cells cultured as in D. (F) After five days of culture with IL2 and IL-33 in control or arginine-depleted media, cells were restimulated with PMA/Iono and amphiregulin was quantified. (G) Representative histograms and quantification of ps6 levels in pS6-positive cells at day 1 of culture. The data (D-G) are representative of at least two independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E), paired T-test (F), or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Expressing, Control

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Expresssion of Th2 cell related genes in scRNAseq dataset of T cells isolated from mAT of Hpoly -infected mice identifying cluster of GATA3+ST2+ Th2 cells (circled). CD4 T cells from Hpoly-infected mice were stimulated with anti-CD3/CD28 and treated with DFMO (B) or cultured in arginine-depleted media (C). Mean fluorescence intestity of GATA3 and cells numbers were quantified. The data (B,C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture, Fluorescence