Journal: Nature Communications

Article Title: Orai inhibition modulates pulmonary ILC2 metabolism and alleviates airway hyperreactivity in murine and humanized models

doi: 10.1038/s41467-023-41065-4

Figure Lengend Snippet: A Block of endogenous SOCE in FACS-sorted pulmonary ILC2s after exposure to compound 5D measured by flow cytometry. Cells were pretreated with thapsigargin to deplete the intracellular Ca 2+ stores before addition of Ca 2+ and 5D exposure. B FACS-sorted pulmonary murine ILC2s were cultured for 24 h with survival cytokines rmIL-2, rmIL-7 and with rmIL-33 or PBS control for 24 h. 5D or the vehicle control were then added to the wells for an additional 48 h. C After 48 h, cells were collected and Ki67 as a measure of proliferation was assessed and quantified by flow cytometry presented as mean ± SEM. n = 3 biologically independent samples. D Supernatant was collected and secreted cytokines were measured by Legendplex (S2A). Presented as mean ± SEM. n = 3 biologically independent samples. E Representative flow cytometry plots of IL-5 + and IL-13 + ILC2s from ILC2s cultured with 5D or the vehicle and corresponding quantitation presented as mean ± SEM. n = 3 biologically independent samples. F Pulmonary ILC2s were sorted from wild-type (WT), Orai1 −/− and Orai1/2 −/− mice and cultured with and without tamoxifen for 48 h. Cells were collected, counted, and cultured for an additional 48 h. Supernatant was collected and proinflammatory cytokines were measured by Legendplex (S2B). Presented as mean ± SEM. n = 3 biologically independent samples. G ILC2s were collected, and intracellular IL-5 and IL-13 production was measured and quantified by flow cytometry (S2C). Presented as mean ± SEM. n = 3 biologically independent samples. Data are representative of 2 independent experiments and are presented as means ± SEM. Source data are provided as a Source data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. ILC2 cell image designed with Servier Medical Art.

Article Snippet: APC anti-mouse IL-13 (85BRD, Thermofisher), PE anti-mouse IL-5 (TRFK5, BioLegend) were used.

Techniques: Blocking Assay, Flow Cytometry, Cell Culture, Quantitation Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Orai inhibition modulates pulmonary ILC2 metabolism and alleviates airway hyperreactivity in murine and humanized models

doi: 10.1038/s41467-023-41065-4

Figure Lengend Snippet: A Schematic of the mitochondrial electron transport chain (mtETC) and the associated complexes. B Heatmap of statistically significant differentially expressed genes associated with the mtETC in ILC2s cultured with 5D or the vehicle. Arrow colors coordinate with the associated complex in ( A ). C Flow cytometry histogram and quantification of mitochondrial mass measured by Mitotracker Green in ILC2s cultured with 5D or the vehicle presented as mean ± SEM. n = 3 biologically independent samples. D Flow cytometry histogram and quantification of mitochondrial membrane potential measured by Mitotracker Red in ILC2s cultured with 5D or the vehicle presented as mean ± SEM. n = 3 biologically independent samples. E Total cellular reactive oxygen species (ROS) in treated and untreated ILC2s measured by microplate fluorescence presented as mean ± SEM. n = 3 biologically independent samples. F Flow cytometry histogram and quantification of mitochondrial ROS in ILC2s cultured with 5D or the vehicle as measured by Mitosox presented as mean ± SEM. n = 3 biologically independent samples. G NADH/NAD+ ratio quantification in ILC2s cultured with 5D or the vehicle measured by microplate reader presented as mean ± SEM. n = 3 biologically independent samples. H ATP production presented as OCR measured by Seahorse Mitostress Test assay presented as mean ± SEM. n = 3 biologically independent samples. I Representative flow cytometry plots and quantification of intracellular IL-5 production in ILC2s cultured with the vehicle, 5D, or 5D and the addition of antioxidant NAC presented as mean ± SEM. n = 3 biologically independent samples. J Representative flow cytometry plots and quantification of intracellular IL-13 production in ILC2s cultured with the vehicle, 5D, or 5D and the addition of antioxidant NAC presented as mean ± SEM. n = 3 biologically independent samples. K Representative flow cytometry plots and quantification of proliferation in ILC2s cultured with the vehicle, 5D, or 5D and the addition of antioxidant NAC presented as mean ± SEM. n = 3 biologically independent samples. Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Figure 5A was created with BioRender.com.

Article Snippet: APC anti-mouse IL-13 (85BRD, Thermofisher), PE anti-mouse IL-5 (TRFK5, BioLegend) were used.

Techniques: Cell Culture, Flow Cytometry, Membrane, Fluorescence, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Orai inhibition modulates pulmonary ILC2 metabolism and alleviates airway hyperreactivity in murine and humanized models

doi: 10.1038/s41467-023-41065-4

Figure Lengend Snippet: A FACS-sorted human blood ILC2s were cultured with rhIL-2, rhIL-7, and rhIL-33 for 24 h. 5D or vehicle were added to the culture for an additional 48 h. B Flow cytometry histogram and quantification of Orai1 expression after 24 h with survival cytokines in ( A ) presented as mean ± SEM. n = 4 biologically independent samples. C Flow cytometry histogram and quantification of Orai2 expression after 24 h with survival cytokines in ( A ) presented as mean ± SEM. n = 4 biologically independent samples. D After 48 h with 5D or vehicle, supernatant was collected and IL-5 and IL-13 levels were measured by ELISA. n = 6 biologically independent samples. E FACS-sorted human blood ILC2s from healthy donors were adoptively transferred into Rag2 −/− γc −/− mice. Mice were intranasally challenged with rhIL-33 (1 μg) or PBS and treated with i.p. injections of 5D or vehicle days 0, 1, and 2. Mice were euthanized on day 3 and AHR ( F ) was measured presented as mean ± SEM. n = 4 biologically independent samples. G Total number of eosinophils infiltrating the BAL presented as mean ± SEM. n = 4 biologically independent samples. H Total number of human ILC2s found in the lung presented as mean ± SEM. n = 4 biologically independent samples. Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse and human ILC2 cell images designed with Servier Medical Art.

Article Snippet: APC anti-mouse IL-13 (85BRD, Thermofisher), PE anti-mouse IL-5 (TRFK5, BioLegend) were used.

Techniques: Cell Culture, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Developmental cell

Article Title: A comprehensive roadmap of murine spermatogenesis defined by single-cell RNA-seq

doi: 10.1016/j.devcel.2018.07.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC anti-mouse IL-13 Antibody , Novus , Cat#011818.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Gene Expression, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease

doi: 10.1111/jcmm.13554

Figure Lengend Snippet: The level of IFN ‐γ produced by iNKT cells was impaired and significantly associated with bone disease in newly diagnosed multiple myeloma ( NDMM ) patients. A,B, The expression of Th1 cytokines ( IFN ‐γ, TNF ) and Th2 cytokines ( IL ‐4, IL ‐13) by iNKT cells in healthy controls ( HC s) and NDMM patients were examined by flow cytometry after stimulation with α‐GalCer. The percentages of IFN ‐γ + (C), TNF + (D) iNKT cells were significantly lower in NDMM and relapsed/refractory MM ( RMM ) patients than in HC s. The percentages of IL ‐4 + (E) and IL ‐13 + (F) iNKT cells were significantly higher in NDMM and RMM patients than in HC s. G, The percentages of IFN ‐γ + iNKT cells were strongly lower in NDMM patients with Stage C bone disease than with Stage A/B bone disease. H, The percentages of IFN ‐γ + iNKT cells was significantly correlated with the level of carboxy‐terminal cross‐linking telopeptide of type I collagen ( CTX ) respectively by Spearman's correlation coefficient. I, The percentages of IFN ‐γ + iNKT cells were significantly correlated with the percentages of osteoclast precursors ( OCP s) respectively by Spearman's correlation coefficient. (Medians of each group were compared using Kruskal‐Wallis test followed by all pairwise multiple comparisons; * P < .05; ** P < .01; *** P < .001)

Article Snippet: Cells were incubated with 20 μL anti‐human IFN‐γ APC (BD Biosciences), anti‐human TNF APC (BD Biosciences), anti‐human IL‐4 APC (BD Biosciences), anti‐human IL‐13 APC (BD Biosciences) in the dark at 4°C for 30 minutes.

Techniques: Produced, Expressing, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease

doi: 10.1111/jcmm.13554

Figure Lengend Snippet: Inhibition of osteoclastogenesis by iNKT cells via IFN ‐γ production. A, In healthy controls ( HC s), the level of IFN ‐γ in osteoclasts ( OC s) culture supernatants at day 7 and day 14 markedly increased in α‐GalCer‐stimulated cultures compared with DMSO control cultures. The differences at day 7 were significant, but at day 14 were no significant. About the level of TNF (B) and IL ‐13 (C) in OC s culture supernatants, no significant differences were found between α‐GalCer‐stimulated cultures and DMSO control cultures at day 0, day 3, day 7 and day 14 in NDMM patients and HC s. Mean ± SEM between the experimental samples were compared using Student's t test. D, Representative tartrate‐resistant acid phosphate ( TRAP )‐positive multinucleated cells ( MNC ) from a NDMM patient in the presence or absence of recombinant IFN ‐γ or α‐GalCer and a HC in the presence or absence of anti‐ IFN ‐γ or α‐GalCer. Original magnification × 100 (Bar = 100 μm). E(a), The number of TRAP + MNC s was significantly increased in the presence of anti‐ IFN ‐γ and α‐GalCer cultures compared with the presence of α‐GalCer cultures (b) The number of TRAP + MNC s was significantly reduced in the presence of IFN ‐γ and α‐GalCer cultures compared with the presence of α‐GalCer cultures. Mean ± SEM of each group were compared using one‐way ANOVA analysis. F, The mRNA expression of osteoclast‐associated genes, such as TRAP , osteoclast‐associated receptor ( OSCAR ) and RANKL was significantly improved in the presence of anti‐ IFN ‐γ and α‐GalCer cultures compared with the presence of α‐GalCer cultures. Medians of each group were compared using Kruskal‐Wallis test followed by all pairwise multiple comparisons. G , The mRNA expression of RANKL was significantly decreased in the presence of IFN ‐γ and α‐GalCer cultures compared with the presence of α‐GalCer cultures. Medians of each group were compared using Kruskal‐Wallis test followed by all pairwise multiple comparisons (* P < .05, ** P < .01, *** P < .001)

Article Snippet: Cells were incubated with 20 μL anti‐human IFN‐γ APC (BD Biosciences), anti‐human TNF APC (BD Biosciences), anti‐human IL‐4 APC (BD Biosciences), anti‐human IL‐13 APC (BD Biosciences) in the dark at 4°C for 30 minutes.

Techniques: Inhibition, Recombinant, Expressing

Journal: Nature Communications

Article Title: Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis

doi: 10.1038/s41467-017-00678-2

Figure Lengend Snippet: NKp30-B7H6 interaction and tumour-derived PGD2 favour IL-13 secretion. a Cytokine concentrations in serum samples of HD and APL patients (APL) ( n = 11). b Representative examples of flow cytometry analysis of cytokine production by ILC in HD and APL patients (APL). c Frequencies of cytokine producing ILC in HD and APL patients (APL), upon co-culture with APL cell lines for 48 h (six independent experiments). d Representative example of flow cytometry analysis of IL-13-positive cells upon co-culture with autologous APL blasts. e Frequencies of IL-13 + ILC2, NKT and Th2 cells ( n = 6). f Expression of B7H6 and g frequency of B7H6-positive cells in APL cell lines (NB4, HL60) and a control cell line (721.221, lymhphoblastoid cell line) (six independent experiments). h Representative example of flow cytometry analysis of the expression of B7H6 and i frequency of B7H6-positive cells in APL blasts in bone marrow (APL BM), in APL blasts in peripheral blood (APL PB), ( n = 11) or in a control cell line (721.221, n = 6). j Representative example of flow cytometry analysis of the expression of NKp30 on ILC2 in peripheral blood of healthy donors (HD PB), in APL BM or in APL PB. k Relative frequencies of NKp30 expressing ILC2 in HD PB, APL BM or APL PB ( n = 11). l Representative example of flow cytometry analysis of IL-13 produced by ILC2 co-cultured with the APL cell line NB4 in the presence of an anti-NKp30 blocking antibody (aNKp30), Ig control or medium. m Frequency of ILC2 IL-13 + in medium ( n = 9), in the presence of an aNKp30 or an Ig control (IgM Ctrl) ( n = 6, three independent experiments). n Quantification of IL-33, IL-25, TSLP and PGD2 in the sera of HD and APL patients (APL) ( n = 11). o Quantification of PGD2 in supernatants of leukaemic AML (KG1, THP1) and APL cell lines (NB4, HL60), in the absence or presence of arachidonic acid (ArAc) (1 experiment). Error bars are s.e.m. Statistical analysis was performed using Mann-Whitney test ( a – c , n ) and Kruskal-Wallis test ( d – i , m ) and ANOVA test ( k )

Article Snippet: Intracellular staining was performed using PE-Cy7-conjugated IFN-γ (cn: 557844, 1:200), APC-conjugated anti-IL-13 (cn: 561162, 1:100) (both from BD Pharmingen) and AlexaFluor700-conjugated anti-IL-17A (cn: 512318, 1:100, BioLegend) after fixation and permeabilization with 0.1% saponin (Sigma).

Techniques: Derivative Assay, Flow Cytometry, Co-Culture Assay, Expressing, Produced, Cell Culture, Blocking Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis

doi: 10.1038/s41467-017-00678-2

Figure Lengend Snippet: ILC2-derived IL-13 expands and activates suppressive M-MDSCs in APL. a Representative example of flow cytometry analysis of cell surface expression of the IL-13Rα1 on CD3 + , CD14 − , CD14 + cells in peripheral blood of an APL patient. b Frequencies of IL-13Rα1 expressing cells in the different compartments (CD3 + , CD14 − , CD14 + n = 11). c Representative example of flow cytometry phenotypic analysis of CD14 + cells in healthy donors (HD) and APL patients (APL). Evaluated are the expression of HLA-DR and CD33, to define phenotypic M-MDSC. d Expression of arginase-1 and e iNOS in purified CD14 + cells from HD and APL, as assessed by qPCR ( n = 11). f Frequency of M-MDSCs in peripheral blood of HD and APL ( n = 22). g , h Expression of arginase-1 ( g ) and iNOS ( h ) as assessed by qPCR, in purified CD14 + cells cultured in medium supplemented or not with recombinant IL-13 (rIL-13), or cultured in supernatants derived from ILC2 cell lines, activated by PDG2 and the NB4 APL cell line (SN ILC2). A blocking anti-IL-13 antibody (aIL-13) or an Ig control (Ig Ctrl) were added where indicated ( n = 6, three independent experiments). i Representative example of CFSE-dilutions of CD3 + T cells co-cultured with CD14 + monocytes, or with in vitro - induced M-MDSCs (CD14 + SN ILC2). j Frequencies of proliferating CD3 + T cells upon co-culture with CD14 + cells or with in vitro induced M-MDSCs (CD14 + SN ILC2) ( n = 6, three independent experiments). Error bars are s.e.m. Statistical analysis was performed using ANOVA test ( b ), t test ( d – f ), Kruskal-Wallis test ( g , h ) and Mann-Whitney test ( j )

Article Snippet: Intracellular staining was performed using PE-Cy7-conjugated IFN-γ (cn: 557844, 1:200), APC-conjugated anti-IL-13 (cn: 561162, 1:100) (both from BD Pharmingen) and AlexaFluor700-conjugated anti-IL-17A (cn: 512318, 1:100, BioLegend) after fixation and permeabilization with 0.1% saponin (Sigma).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Purification, Cell Culture, Recombinant, Blocking Assay, In Vitro, Co-Culture Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis

doi: 10.1038/s41467-017-00678-2

Figure Lengend Snippet: ATRA reverses the PGD2-ILC2-IL-13-M-MDSC immunosuppressive axis. a Schematic representation of the hypothetical immunosuppressive chain established in APL patients at diagnosis. b Comparison of serum concentrations of PGD2 (APL = 11; Rem = 9), c frequencies of ILC2 NKp30 + (APL = 11, APL Rem = 9), d frequencies of total ILC2 (APL = 22; APL Rem n = 9), e serum concentrations of IL-13 (APL n = 11; APL Rem n = 9) and f frequencies of M-MDSC (APL n = 22; APL Rem = 9) in peripheral blood of APL patients at diagnosis (APL) or in remission after ATRA treatment (APL Rem). Dashed lin es represent mean values of all the parameters in HD. g Schematic representation of APL establishment and ATRA treatment schedule in FVB/NJ mice upon injection of APL splenocytes (three independent experiments), as assessed by quantification of the normal composition of the bone marrow (Gr1 + CD11b + cells) h , as previously described . i Survival curves of untreated (APL, n = 13) and ATRA-treated (ATRA) APL mice ( n = 6). j Quantification of PGD2 concentrations (CTR n = 15, APL n = 13, ATRA day 14 n = 9, ATRA day 25 n = 6), k ILC2 frequencies and l M-MDSCs frequencies in control FVB/NJ mice (CTR), APL mice before (APL) and after ATRA treatment (ATRA day14; ATRA day25) (CTR n = 12, APL n = 10, ATRA day 14 n = 9, ATRA day 25 n = 6). m Schematic representation of APL establishment and ATRA treatment schedule in HIS mice upon injection of an APL cell line (two independent experiments). n Survival curves of untreated (APL, n = 5) and ATRA-treated (ATRA, n = 3) APL HIS mice. o Quantification of PGD2 concentrations (CTR n = 8, APL n = 8, ATRA n = 5), p ILC2 frequencies (CTR n = 11, APL n = 13, ATRA n = 9) and q M-MDSCs frequencies (CTR n = 8, APL n = 8, ATRA n = 5) in APL HIS mice before (APL) and after ATRA treatment at day 25 (ATRA) (up to two independent experiments). Error bars are s.e.m. Statistical analysis was performed using Mann-Whitney test ( b – f ), Kruskal-Wallis test ( h , j – l , o – q ) and Log-rank test (Mantel-Cox) ( i , n )

Article Snippet: Intracellular staining was performed using PE-Cy7-conjugated IFN-γ (cn: 557844, 1:200), APC-conjugated anti-IL-13 (cn: 561162, 1:100) (both from BD Pharmingen) and AlexaFluor700-conjugated anti-IL-17A (cn: 512318, 1:100, BioLegend) after fixation and permeabilization with 0.1% saponin (Sigma).

Techniques: Injection, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis

doi: 10.1038/s41467-017-00678-2

Figure Lengend Snippet: Therapeutic interference of the PGD2-ILC2-IL-13-M-MDSC immunosuppressive axis. a Schematic representation of DTx treatment schedule in iCOS-T mice upon injection of APL splenocytes (one independent experiment). b Quantification of ILC2 and c M-MDSCs frequencies in control mice (CTRL), APL mice (APL) and iCOS-T APL mice (DTx) ( n = 5). d Schematic representation of the schedule of PGD2 inhibitor (TM30089) and anti-IL-13 neutralizing antibody treatment in FVB/NJ mice injected with APL blasts (two independent experiments). e Comparison of serum concentrations of PGD2 (APL n = 13, PGD2i + αIL-13 n = 12), f ILC2 frequencies (APL n = 12, PGD2i + αIL-13 n = 12), g M-MDSC frequencies (APL n = 12, PGD2i + a IL-13 n = 12). h Survival curves of untreated (APL) and PGD2i + αIL-13-treated FVB/NJ mice injected with APL blasts (APL n = 12, PGD2i + αIL-13 n = 12). i Schematic representation of the schedule of PGD2 inhibitor (TM30089), anti-NKp30 blocking antibody and anti-IL-13 neutralizing antibody treatment in HIS mice injected with APL blasts (one experiment). j Representative examples and k cumulative data of luminescence measurement in untreated APL HIS mice (APL) and mice treated with PGD2 inhibitor (TM30089), anti-NKp30 blocking antibody and anti-IL-13 neutralizing antibody (COMBO) for quantification of leukaemic engraftment ( n = 8). l Quantification of PGD2 concentrations ( n = 8), m ILC2 frequencies (APL n = 13, COMBO n = 8) and n M-MDSCs frequencies in untreated APL HIS mice (APL) and mice treated with PGD2 inhibitor (TM30089), anti-NKp30 blocking antibody and anti-IL-13 neutralizing antibody (COMBO) ( n = 8). o Survival curves of untreated APL HIS mice and APL HIS mice treated with PGD2 inhibitor (TM30089), anti-NKp30 blocking antibody and anti-IL-13 neutralizing antibody (COMBO; n = 5). Error bar s are s.e.m. Statistical analysis was performed using Kruskal-Wallis test ( b , c ), Mann-Whitney test ( e – g , k , l – n ) and Log-rank test (Mantel-Cox) ( h , o )

Article Snippet: Intracellular staining was performed using PE-Cy7-conjugated IFN-γ (cn: 557844, 1:200), APC-conjugated anti-IL-13 (cn: 561162, 1:100) (both from BD Pharmingen) and AlexaFluor700-conjugated anti-IL-17A (cn: 512318, 1:100, BioLegend) after fixation and permeabilization with 0.1% saponin (Sigma).

Techniques: Injection, Blocking Assay, MANN-WHITNEY