immunocult human th2 differentiation supplement  (STEMCELL Technologies Inc)

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Reverse transcriptase PCR gel for CFTR mRNA (predicted size of 125 bp; arrow) in Cftr +/+ and Cftr −/− mouse CD4 + T cells. Lane 1 is a 100 bp ladder. ( B ) qPCR analysis of Cftr expression in Cftr +/+ and Cftr −/− mouse CD4 + T cells at 0 and 24 hours following TCR ligation with anti-CD3 and anti-CD28 mAbs ( n = 4 per genotype and time point). ( C ) Immunostaining of CFTR (green), plasma membrane lectin (red), and nuclei (blue) in 24-hour–cultured mouse CD4 + cells (3 different biologic replicates per genotype, Cftr +/+ and Cftr −/− ). Scale bar: 10 μm. ( D ) Immunoprecipitated CFTR protein in immortalized human CD4 + T cells (Jurkat) using UNC-450 anti-CFTR monoclonal antibody (mAb) for pulldown and UNC-596 anti-CFTR mAb for detection compared with immunoprecipitation isotype control. ( E – H ) qPCR analysis of Cftr expression in Cftr +/+ and Cftr −/− cultured mouse CD4 + T cells at 0, 6, 18, 48, 72, and 78 hours in Th2 ( E ), Th1 ( F ), Th17 ( G ), and Tregs ( H ) ( n = 3 per genotype per time point). Dotted lines represent TCR ligation with anti-CD3 (1 μg/mL) and anti-CD28 (0.5 μg/mL) mAbs and (a) anti–IFN-γ (10 μg/mL) and mouse IL-4 (10 ng/mL) for Th2 cells, (b) anti–IL-4 (10 μg/mL) and mouse IL-12 (10 ng/mL) for Th1 cells, and (c) human TGF-β (0.5 ng/mL), mouse IL-23 (10 ng/mL), mouse IL-6 (40 ng/mL), mouse IL-1b (10 ng/mL), anti–IL-4 (10 μg/mL), and anti–IFN-γ (10 μg/mL) for Th17 cells. Tregs were polarized and restimulated with anti-CD3 (1 μg/mL), human IL-2 (100 IU/mL), and recombinant human TGF-β (1ng/mL). Data are shown as mean ± SD. Statistical analysis were done in B and E – H by 1-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Reverse Transcription, Expressing, Ligation, Immunostaining, Clinical Proteomics, Membrane, Cell Culture, Immunoprecipitation, Control, Recombinant

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Schematic diagram showing isolation and polarization of naive CD4 + T cells to Th2 cells for whole transcriptome analysis. ( B ) PCA plot of gene expression data for 3 biological replicates used for bulk RNA-Seq of Cftr +/+ (black dots) and Cftr −/− (red dots) Th2 cells. ( C ) Volcano plot depicting DESeq2 analysis of differentially expressed genes in Cftr +/+ and Cftr −/− Th2 cells. Red dots represent genes expressed at higher levels in Cftr −/− Th2 cells, while black dots represent genes with higher expression levels in Cftr +/+ Th2 cells. The y axis denotes −log 10 FDR values while the x axis shows log 2 fold change values. Select genes indicated. ( D ) Advanced bubble plot showing KEGG pathways enriched in Cftr −/− Th2 cells. The y axis denotes enrichment score, and −log 10 FDR values are shown on the x axis. The size of the bubble represents the number of genes enriched in each pathway. Select pathways indicated. ( E – H ) Heatmaps showing the differential gene expression profile of core Th2 associated genes including surface markers ( E ), cytokines ( F ), transcription factors ( G ), and CD4 + pan markers ( H ) in Cftr −/− versus Cftr +/+ Th2 cells. Normalized log 2 gene expression determined by RNA-Seq shown to the right of each heatmap with statistically significant DEGs denoted (*) in Cftr +/+ (gray bars) and Cftr −/− (red bars) Th2 cells. RNA-Seq data were generated in biological triplicates from 3 mice and analyzed via DESeq2. KEGG was used for pathway analysis.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Isolation, Gene Expression, RNA Sequencing, Expressing, Generated

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Schematic diagram showing isolation and polarization conditions of naive CD4 + T cells to Th2 cells for IL-4 studies. ( B ) Representative flow cytometry histogram showing the median fluorescence intensity (MFI) of IL-4Rα at 72 hours for Cftr +/+ and Cftr −/− Th2 cells. ( C ) The quantified MFI of IL-4Rα in Cftr +/+ and Cftr −/− Th2 cells at 72 hours ( n = 5 mice per genotype). ( D ) Representative flow cytometry histogram showing the MFI of GATA3 at 72 hours for Cftr +/+ and Cftr −/− Th2 cells. ( E ) The quantified MFI of GATA3 in Cftr +/+ and Cftr −/− Th2 cells at 72 hours ( n = 5 mice per genotype). ( F ) Representative CD4 + populations showing the MFI of GATA3 at 72 hours in the presence of increasing doses of polarizing IL-4 (0–40 ng/mL) for Cftr +/+ and Cftr −/− Th2 cells. ( G and H ) GATA3 MFI and secreted IL-13 from cellular supernatant in cultured Cftr +/+ (black) and Cftr −/− (red) Th2 cells in the presence of increasing doses of IL-4 (0–40 ng/mL). ( C and E ) Data are shown as mean ± SD. Statistical analysis in C and E were performed using unpaired Student’s t test and, in G and H , by 4-parameter logistic regression algorithm (sigmoidal curve fit) to fit. For G and H , data are shown as mean values with the accompanying curve fit (solid line), the 95% CI displayed as a band as well as mean data points for each genotype and concentration. ** P < 0.01 and **** P < 0.0001.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Isolation, Flow Cytometry, Fluorescence, Cell Culture, Concentration Assay

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Schematic diagram detailing the isolation and Th2 polarization of naive human CD4 + T cells used for flow cytometry and cytokine analysis. ( B ) Viability of ivacaftor (IVA) or DMSO (control) cultured human Th2 cells at 7 days. ( C ) Representative flow cytometry histogram showing the median fluorescence intensity (MFI) of GATA3 at 7 days for DMSO- and IVA-treated Th2 cells. ( D ) The quantified MFI of GATA3 in DMSO- and IVA-treated Th2 cells at 72 hours ( n = 6 paired human samples). ( E ) Representative flow cytometry histogram showing the median fluorescence intensity (MFI) of IL-13 at 7 days for DMSO- and IVA-treated Th2 cells. ( F ) The quantified MFI of IL-13 in DMSO- and IVA-treated Th2 cells at 72 hours ( n = 6 paired human samples). ( G ) IL-13 by ELISA in cellular supernatant from cultured DMSO- and IVA-treated Th2 cells. Statistical analysis in B , D , F , and G was performed using paired Student’s t test. * P < 0.05.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Isolation, Flow Cytometry, Control, Cell Culture, Fluorescence, Enzyme-linked Immunosorbent Assay