mouse anti human ifn γ  (Mabtech Inc)

Journal: Molecular Therapy Advances

Article Title: Systems vaccinology analysis of saRNA immunization identifies an acute innate immune signature correlated with adaptive immunity

doi: 10.1016/j.omta.2026.201706

Figure Lengend Snippet: Adaptive immune responses to vaccination correlate weakly with innate responses 24 h after prime immunization Adaptive immune response data are presented from all participants pooled (A–C) or from participants separated into those who received 1 or 5 μg doses (D–F). (A and D) Anti-Spike (S) IgG (ng/mL) in sera from participants receiving two doses of LNP-saRNA at various time points after enrollment. (B and E) Pseudoneutralizing antibody IC50 from participants receiving two doses of LNP-saRNA; responses are shown at 6 weeks after enrollment against Wuhan, Delta, and Omicron spike-expressing pseudoviruses. (C and F) IFN-γ spot forming units (SFU) per million cells (ELISpot) from PBMC stimulated with SARS-CoV-2 spike peptide pools. (G) Correlation between V2a chemokine levels and the last visit antibody response. (H) Correlation between V2a cell levels and the last visit antibody response. Points represent individual participants, bars represent median ± interquartile range (A–F); points represent individual participants.

Article Snippet: Plates were then washed and incubated for 2 h at room temperature with 1 μg/mL mouse-anti-human IFN-γ (Mabtech).

Techniques: Expressing, Enzyme-linked Immunospot

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