Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: The relative production of IFN-γ and IL-4 by ConA-stimulated BALB/c splenocytes exposed to OdDHL in vivo. Splenocytes from OVA-immunized BALB/c mice, given either an OdDHL or control injection, were stimulated with 5 μg of ConA/ml. An ELISA was used to quantify IFN-γ (A) and IL-4 (B) protein levels following 48 h of incubation. Data are presented as the relative production of each cytokine by splenocytes from mice injected with OdDHL compared to the production of each cytokine by splenocytes from mice injected with control. Data are represented as the median ± 25th and 75th percentiles (boxes). Error bars represent the maximum and minimum values; n = 7 to 10. ***, P

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: Relative production of IFN-γ and IL-4 by C57Bl/6 splenocytes exposed to OdDHL in vivo. Splenocytes from OVA-immunized C57Bl/6 mice, given either an OdDHL or control injection, were stimulated with 5 μg of ConA/ml. An ELISA was used to quantify IFN-γ (A) and IL-4 (B) protein levels following 48 h of incubation. Data are presented as the relative production of each cytokine by splenocytes from mice injected with OdDHL compared to the production of each cytokine by splenocytes from mice injected with control. Data represented as the median ± 25th and 75th percentiles (boxes). Error bars represent the maximum and minimum values; n = 8 to 10. *, P

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: The production of IFN-γ and IL-4 by BALB/c splenocytes exposed to OdDHL in vitro. Splenocytes from unimmunized BALB/c mice were stimulated with 5 μg of ConA/ml and were exposed to various concentrations of OdDHL. An ELISA was used to quantify IFN-γ (A) and IL-4 (B) protein levels following 48 h of incubation. Data are represented as the median ± 25th and 75th percentiles (boxes). Error bars represent the maximum and minimum values; n = 10. *, P

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: In Vitro, Mouse Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: The production of IFN-γ and IL-4 by C57Bl/6 splenocytes exposed to OdDHL in vitro. Splenocytes from unimmunized C57Bl/6 mice were stimulated with 5 μg of ConA/ml and were exposed to various concentrations of OdDHL. An ELISA was used to quantify IFN-γ (A) and IL-4 (B) protein levels following 48 h of incubation. Data are represented as the medians ± 25th and 75th percentiles (boxes). Error bars represent the maximum and minimum values; n = 10 (A) or 9 (B). *, P

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: In Vitro, Mouse Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: The production of IFN-γ and IL-4 by TCR transgenic splenocytes exposed to OdDHL in vitro. Splenocytes from unimmunized TCR transgenic mice were stimulated with 5 μM MCC T102S and were exposed to various concentrations of OdDHL. An ELISA was used to quantify IFN-γ (A) and IL-4 (B) protein levels following 48 h of incubation. Data are represented as the median ± 25th and 75th percentiles (boxes). Error bars represent the maximum and minimum values. ND, all values below the limit of detection. The dotted line represents the limit of detection for each assay; n = 10. *, P

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: Transgenic Assay, In Vitro, Mouse Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: Expression of IFN-γ but not IL-4 mRNA is inhibited by OdDHL. TCR transgenic splenocytes were stimulated with antigen, and the cells were harvested at 4, 8, and 12 h poststimulation. Total RNA was extracted and subjected to RT-PCR for β-actin (lower panel), IL-4 (middle panel), and IFN-γ (upper panel). Products were visualized on an 8% polyacrylamide gel. Products were the expected sizes for cDNA. Untreated, control cells stimulated in the absence of OdDHL; treated, cells stimulated in the presence of 4 (A) or 10 μM (B) OdDHL. All cDNA products following RT were either undiluted or diluted 1:10 before undergoing PCR (*, IL-4 results do not include the 1:10 dilution). Results are from one experiment representative of two.

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: Expressing, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Infection and Immunity

Article Title: Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

doi: 10.1128/IAI.71.8.4421-4431.2003

Figure Lengend Snippet: The relative production of IFN-γ and IL-4 by OVA-stimulated BALB/c splenocytes exposed to OdDHL in vivo. Splenocytes from OVA-immunized BALB/c mice, given either an OdDHL or control injection, were stimulated with 200 μg of OVA/ml. An ELISA was used to quantify IFN-γ (A) and IL-4 (B) protein levels following 48 h of incubation. Data are presented as the relative production of each cytokine by splenocytes from mice injected with OdDHL compared to the production of each cytokine by splenocytes from mice injected with control. Data are represented as the median ± 25th and 75th percentiles (boxes). Error bars represent the maximum and minimum values; n = 8 to 10.

Article Snippet: The IFN-γ and β-actin primers were as described by Overbergh et al. ( ) and were purchased from Sigma Aldrich (St. Louis, Mo.).

Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Nucleic Acids Research

Article Title: Expression of RAB4B, a protein governing endocytic recycling, is co-regulated with MHC class II genes

doi: 10.1093/nar/gkl980

Figure Lengend Snippet: Expression of RAB4B is controlled by CIITA and is induced by IFN-γ. mRNA levels for the RAB4B , HLA-DRA and RAB4A genes were determined for ( A ) wt B cells (Raji), CIITA-/- B cells (RJ2.2.5) and CIITA-/- cells complemented with expression vectors encoding the three isoforms of CIITA (CIITA I, III and IV), ( B ) Me67.8 melanoma cells (Me) induced with IFN-γ for 0, 12, 24 and 48 h and ( C ) HUVEC cells induced with IFN-γ for 0, 12, 24 and 48 h. Results were normalized using TBP mRNA. Values for RAB4B and RAB4A are expressed as % of the levels found in wt B cells ( A ) or relative to uninduced cells ( B and C ). Values for HLA–DRA mRNA are expressed as % of the levels found in wt B cells ( A ) or cells induced with IFN-γ for 48 h ( B and C ).

Article Snippet: HUVEC were prepared as described ( ) and induced with 1000 U/ml of IFN-γ (Invitrogen).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Expression of RAB4B, a protein governing endocytic recycling, is co-regulated with MHC class II genes

doi: 10.1093/nar/gkl980

Figure Lengend Snippet: Occupation of the RAB4B promoter by the MHC-II regulatory machinery in DC and IFN-γ induced cells. ( A ) Binding of CIITA to the RAB4B , HLA-DRA and TBP promoters was analyzed by ChIP in DC before (iDC) and after (mDC) maturation with LPS. Results are represented as % of the occupation observed at the HLA-DRA promoter in iDC. The TBP promoter was used as a negative control. ( B and C ) ChIP was used to measure occupancy of the RAB4B promoter by CIITA ( B ) and RFX5 ( C ) in Me67.8 melanoma cells induced with IFN-γ for 0, 2, 4, 6 and 12 h. Results are represented as % of the occupation observed at the HLA–DRA promoter after 12 h of induction.

Article Snippet: HUVEC were prepared as described ( ) and induced with 1000 U/ml of IFN-γ (Invitrogen).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Bet v 1 from Birch Pollen Is a Lipocalin-like Protein Acting as Allergen Only When Devoid of Iron by Promoting Th2 Lymphocytes *

doi: 10.1074/jbc.M114.567875

Figure Lengend Snippet: Apo-, but not holo-lipocalin allergen promotes secretion of IL13. a and b , IL13 levels ( a ) and IFNγ levels ( b ) of stimulated PBMCs. Statistical analyses were conducted with repeated measures analysis of variance following Newman-Keuls multiple comparison test. **, p

Article Snippet: ELISAs for human IL13 and IFNγ were from eBioscience, both assays having a reported sensitivity of 4 pg/ml.

Techniques:

Journal: PLoS ONE

Article Title: Excretory/Secretory Products from Trichinella spiralis Adult Worms Ameliorate DSS-Induced Colitis in Mice

doi: 10.1371/journal.pone.0096454

Figure Lengend Snippet: Treatment with the T. spiralis AES reduced the levels of the DSS-induced pro-inflammatory cytokines IFN-γ, IL-6 and IL-17 in the spleens, MLN and colon lymphocytes. The data are presented as the mean ± SE. The asterisks* indicate statistical significance at P

Article Snippet: The cells were subjected to a cytokine assay using IFN-γ, IL-4, IL-6, IL-10 (BD Biosciences, USA) and IL-17 (R & D Systems, USA) ELISPOT sets according to the manufacturer's instructions.

Techniques:

Journal: PLoS Pathogens

Article Title: UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

doi: 10.1371/journal.ppat.1001071

Figure Lengend Snippet: UNC93B1 mediates host cell resistance to infection with T. gondii through an IFNγ–independent mechanism. (A) Immortalized macrophages were treated overnight with indicated concentrations of IFNγ, and subsequently infected with ME-49 tachyzoites in a multiplicity of infection of 1. The growth of intracellular parasites was monitored by uracil incorporation assay. (B) Immortalized macrophages from WT, 3d and MyD88/TRIF double deficient mice were infected for 48 h with a 1∶1 ratio of luciferase expressing tachyzoites per cell and the relative luciferase units (RLU) were calculated by normalizing the raw luminescence values to the background (error bars, s.e.m.). Asterisk indicates that p

Article Snippet: Cytotoxicity assays Murine immortalized macrophages were stimulated with IFNγ (BD Pharmigen) at 10–100 U/ml as indicated for 24 h prior to infection while control cultures were left untreated.

Techniques: Infection, Mouse Assay, Luciferase, Expressing

Journal: PLoS Pathogens

Article Title: UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

doi: 10.1371/journal.ppat.1001071

Figure Lengend Snippet: UNC93B1 mutation affects IL-12 and early IFNγ production. (A) Levels of IL-12p40 and IFNγ produced by splenocytes collected from non-infected controls, as well as mice at 5 and 8 days after infection with T. gondii . Spleen cells were cultured for 24 h in absence of exogenous stimuli. (B) Levels of cytokines present in the peritoneal cavity exudate of non-infected controls, as well as mice at 3, 5 or 7 days after intraperitoneal infection with T. gondii cysts. (C) Wild-type, 3d and MyD88 knockout bone marrow-derived macrophages were stimulated overnight with various TLR agonists or infected with T. gondii (MOI 5∶1), and cytokine levels in supernatants quantified by ELISA. (D) Immunoblot analysis of STAT-1 phosphorylation in splenocytes collected from animals either non-infected or at 8 days after infection. Asterisk indicates that p

Article Snippet: Cytotoxicity assays Murine immortalized macrophages were stimulated with IFNγ (BD Pharmigen) at 10–100 U/ml as indicated for 24 h prior to infection while control cultures were left untreated.

Techniques: Mutagenesis, Produced, Infection, Mouse Assay, Cell Culture, Knock-Out, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

doi: 10.1371/journal.ppat.1001071

Figure Lengend Snippet: Upon infection with T. gondii , 3d mice display a significant number of T cells expressing activation markers in the surface. (A) Flow cytometry analysis of splenocytes isolated from non-infected animals or at 8 days after infection with T. gondii . Cells were stained with fluorescent antibodies anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD69 and anti-CD154. (B–C) IFNγ-producing CD4 + and CD8 + -T cells present in splenocytes of C57BL/6 and 3d mice non-infected or at 8 days post-infection, as measured by flow cytometry. A panel with results for IFNγ -positive CD8 + -T cells from representative individual wild-type and 3d mice is shown in B. One asterisk indicates that p

Article Snippet: Cytotoxicity assays Murine immortalized macrophages were stimulated with IFNγ (BD Pharmigen) at 10–100 U/ml as indicated for 24 h prior to infection while control cultures were left untreated.

Techniques: Infection, Mouse Assay, Expressing, Activation Assay, Flow Cytometry, Cytometry, Isolation, Staining

Journal: PLoS Pathogens

Article Title: UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

doi: 10.1371/journal.ppat.1001071

Figure Lengend Snippet: Unimpaired production of pro-inflammatory cytokines in 3d mice infected with T. gondii . (A–C) Levels of IL-6, MCP-1, IFNγ, and TNFα measured in sera of mice at 0 and 8 days after infection, employing the BD Cytometric Bead Assay (CBA) Mouse Inflammation Kit. IL-12p40 levels in sera of mice were measured by ELISA. A panel with results from representative individual wild-type, 3d, MyD88-deficient and MyD88/TRIF double deficient animals is shown in A. One asterisk indicates that p

Article Snippet: Cytotoxicity assays Murine immortalized macrophages were stimulated with IFNγ (BD Pharmigen) at 10–100 U/ml as indicated for 24 h prior to infection while control cultures were left untreated.

Techniques: Mouse Assay, Infection, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay

Journal: Cytotechnology

Article Title: Reciprocal immuno-biological alterations occur during the co-culture of natural killer cells and adipose tissue-derived mesenchymal stromal cells

doi: 10.1007/s10616-019-00294-6

Figure Lengend Snippet: Effect of AT-MSCs on NK cell cytokine secretion. IL-2-activated NK cells were cultivated, for 5 days, in the absence or presence of AT-MSCs. Cell-free supernatants were collected and assayed by ELISA for their content of IFN-γ and TNFα. Data represent mean ± SEM of the cytokine concentration from seven independent experiments, each performed in triplicate. ** p

Article Snippet: The levels of IFN-γ (R & D Systems; Minneapolis, USA; the minimum detectable concentration is less than 8.0 pg/mL), TNFα (R & D Systems; Minneapolis, USA; 1.6 pg/mL sensitivity), perforin (Abcam; France; 40 pg/mL), and granzymes A and B (Biovendor; Germany; 0.4 pg/mL and 0.2 pg/mL, respectively) were measured in the different cell culture supernatants issued from the co-culture models using the ELISA techniques according to the manufacturer’s guidelines.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Gut Pathogens

Article Title: The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis

doi: 10.1186/1757-4749-6-19

Figure Lengend Snippet: Flow cytometry analysis of lymphocyte populations in mesenteric lymph nodes following ileitis induction. Lymphocyte populations were isolated from mesenteric lymph nodes (MLN) derived from C57BL/6 wildtype (WT) and TLR-9 deficient (TLR-9-/-) mice before (naïve) and seven days following ileitis induction (ILE) and analyzed by flow cytometry. Frequencies of CD4+ cells producing IFN-γ ( right panel ), CD4+ CD3+ cells ( left panel ), as well as CD69+ CD4+ cells ( middle panel ), are indicated in representative FACS plots (in %). Data shown are representative for three independent experiments.

Article Snippet: Stainings and cell sorting with anti-CD3 (clone 145-2C11, isotype hamster IgG1; BD Pharmingen), anti-CD4 (clone GK1.5 isotype rat IgG2b; BD Pharmingen), anti-CD69 (clone H1.2 F3, isotype Hamster IgG1*, Lamda 3; BD Pharmingen), and anti-IFN-γ (clone 145-2C11, isotype hamster IgG1; BD Pharmingen) were performed.

Techniques: Flow Cytometry, Cytometry, Isolation, Derivative Assay, Mouse Assay, FACS

Journal: Gut Pathogens

Article Title: The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis

doi: 10.1186/1757-4749-6-19

Figure Lengend Snippet: Systemic and extra-intestinal pro-inflammatory cytokine responses following ileitis induction. Serum (A) IL-6 and (B) IFN-γ, splenic (C) TNF-α and (D) IFN-γ as well as hepatic (E) TNF-α and (F) IL-6 protein concentrations were determined in respective ex vivo biopsies derived from C57BL/6 wildtype (WT; black circles) and TLR-9 deficient (TLR-9-/-; white circles) mice seven days following ileitis induction (ILE). Naïve mice served as negative controls (N). Numbers of analyzed mice (in parentheses), means (black bars) and levels of significance ( P -values) as compared to the respective groups (determined by Mann–Whitney- U test) are indicated. Data shown were pooled from three independent experiments.

Article Snippet: Stainings and cell sorting with anti-CD3 (clone 145-2C11, isotype hamster IgG1; BD Pharmingen), anti-CD4 (clone GK1.5 isotype rat IgG2b; BD Pharmingen), anti-CD69 (clone H1.2 F3, isotype Hamster IgG1*, Lamda 3; BD Pharmingen), and anti-IFN-γ (clone 145-2C11, isotype hamster IgG1; BD Pharmingen) were performed.

Techniques: Ex Vivo, Derivative Assay, Mouse Assay, MANN-WHITNEY

Journal: Gut Pathogens

Article Title: The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis

doi: 10.1186/1757-4749-6-19

Figure Lengend Snippet: Ileal cytokine secretion following ileitis induction. (A) IFN-γ, (B) TNF-α, (C) nitric oxide, (D) IL-6, and (E) IL-10 protein levels were determined in ex vivo ileal biopsies derived from C57BL/6 wildtype (WT; black circles) and TLR-9 deficient (TLR-9-/-; white circles) mice seven days following ileitis induction (ILE) as described in methods. Naïve mice served as negative controls (N). Numbers of analyzed mice (in parentheses), means (black bars) and levels of significance ( P -values) as compared to the respective groups (determined by Mann–Whitney- U test) are indicated. Data shown were pooled from three independent experiments.

Article Snippet: Stainings and cell sorting with anti-CD3 (clone 145-2C11, isotype hamster IgG1; BD Pharmingen), anti-CD4 (clone GK1.5 isotype rat IgG2b; BD Pharmingen), anti-CD69 (clone H1.2 F3, isotype Hamster IgG1*, Lamda 3; BD Pharmingen), and anti-IFN-γ (clone 145-2C11, isotype hamster IgG1; BD Pharmingen) were performed.

Techniques: Ex Vivo, Derivative Assay, Mouse Assay, MANN-WHITNEY

Journal: Gut Pathogens

Article Title: The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis

doi: 10.1186/1757-4749-6-19

Figure Lengend Snippet: Pro-inflammatory cytokine responses in mesenteric lymph nodes following ileitis induction. (A) IFN-γ, B) TNF-α, and (C) nitric oxide protein concentrations were determined in ex vivo biopsies of mesenteric lymph nodes (MLN) derived from C57BL/6 wildtype (WT; black circles) and TLR-9 deficient (TLR-9-/-; white circles) mice seven days following ileitis induction (ILE). Naïve mice served as negative controls (N). Numbers of analyzed mice (in parentheses), means (black bars) and levels of significance ( P -values) as compared to the respective groups (determined by Mann–Whitney- U test) are indicated. Data shown were pooled from three independent experiments.

Article Snippet: Stainings and cell sorting with anti-CD3 (clone 145-2C11, isotype hamster IgG1; BD Pharmingen), anti-CD4 (clone GK1.5 isotype rat IgG2b; BD Pharmingen), anti-CD69 (clone H1.2 F3, isotype Hamster IgG1*, Lamda 3; BD Pharmingen), and anti-IFN-γ (clone 145-2C11, isotype hamster IgG1; BD Pharmingen) were performed.

Techniques: Ex Vivo, Derivative Assay, Mouse Assay, MANN-WHITNEY

Journal: Gut Pathogens

Article Title: The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis

doi: 10.1186/1757-4749-6-19

Figure Lengend Snippet: Cerebral histopathological changes following acute ileitis induction. C57BL/6 wildtype (WT; black circles) and TLR-9 deficient (TLR-9-/-; white circles) mice were perorally infected with 100 cysts of T. gondii ME49 strain on day 0 to induce acute ileitis. Naïve mice served as negative controls (N). Intracerebral (A) IFN-γ, (B) TNF-α, and (C) IL-6 mRNA expression levels were measured in ex vivo brain biopsies applying quantitative RT-PCR. Numbers of analyzed mice (in parentheses), means (black bars), and levels of significance ( P -values) determined by Mann–Whitney- U test are indicated. Data shown were pooled from three independent experiments.

Article Snippet: Stainings and cell sorting with anti-CD3 (clone 145-2C11, isotype hamster IgG1; BD Pharmingen), anti-CD4 (clone GK1.5 isotype rat IgG2b; BD Pharmingen), anti-CD69 (clone H1.2 F3, isotype Hamster IgG1*, Lamda 3; BD Pharmingen), and anti-IFN-γ (clone 145-2C11, isotype hamster IgG1; BD Pharmingen) were performed.

Techniques: Mouse Assay, Infection, Expressing, Ex Vivo, Quantitative RT-PCR, MANN-WHITNEY

Journal: Oncology Letters

Article Title: Mechanisms for enhanced antitumor immune responses induced by irradiated hepatocellular carcinoma cells engineered to express hepatitis B virus X protein

doi: 10.3892/ol.2018.8430

Figure Lengend Snippet: Detection of specific T responses after priming with DCs. Naïve T-cells were isolated from spleens of C57BL/6 mice and co-cultured with DCs pulsed by NS, irradiated Adnull-infected Hepa1-6 cells, irradiated Hepa1-6 cells, irradiated AdHBx-infected Hepa1-6 cells or irradiated AdHBx-infected Hepa1-6 cells co-treated with 3-MA. The Golgi Stop was added during the last 4–6 h. The cells were then harvested and stained by anti-CD4-APC and anti-IFN-γ-PE or anti-CD8-FITC and anti-IFN-γ-PE. Fluorescence profiles were acquired on a FACScan and analyzed using FlowJo software version 7.6.1 (A) Percentage of the IFN-γ expressing CD4 + T cells (B) and IFN-γ expressing CD8 + T cells (C) were shown as mean ± SEM (*P

Article Snippet: The T cells were then harvested and stained by anti-CD4-APC and anti-IFN-γ-PE or anti-CD8-FITC and anti-IFN-γ-PE (BD Biosciences).

Techniques: Isolation, Mouse Assay, Cell Culture, Irradiation, Infection, Staining, Fluorescence, Software, Expressing

Journal: PLoS ONE

Article Title: Th17 Effector Cells Support B Cell Responses Outside of Germinal Centres

doi: 10.1371/journal.pone.0049715

Figure Lengend Snippet: Differential expansion of Th1 and Th17 populations after adoptive transfer. Th1 or Th17 recipient mice were immunised with HEL-OVA in CFA or PBS and the expansion of transgenic T cells and their ability to produce IL-17 and IFNγ was analysed by FACS. Representative FACS plots demonstrating gating strategy are demonstrated (A). Collective data demonstrating the percentage (B) and number of (C) of transgenic T cells at days 3, 7 and 10 post-immunisation amongst draining lymph node cells was assessed by flow cytometry, based on the expression of CD4 and the clonotypic TcR recognised by the KJ1.26 antibody (A). Unimmunised controls from each time point were averaged and represented as day 0. At the same days cells from the dLNs were stimulated with PMA and ionomycin and the expression of IL-17 (D) and IFNγ (E) by the transferred transgenic T cells was assessed by flow cytometry. Day 0 represents the proportion of IL-17 or IFNγ population in the transferred polarised populations. Grey line represents Th17 immunised recipients, grey dotted line unimmunised Th17 recipients, black line Th1 immunised recipients and black dotted line Th1 unimmunised recipients. Data represent mean ±SEM. *: Th1/HEL-OVA vs. Th17/HEL-OVA, *p

Article Snippet: Th17 differentiation was induced by culturing CD4+ T cells and APCs in the presence of anti-IL-4 (10 µg/ml), anti-IFNγ (10 µg/ml, BD Biosciences, Oxford, UK), IL-6 (20 ng/ml), TGFβ (1 ng/ml,), IL-23 (10 ng/ml), IL-1β (10 ng/ml) (R & D Systems) and OVA323–339 (0.5 µg/ml).

Techniques: Adoptive Transfer Assay, Mouse Assay, Transgenic Assay, FACS, Flow Cytometry, Cytometry, Expressing

Journal: PLoS ONE

Article Title: Role of Lactobacillus pentosus Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement

doi: 10.1371/journal.pone.0091857

Figure Lengend Snippet: Important factor for the enhancement IgA production from PP cells by b240. (A) PP cells (1.5×10 6 cells) were cultured with saline (open circles), 1.2×10 6 counts of heat-killed b240 (closed squares), or 1.2 × 10 7 counts of heat-killed b240 (closed circles) for 1, 3, 5, and 7 days. (B, C) In the presence or absence of heat-killed b240 (4.7×10 6 counts), PP cells (5.8×10 5 cells) were cultured with (B) anti-IL-6 mAb (10 μg/ml), anti-IFN-γ mAb (10 μg/ml), anti-TNF mAb (10 μg/ml), rat IgG1 k isotype control (10 μg/ml), (C) LE540 (1 μM), BCMA-Ig+ TACI-Ig (5 μg/ml each), dimethyl sulfoxide, or human IgG1 Fc antibody (10 μg/ml) for 4 days. The stimulation index of each sample was calculated (for example, (b240-treatment and anti-IL-6 Ab treatment)/(saline-treatment and anti-IL-6 Ab treatment) is the stimulation index for anti-IL-6 Ab treatment). (D) PP cells (5.8×10 5 cells) were cultured with a low dose (light gray), medium dose (dark gray), and high dose (black) of rIL-6 (0.4, 2, or 10 ng/ml), rIFN-γ (0.6, 3, or 15 ng/ml), rTNF (0.08, 0.4, or 2 ng/ml), or heat-killed b240 (4.7×10 6 counts) for 4 days. IgA or cytokine in the culture supernatants was determined by ELISA or CBA. Data are expressed as mean ± SEM (n = 3). (A, B) * P

Article Snippet: PP cells were cultured with or without heat-killed b240 in 630 μl complete medium in a 48-well culture plate (PP cells: 5.8×105 cells/well; b240: 4.7×106 counts/well) in the presence or absence of 1 μM LE540 (Wako), which is an inhibitor of RA receptors, B cell maturation (BCMA)-Ig and transmembrane activator + CAML-interactor (TACI)-Ig (5 μg/ml each; these are Fc chimeras of the receptors of APRIL and BAFF; R & D Systems), neutralizing antibodies specific for IL-6 (MP5-20F3; anti-IL-6 mAb, 10 μg/ml; BD Biosciences), 10 μg/ml anti-IFN-γ mAb (R4-6A2; BD Biosciences), or 10 μg/ml anti-TNF mAb (MP6-XT3; BD Biosciences).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

Journal: Nutrients

Article Title: Annona muricata L.-Derived Polysaccharides as a Potential Adjuvant to a Dendritic Cell-Based Vaccine in a Thymoma-Bearing Model

doi: 10.3390/nu12061602

Figure Lengend Snippet: Immunization with ALP-treated DCs induces a robust anti-tumor effect and immunity. ( A ) Two weeks after the final immunization, single cells obtained from PBS-, iDC-, OVAs-DC-, and ALP/OVAs-DC-immunized mice were stimulated ex vivo with OVA peptides for 6 h. The percentage of OVA-specific CD3 + CD4 + and CD3 + CD8 + T cells producing CD107a, IFN-γ, IL-2, or TNF-α was analyzed in accord with the gating strategy described in Figure S1 ( Supplementary Materials ). Pie charts indicate the mean frequencies of CD4 + and CD8 + T cells co-expressing IFN-γ, TNF-α, IL-2, and CD107a. ( B ) Serum samples were analyzed using ELISA for OVA 323–339 -specific IgG2a and IgG1 antibodies in each immunized mouse. The mean ± SD ( n = 6 mice/group) shown are representative of 2 independent experiments. ( C ) Tumor volumes in mice ( n = 10 mice/group) that received PBS (G1: white solid dot), iDC (G2: green solid dot), OVAs-DC (G3: red solid dot), and ALP/OVA-DCs (G4: blue solid dot) vs. E.G7. Tumor growth was identified by calculating the diameter of the tumor every 3 days for 30 days. The mean ± SD shown are representative of 2 independent experiments. SD; standard deviation.

Article Snippet: After washing the cells by adding BD Perm/Wash buffer, the cells were incubated with anti-IFN-γ (PE, eBioscience), anti-TNF-α (APC, eBioscience), and anti-IL-2 (PE-Cy7, eBioscience) mAbs for 30 min at room temperature.

Techniques: Mouse Assay, Ex Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation

doi: 10.3390/ijms19113693

Figure Lengend Snippet: Tumor-infiltrating immune cells and their role in tumor growth. Analysis of tumor-infiltrating cells was performed by flow cytometry ( A , B ). Mice ( n = 4) were injected with tumor cells and immunized by a gene gun. Vaccine adjuvants and anti-Tim-3 were administered on the same days as the DNA vaccines. Tumor cells were isolated on day 12 from non-treated tumors and on days 14–18 from treated tumors and stained with fluorochrome-labeled antibodies. ( A ) Frequencies of CD45 + and CD3 + cells, Treg (CD4 + CD25 + Foxp3 + ) and Nrp1 + Treg cells. Statistical significance refers to the comparison with the non-treated (pBSC) group. ( B ) Overview of the mean percentages of the major subpopulations of tumor-infiltrating cells in total cells. ( C ) The effect of in vivo depletion of immune cells and neutralization of IFN-γ on the anti-tumor response induced by immunotherapies with ODN1826 or α-GalCer in the immunized mice ( n = 5). Vaccine adjuvants were injected on the days of immunization. Statistical significance refers to the comparison with the group that was immunized with the PADRE.E7GGG gene and received an adjuvant. Bars: ±SEM; * p

Article Snippet: Different variants of cell activation were tested with the following concentrations of reagents: 5 μg/mL of ODNs, 10 μg/mL of LMS, 10 μg/mL of anti-Tim-3, 10 ng/mL of LPS (Sigma-Aldrich, St. Louis, MO, USA, L4391) and 200 U/mL IFN-γ (PeproTech, Rocky Hill, NJ, USA, 315-05).

Techniques: Flow Cytometry, Cytometry, Mouse Assay, Injection, Isolation, Staining, Labeling, In Vivo, Neutralization

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: PTPN2 deficiency enhances the generation and the antigen‐induced activation of CAR T cells Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl HER‐2 CAR T cells were generated with varying concentrations of α‐CD3 (0.05, 0.125 and 0.5 μg/ml) in the presence of α‐CD28 (0.5 μg/ml) and IL‐2 (2 ng/ml). After 6 days in culture, the generation of effector/memory (CD44 hi CD62L lo ) CD8 + HER‐2 CAR T cells was determined by flow cytometry. Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl HER‐2 CAR T cells were incubated with HER‐2‐expressing 24JK sarcoma cells (24JK‐HER‐2) and HER‐2‐negative 24JK sarcoma cells. (B) CD44, CD25, PD‐1 and LAG‐3 MFIs, (C) the proportion of CD8 + IFNγ + versus CD8 + TNF + CAR T cells and GrzB mean fluorescence intensity (MFI) were determined by flow cytometry. Data information: Significance in (A–C) was determined using 2‐tailed Mann–Whitney U ‐test. * P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: Activation Assay, Generated, Flow Cytometry, Cytometry, Incubation, Expressing, Fluorescence, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: PTPN2 deletion enhances the LCK‐dependent activation of CAR T cells CD8 + HER‐2 CAR T cells generated from Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− splenocytes were stained for intracellular p(Y418)‐SFK, and p(Y418)‐SFK MFIs were determined by flow cytometry. HER‐2‐specific Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− CAR T cells were incubated with HER‐2‐expressing 24JK sarcoma cells (24JK‐HER‐2) or HER‐2‐negative 24JK sarcoma cells, and CD44, CD25, PD‐1 and LAG‐3 MFIs on CD8 + CAR T cells were determined by flow cytometry. Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl or Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− HER‐2 CAR T cells were incubated with 24JK‐HER‐2 or 24JK sarcoma cells and the proportion of CD8 + IFNγ + versus CD8 + IFNγ + TNF + CAR T cells determined by flow cytometry. Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl or Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− HER‐2 CAR T cells were incubated with 5 μM CTV‐labelled (CTV bright ) 24JK‐HER‐2 and 0.5 μM CTV‐labelled (CTV dim ) 24JK sarcoma cells. Antigen‐specific target cell lysis was monitored for the depletion of CTV bright 24JK‐HER‐2 cells by flow cytometry. HER‐2‐specific Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl and Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− CAR T cells were incubated with plate‐bound α‐CD3 and CD25 MFIs on CD8 + CD44 hi CD62L lo versus CD8 + CD44 hi CD62L hi CAR T cells determined by flow cytometry. Data information: Representative histograms and results (means ± SEM) from at least two independent experiments are shown. In (A–C, E) significance determined using 2‐tailed Mann–Whitney U ‐test. In (D), significance was determined using 2‐way ANOVA test. * P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: Activation Assay, Generated, Staining, Flow Cytometry, Cytometry, Incubation, Expressing, Lysis, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: PTPN2 deletion in T cells increases tumour immunosurveillance 12‐month‐old Ptpn2 fl / fl ; p53 +/− and Lck ‐Cre; Ptpn2 fl / f ;p53 +/− mice were assessed for (A) disease and tumour incidence. (B) Lymphocyte subsets from 12‐month‐old Ptpn2 fl / fl ; p53 +/− and Lck ‐Cre; Ptpn2 fl / f ;p53 +/− mice were analysed by flow cytometry. Significance in (A) was determined using two‐sided Fisher's exact test. AT‐3‐OVA mammary tumour cells were injected into the fourth inguinal mammary fat pads of female Ptpn2 fl / fl and Lck ‐Cre; Ptpn2 fl / fl mice and (C) tumour growth monitored over 26 days. (D) At day 26 (d26), the numbers of activated tumour‐infiltrating lymphocytes (TILs) were determined. (E) The proportion of IFNγ + versus IFNγ + TNF + d26 TILs was determined by flow cytometry. (F) d26 TILs were incubated with AT‐3‐OVA tumour cells isolated from tumour‐bearing C57BL/6 mice, and the proportion of IFNγ + T cells was determined. Data information: Representative flow cytometry profiles and results (means ± SEM) from at least two independent experiments are shown. In (C), significance was determined using 2‐way ANOVA test and in (D–F) significance determined using 2‐tailed Mann–Whitney U ‐test. ** P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: Mouse Assay, Flow Cytometry, Cytometry, Injection, Incubation, Isolation, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: Targeting Ptpn2 with siSTABLE™ siRNAs or using CRISPR‐Cas9 RNP enhances tumour‐specific CAR T‐cell responses Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl CD8 + HER‐2 CAR T cells were treated with PTPN2‐inhibitor (+) or vehicle (−) followed by incubation with 24JK‐HER‐2 versus 24JK sarcoma cells. The proportion of CD8 + IFNγ + versus CD8 + IFNγ + TNF + CAR T cells was determined by flow cytometry. Human PBMCs were pretreated with PTPN2‐inhibitor (+) or vehicle (−) and stimulated with α‐CD3. MFIs for CD69 on CD8 + CCR7 + CD45RA + T cells and CD8 + CCR7 + CD45RA + T‐cell numbers were determined by flow cytometry. HER‐2 CAR T cells transfected with GFP versus Ptpn2 siSTABLE™ siRNAs were incubated with 24JK‐HER‐2 versus 24JK sarcoma cells. CD44, CD25, PD‐1 and LAG‐3 MFIs on CD8 + CAR T cells were determined by flow cytometry. HER‐2 CAR T cells transfected with GFP versus Ptpn2 siSTABLE™ siRNAs were incubated with 24JK‐HER‐2 versus 24JK sarcoma cells. The proportion of CD8 + IFNγ + CAR T cells was determined by flow cytometry. HER‐2 CAR T cells transfected with GFP versus Ptpn2 siSTABLE™ siRNAs were incubated with 5 μM CTV‐labelled (CTV bright ) 24JK‐HER‐2 and 0.5 μM CTV‐labelled (CTV dim ) 24JK sarcoma cells. Antigen‐specific target cell lysis (24JK‐HER‐2 versus 24JK response) was monitored for the depletion of CTV bright 24JK‐HER‐2 cells by flow cytometry. mCherry + CAR T cells isolated from HER‐2‐E0771 tumours 21 days post‐adoptive transfer were assessed for the proportion of CD8 + IFNγ + versus CD8 + IFNγ + TNF + CAR T cells by flow cytometry. HER‐2 CAR T cells were transfected with control versus Ptpn2 sgRNAs plus Cas9 using the Lonza 4D‐Nucleofector to delete PTPN2 by CRISPR‐Cas9 RNP. (G) Control and PTPN2‐deleted HER‐2 CAR T cells were incubated with 24JK‐HER‐2 versus 24JK sarcoma cells and the proportion of CD8 + IFNγ + CAR T cells determined by flow cytometry. (H) Alternatively, FACS‐purified CD8 + CD44 hi CD62L hi CAR T cells and CD8 + CD44 hi CD62L lo CAR T control and PTPN2‐deleted HER‐2 CAR T cells were incubated with 5 μM CTV‐labelled (CTV bright ) 24JK‐HER‐2 cells and 0.5 μM CTV‐labelled (CTV dim ) 24JK sarcoma cells. Antigen‐specific target cell lysis (24JK‐HER‐2 versus 24JK response) was assessed by monitoring for the depletion of CTV bright 24JK‐HER‐2 cells by flow cytometry. Data information: Representative results (means ± SEM) from at least two independent experiments are shown. In (A–D, F, G), significance was determined using 2‐tailed Mann–Whitney U ‐test. In (E, H), significance was determined using 2‐way ANOVA test. * P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: CRISPR, Incubation, Flow Cytometry, Cytometry, Transfection, Lysis, Isolation, Adoptive Transfer Assay, FACS, Purification, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: PTPN2 targeting enhances murine and human CAR T‐cell responses CD8 + LY CAR T cells generated from human PBMCs were treated with PTPN2‐inhibitor (+) or vehicle (−) followed by incubation with plate‐bound α‐LY, LY‐negative MDA‐MB‐435 cells and LY‐expressing OVCAR‐3 cells. The proportion of IFNγ + versus IFNγ + TNF + CD8 + CAR T cells was determined by flow cytometry. HER‐2 CAR T cells generated from C57BL/6 and Lck ‐Cre; Ptpn2 fl / fl splenocytes were transfected with GFP versus Ptpn2 siSTABLE™ FITC‐conjugated siRNAs, and intracellular PTPN2 levels were determined by flow cytometry. HER‐2‐E0771 mammary tumour cells (2 × 10 5 ) were injected into the fourth inguinal mammary fat pads of female HER‐2 TG mice. Six days after tumour injection, HER‐2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10 × 10 6 HER‐2 CAR T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC‐conjugated siRNAs 2 days before adoptive CAR T‐cell transfer. Mice were injected with IL‐2 (50,000 IU/day) on days 0–4 after adoptive CAR T‐cell transfer, and (C) tumour growth was monitored. (D) CD45 + CD3 + CD8 + mCherry + CAR T cells numbers were determined in HER‐2‐E0771‐positive tumours and spleens by flow cytometry 21 days post‐adoptive transfer. HER‐2 CAR T cells generated from C57BL/6 and Lck ‐Cre; Ptpn2 fl / fl splenocytes were transfected with Cas9 and control or Ptpn2 sgRNAs using the Lonza 4D‐Nucleofector and after 2 days intracellular PTPN2 levels determined by flow cytometry. HER‐2‐E0771 mammary tumour cells (2 × 10 5 ) were injected into the fourth inguinal mammary fat pads of female HER‐2 TG mice. Six days after tumour injection, HER‐2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10 × 10 6 control HER‐2 CAR T cells or those in which PTPN2 had been deleted by CRISPR RNP. Mice were injected with IL‐2 (50,000 IU/day) on days 0–4 post‐adoptive CAR T‐cell transfer and (F) tumour growth monitored. (G) CD45 + CD8 + mCherry + CAR T‐cell numbers were determined in HER‐2‐E0771 tumours by flow cytometry 19 days post‐adoptive transfer. Data information: Representative flow cytometry profiles and results (means ± SEM) from two independent experiments are shown. In (A, D, G), significance was determined using 2‐tailed Mann–Whitney U ‐test. In (C, F), significance was determined using 2‐way ANOVA test. * P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: Generated, Incubation, Multiple Displacement Amplification, Expressing, Flow Cytometry, Cytometry, Transfection, Injection, Mouse Assay, Irradiation, Adoptive Transfer Assay, CRISPR, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: PTPN2 deletion enhances CD8 + T‐cell‐mediated immunosurveillance AT‐3‐OVA mammary tumour cells (1 × 10 6 ) were injected into the fourth inguinal mammary fat pads of female Ly5.1 + mice. Seven days after tumour injection, FACS‐purified 2 × 10 6 naïve CD8 + CD44 lo CD62L hi lymph node T cells from Ly5.2 + ;OT‐1; Ptpn2 fl / fl versus Ly5.2 + ;OT‐1; Lck ‐Cre; Ptpn2 fl / fl mice were adoptively transferred into tumour‐bearing Ly5.1 + mice. Tumour‐bearing Ly5.1 + mice were monitored for (A) tumour growth over 21 days and (D) for survival over 86 days. (B) After 21 days, TILs were processed for flow cytometry and donor T‐cell numbers (Ly5.1 − Ly5.2 + ) determined. (C) After 9 days, the proportion of Ly5.2 + IFNγ + TNF + versus Ly5.2 + GrzB + TILs was determined. Gene expression in tumours from mice treated with Ly5.2 + ;OT‐1; Ptpn2 fl / fl T cells 21 days post‐adoptive transfer versus those re‐emerging in mice treated with Ly5.2 + ;OT‐1; Lck ‐Cre; Ptpn2 fl / fl T cells. B16.F10‐OVA melanoma cells (1 × 10 5 ) were engrafted onto the abraded skin in the flanks of Ly5.1 + mice. 24 h after tumour cell engraftment, naïve CD8 + CD44 lo CD62L hi lymph node T cells from Ly5.2 + ;OT‐1; Ptpn2 fl / fl versus Ly5.2 + ;OT‐1; Lck ‐Cre; Ptpn2 fl / fl mice were adoptively transferred and tumour incidence monitored. (G) Epidermal lymphocytes from tumour‐free mice were stained for CD69 hi CD103 hi and donor‐derived (Ly5.2 + vα2 + ) tissue‐resident memory T cells (T RMs ) determined by flow cytometry. (H) Tumour sizes in B16.F10‐OVA melanoma bearing mice were determined between days 21 and 23. (I) TILs were assessed for Ly5.2 + ;OT‐1; Ptpn2 fl / fl and Ly5.2 + ;OT‐1; Lck ‐Cre; Ptpn2 fl / fl donor T‐cell numbers by flow cytometry. Data information: Representative flow cytometry profiles and results (means ± SEM) from at least two independent experiments are shown. In (A), significance was determined using 2‐way ANOVA test and in (B, C, E, G, H, I) significance determined using 2‐tailed Mann–Whitney U ‐test. In (D, F), significance was determined using log‐rank (Mantel–Cox) test. * P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: Injection, Mouse Assay, FACS, Purification, Flow Cytometry, Cytometry, Expressing, Adoptive Transfer Assay, Staining, Derivative Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours

doi: 10.15252/embj.2019103637

Figure Lengend Snippet: PTPN2 deficiency enhances CAR T‐cell efficacy in vivo by promoting STAT‐5‐mediating homing to CXCL9/10‐expressing tumours HER‐2‐E0771 mammary tumours cells (2 × 10 5 ) were injected into the fourth inguinal mammary fat pads of female HER‐2 TG mice. Six days after tumour injection, HER‐2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6 × 10 6 FACS‐purified CD8 + CD62L hi CD44 hi central memory HER‐2 CAR T cells generated from Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl ; Stat5 fl /+ or Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− splenocytes. Mice were injected with IL‐2 (50,000 IU/day) on days 0–4 after adoptive CAR T‐cell transfer and (B) monitored for tumour growth. (A, C) Lymphocytes were isolated from the tumours on (A) day 3 or (C) day 16 post‐adoptive transfer, and mCherry + CD45 + CD8 + CAR T‐cell numbers were determined by flow cytometry. In (C), TILs were stained for intracellular IFNγ and TNF after PMA/ionomycin treatment. HER‐2‐E0771 cells generated to inducibly overexpress PTPN2 in response to doxycycline (E0771‐HER‐2‐PTPN2 hi ) were pre‐incubated (24 h) with vehicle or doxycycline (DOX) subsequently stimulated with IFNγ for the indicated times. STAT‐1 Y701 phosphorylation (p‐STAT‐1) and PTPN2 levels were assessed by immunoblotting. Cxcl9 and Cxcl10 mRNA levels in vehicle versus DOX‐treated and IFNγ‐stimulated HER‐2‐E0771 cells were assessed by quantitative real‐time PCR. E0771‐HER‐2‐PTPN2 hi mammary tumour cells (2 × 10 5 ) were injected into the fourth inguinal mammary fat pads of female HER‐2 TG mice. Five days after tumour injection, mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6 × 10 6 FACS‐purified central memory Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl HER‐2 CAR T cells. Mice were then injected with IL‐2 (50,000 IU/day) on days 0–4 post‐adoptive CAR T‐cell transfer, and (H) tumour growth was monitored. In (G), CD45 + CD8 + mCherry + TILs were quantified by flow cytometry at day 4 post‐adoptive transfer. Data information: Representative flow cytometry profiles and results (means ± SEM) from two independent experiments are shown. In (A, E), significance was determined using 1‐way ANOVA test. In (B, G, H), significance was determined using 2‐way ANOVA test. In (C), significance was determined using 2‐tailed Mann–Whitney U ‐test. * P

Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.

Techniques: In Vivo, Expressing, Injection, Mouse Assay, Irradiation, Adoptive Transfer Assay, FACS, Purification, Generated, Isolation, Flow Cytometry, Cytometry, Staining, Incubation, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Lung India : Official Organ of Indian Chest Society

Article Title: Effect of mycobacterial secretory proteins on the cellular integrity and cytokine profile of type II alveolar epithelial cells

doi: 10.4103/0970-2113.102796

Figure Lengend Snippet: Cytokine release from the A549 cells treated with narrow molecular mass fractions of mycobacterial STCF (a) IFN-γ, (b) TNF-β, (c) IL-6, and (d) IL-12. Bars represent mean values ± SD; n = 3

Article Snippet: Cytokines Release Assay 4 × 105 A549 cells/mL were cultured, in 96-well plates in 5% CO2 at 37° C. Concentration of various cytokines; interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-6, IL-12(p40) was measured in the culture supernatants removed from the cell monolayers stimulated with 20 μg/mL of each of the fractions for 72 hr by ELISA kits (BD Pharmingen) according to manufacturer's instructions.

Techniques:

Journal: Immunology

Article Title: Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-? via CXCR3 chemokines

doi: 10.1111/j.0019-2805.2004.01818.x

Figure Lengend Snippet: Keyhole limpet haemocyanin (KLH) delayed-type hypersensitivity (DTH) is dependent on interferon-γ (IFN-γ) on day 3 and day 5 after immunization. Left column: DTH was elicited on day 3 and 5 in IFN-γ +/+ mice (Group C and E), but

Article Snippet: Quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed on ear extracts for IL-4, eotaxin, IL-5, interferon-γ (IFN-γ) and IP-10, using two different specific mAbs [all BD PharMingen, San Diego, CA, except for eotaxin (R & D, Minneapolis, MN) and IP-10 (Leinco Technologies, St Louis, MO)], in 0·1 m NaHCO3 (pH 8–9) at 4°.

Techniques: Mouse Assay

Journal: Immunology

Article Title: Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-? via CXCR3 chemokines

doi: 10.1111/j.0019-2805.2004.01818.x

Figure Lengend Snippet: Production of interferon-γ (IFN-γ) and IP-10 [IFN-γ-inducible protein chemokine of 10 000 molecular weight (CXCL10)] in 24-hr keyhole limpet haemocyanin (KLH) delayed-type hypersensitivity (DTH) ear extracts, and dependency of

Article Snippet: Quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed on ear extracts for IL-4, eotaxin, IL-5, interferon-γ (IFN-γ) and IP-10, using two different specific mAbs [all BD PharMingen, San Diego, CA, except for eotaxin (R & D, Minneapolis, MN) and IP-10 (Leinco Technologies, St Louis, MO)], in 0·1 m NaHCO3 (pH 8–9) at 4°.

Techniques: Molecular Weight

Journal: Immunology

Article Title: Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+

doi: 10.1046/j.1365-2567.2003.01631.x

Figure Lengend Snippet: Chemical structures of BPA and NP, and their effects on IL-4 production in KLH-primed lymph node cells and PMA-activated EL4 cells. (a) Chemical structures of test chemicals: bisphenol A and 4-nonylphenol. (b, c) Mice were injected into the footpad with KLH (100 µg) in alum. Seven days later, the lymph node cells were collected and stimulated in vitro for 4 days with KLH (50 µg/ml) in the presence of varying amounts of BPA or NP. (d) EL4 thymoma cells were treated with PMA (1 ng/ml), and then exposed to varying amounts of BPA or NP for 2 days. The cell culture supernatants were harvested and assayed for IL-4 or IFN-γ by ELISA. The values represent the mean ± SEM ( n = 4). * P

Article Snippet: Anti-mIL-4 (BVD4 and BVD6), anti-mouse-interferon-γ (IFN-γ) (R46A2 and XMG1.2), anti-mouse IgE, purified mouse IgE, biotinylated anti-mouse IgE, recombinant murine IL-4 and IFN-γ were from PharMingen Co. (San Diego, CA).

Techniques: Mouse Assay, Injection, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: The immune paradox of sarcoidosis and regulatory T cells

doi: 10.1084/jem.20050648

Figure Lengend Snippet: Most cells that coproduce TNF-α and IFN-γ do not secrete IL-2. Cytokine production profile of CD4 + T cells after stimulation with PMA. Fresh PBMCs from healthy controls or patients were cultured for 24 h in the presence of PMA. Cells were permeabilized, stained with a cocktail of anti-CD4–PerCP, anti–IL-2–PE, anti–TNF-α–allophycocyanin, and anti–IFN-γ–FITC and analyzed on a FACScalibur flow cytometer. Analysis on gated CD4 cells is presented (left). Proportions of IFN-γ and/or TNF-α–secreting cells are indicated. IL-2 detection in the indicated gated subsets is presented (right). Representative results of independent experiments in three sarcoidosis patients and five healthy controls are shown.

Article Snippet: Stimulated cells were permeabilized and stained with anti-CD4–perCP, anti-IL-2-PE, anti–TNF-α–allophycocyanin, and anti–IFN- γ–FITC (all obtained from BD Biosciences).

Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

Journal: AIDS Research and Human Retroviruses

Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

doi: 10.1089/aid.2009.0149

Figure Lengend Snippet: HIV-specific TGF-β-positive CD4 + T cells do not produce IL-10 or IFN-γ. PBMCs were stimulated with HIV peptides and then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β, IL-10, and IFN-γ-positive CD4 + T cells was determined. Representative plots of the number of TGF-β, IL-10, and IFN-γ-positive CD4 + T cells after subtraction of the background values.

Article Snippet: PBMCs were permeabilized and stained with different combinations of the following antibodies: CTLA-4 APC, IL-10 PE, IL-10 APC, IFN-γ FITC (BD Pharmingen), TGF-β PE (Biotest Diagnostics, Denville, NJ) and analyzed by flow cytometry.

Techniques: Staining, Flow Cytometry, Cytometry

Journal: AIDS Research and Human Retroviruses

Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

doi: 10.1089/aid.2009.0149

Figure Lengend Snippet: TGF-β receptor II blockade increases IFN-γ expression by HIV-specific T cells. PBMCs were stimulated with Gag peptides in the presence of anti-TGF-β R II (or isotype control). PBMCs were then stained with anti-IFN-γ FITC, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7 and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte populations and then the percent of IFN-γ-positive cells was determined after subtraction of the background values. Plots are from three independent experiments yielding similar results.

Article Snippet: PBMCs were permeabilized and stained with different combinations of the following antibodies: CTLA-4 APC, IL-10 PE, IL-10 APC, IFN-γ FITC (BD Pharmingen), TGF-β PE (Biotest Diagnostics, Denville, NJ) and analyzed by flow cytometry.

Techniques: Expressing, Staining, Flow Cytometry, Cytometry

Journal: AIDS Research and Human Retroviruses

Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

doi: 10.1089/aid.2009.0149

Figure Lengend Snippet: CTLA-4 blockade decreases TGF-β expression by HIV-specific CD4 + T Cells. PBMCs ( n = 6) were stimulated with Gag peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β- and IFN-γ-positive cells was determined. Results were expressed as percent of Gag-specific CD4 + T cells expressing TGF-β or IFN-γ after subtraction of the background values. ( A ) Representative plots of Gag-specific CD4 + T cells expressing TGF-β or IFN-γ in the presence or absence of anti-CTLA-4. ( B ) Dashed line represents the cutoff for significant cytokine expression. Percentages in parentheses are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t test.

Article Snippet: PBMCs were permeabilized and stained with different combinations of the following antibodies: CTLA-4 APC, IL-10 PE, IL-10 APC, IFN-γ FITC (BD Pharmingen), TGF-β PE (Biotest Diagnostics, Denville, NJ) and analyzed by flow cytometry.

Techniques: Expressing, Staining, Flow Cytometry, Cytometry

Journal: AIDS Research and Human Retroviruses

Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

doi: 10.1089/aid.2009.0149

Figure Lengend Snippet: CTLA-4 blockade increases IFN-γ expression by CMV-specific CD4 + T cells. PBMCs ( n = 6) were stimulated with CMV PP65 peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and the percent of IFN-γ-positive cells was determined. Results were expressed as percent of CMV-specific CD4 + T cells expressing IFN-γ after subtraction of the background values. The dashed line represents the cutoff for significant cytokine expression. Percentages in parentheses are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t test.

Article Snippet: PBMCs were permeabilized and stained with different combinations of the following antibodies: CTLA-4 APC, IL-10 PE, IL-10 APC, IFN-γ FITC (BD Pharmingen), TGF-β PE (Biotest Diagnostics, Denville, NJ) and analyzed by flow cytometry.

Techniques: Expressing, Staining, Flow Cytometry, Cytometry

Journal: Cell Death and Differentiation

Article Title: NOX2-dependent ATM kinase activation dictates pro-inflammatory macrophage phenotype and improves effectiveness to radiation therapy

doi: 10.1038/cdd.2017.91

Figure Lengend Snippet: Classical macrophage activation is dependent on ATM. ( a – c ) Representative confocal micrographs and frequencies of murine RAW264.7 macrophages showing γ -H2AX + nuclear foci ( a , b ) or ATMS1981* phosphorylation (ATMS1981* + ) ( a , c ) in control cells or after 24 h treatments with 20 ng/ml of recombinant murine IFN- γ (mIFN- γ ), 100 ng/ml of lipopolysaccharide (LPS) or 10 μ M of cisplatinium (CDDP) are shown (scale bar, 20 μ m). Representative macrophages with ATMS1981* + and γ -H2AX + nuclear foci are shown in inserts (scale bar, 5 μ m). Results are expressed as mean value±S.E.M. P -values (* P

Article Snippet: Recombinant Human IFN- γ (IFN- γ , #285-IF/CF) was from R & D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Recombinant

Journal: Journal of Virology

Article Title: Differential Effect of Human Herpesvirus 6A on Cell Division and Apoptosis among Na?ve and Central and Effector Memory CD4+ and CD8+ T-Cell Subsets ▿

doi: 10.1128/JVI.00106-09

Figure Lengend Snippet: HHV-6A-induced cytokine production. MNCs were cultured with controls (Cont) and HHV-6A for 2 to 5 days, and culture supernatants were collected and analyzed by ELISA. HHV-6A induced significant amounts of IL-6, IL-8, TNF-α, and IFN-γ.

Article Snippet: The cytokines interleukin-8 (IL-8), IL-6, TNF-α, and gamma interferon (IFN-γ) were assayed in accordance with BD Pharmingen protocols.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: CD40L membrane retention enhances the immunostimulatory effects of CD40 ligation

doi: 10.1038/s41598-019-57293-y

Figure Lengend Snippet: T-cell proliferation and cytotoxic response to mCD40L-activated DC compared with sCD40L. DC co-cultured for 24 hours with CFPAC-1 cells (CFPAC-1 CNT) alone or CFPAC-1 cells pre-transduced with 50 MOI RAdMock (AdM) or RAdnCD40L (AdnL) or treated with sCD40L (sL; 1 µg/ml) or the MC were retrieved and loaded with CFPAC-1 tumour lysate. ( A ) CFSE-labelled autologus CD3+ T cells were incubated with tumour cell lysate-loaded DC at a responder to-stimulator (R:S) T-cell/DC ratio of 10:1 for 5 days or cultured alone as a negative control. Retrieved CD3+ T cells were examined for CD8+ T cells by gating CD3 + CD8+ T cells population utilizing anti-CD3-Pacific blue and anti-CD8-Alexa Fluor 700. CD8+ T cells were selected by gating CD3 and CD8 double stained cells with negative or low CFSE. The results were expressed as the percentage of CFSE negative or low cells with Pacific blue and Alexa Fluor 700 positive staining and represent the mean of three biological experiments ± SD. Two-tailed t-test analysis comparing different treatments including sL/CNT*(p = 0.1515, p = 0.0334), AdnL/sL**(p = 0.0059, p = 0.0148), AdnL/AdM*** (p = 0.0083,p = 0.0132) and MC/CNT****(p = 0.0091, p = 0.0024). ( B) In vitro expanded T cells obtained from co-culture with DC loaded with tumour lysate for 7 days were stimulated for 5 hours with irradiated CFPAC-1 cell lysate-loaded autologous PBMCs. Unstimulated CD3+ T cells were used as a negative control (unstimulated T cells). Protein transport inhibitor, GolgiStop and anti-CD107a PE Ab were added 1 hours after stimulation. Cells were stained with anti-CD3-Pacific blue, anti-CD8-AlexaFluor 700 and anti-IFN-γ APC. Anti-CD3, -CD8 positive stained were gated by flow cytometery and analyzed for CD1017a and IFN-γ positive staining cells. Results represent the mean of three biological experiments ± SD. Two-tailed t-test analysis comparing different treatments including sL/CNT* (p = 0.3671, p = 0.5739), AdnL/sL** (p = 0.0043, p = 0.0025), AdnL/AdM*** (p = 0.0008, p = 0.0068) and MC/CNT**** (p = 0.0066, p = 0.0026) for IFN-γ and CD1017a positive cells respectively.

Article Snippet: Following PBS washing, cells were fixed, permeabilized with Cytofix/Cytoperm solution and stained with anti-IFN-γ APC (BD Biosciences) at 4 °C for 20 min then analysed by flow cytometry.

Techniques: Cell Culture, Transduction, Incubation, Negative Control, Staining, Two Tailed Test, In Vitro, Co-Culture Assay, Irradiation, Flow Cytometry

Journal: Oncotarget

Article Title: SHP2 negatively regulates HLA-ABC and PD-L1 expression via STAT1 phosphorylation in prostate cancer cells

doi: 10.18632/oncotarget.18591

Figure Lengend Snippet: SHP2 depletion upregulated the expression of HLA-ABC and PD-L1 in PC3 and DU145 cells PC3 and DU145 cells were transfected with SHP2 siRNA (50 nmol/mL) for 48 hours. IFN-γ was used as a positive control to induce the expression of HLA-ABC and PD-L1. (A) A representative flow histogram of HLA-ABC is shown. (B) HLA-ABC MFI fold change of PC3 and DU145 after treatment with SHP2 siRNA or IFN-γ for 48 hours. Bar graph shows the MFI results represented as mean ±SE. (C) A representative flow histogram of PD-L1 of PC3 cells is shown. (D) PD-L1 MFI fold change of PC3 and DU145 after transfection of SHP2 siRNA or treatment with IFN-γ for 48 hours. Bar graphs represent mean ±SD from duplicate samples in three independent experiments. Full black line depicts isotype group. Full gray line depicts control siRNA group. Black line depicts SHP2 siRNA group. Dashed line depicts IFN- γ group.

Article Snippet: Recombinant human IFN-γ was purchased from PeproTech (Princeton, NJ, USA).

Techniques: Expressing, Transfection, Positive Control, Flow Cytometry

Journal: PLoS ONE

Article Title: Influence of Maternal Gestational Treatment with Mycobacterial Antigens on Postnatal Immunity in an Experimental Murine Model

doi: 10.1371/journal.pone.0009699

Figure Lengend Snippet: Distribution of Qdot-ITK-Ag85A in mother and fetuses. Pregnant mice (3 animals per group) were injected with Qdot-ITK-Ag85A (120 pmol/mouse) via s.c. Mice were sacrificed 48 h post inoculation and mother organs as well as fetuses and placentas were removed and embedded in OCT. Sections were fixed, mounted and observed under green light in a fluorescence microscope. Qdot-ITK-Ag85A fluorescence was detected in the skin (a), placenta (b) and fetuses (c, d). Magnification 100×. Prior to the inoculation, Qdot-ITK-Ag85A were run in a 0.5% agarose gel (e) to check conjugation. Lane 1, unconjugated Qdot-ITK; lane 2, Qdot-ITK-Ag85A. Recall IFN-γ responses after in vitro restimulation of lung cells with Ag85A were measured in the offspring born to the mother that received Qdot-ITK-Ag85A or Qdot-ITK (f).

Article Snippet: IFN-γ ELISA A commercially available kit for IFN-γ (Mabtech, Stockholm, Sweden) was used to determine the cytokine levels in the culture supernatants according to the manufacturer's recommendations, with slight modifications.

Techniques: Mouse Assay, Injection, Fluorescence, Microscopy, Agarose Gel Electrophoresis, Conjugation Assay, In Vitro

Journal: PLoS ONE

Article Title: Influence of Maternal Gestational Treatment with Mycobacterial Antigens on Postnatal Immunity in an Experimental Murine Model

doi: 10.1371/journal.pone.0009699

Figure Lengend Snippet: Maternal treatment increases postnatal cellular immune responses. Pregnant mice at 2 nd week of gestation were treated with rHBHA via s.c. Following birth, neonates were immunized twice i.n. with rHBHA formulated with CT at week 1 (W1) and week 4 (W4). One week after the last immunization, lung cells from the rHBHA-immunized animals were re-stimulated in vitro with rHBHA to measure recall IFN-γ responses. Data show mean IFN-γ ± s.d. of triplicate wells prepared from pooled samples (3–4 mice/group). Results were analyzed from two independent experiments. p value (s) was calculated by comparing two groups using t test. * significant when p

Article Snippet: IFN-γ ELISA A commercially available kit for IFN-γ (Mabtech, Stockholm, Sweden) was used to determine the cytokine levels in the culture supernatants according to the manufacturer's recommendations, with slight modifications.

Techniques: Mouse Assay, In Vitro

Journal: The Journal of Experimental Medicine

Article Title: Chronic Inflammation and Susceptibility to Bacterial Infections in Mice Lacking the Polypeptide (p)105 Precursor (NF-?B1) but Expressing p50

doi:

Figure Lengend Snippet: Cytokine production in T cells or macrophages. ( A ) Secretion of IL-2, IL-4, and TNF-α is decreased in p105 −/− T cells. Splenic T cells purified from 3-wk-old control ( closed bars ) or p105 −/− ( open bars ) untreated ( 1 ) or treated with anti-CD3 ( 2 ) or anti-CD3 plus anti-CD28 ( 3 ) for 24 h. The cytokine levels in the supernatants were determined by ELISA. ( B ) Cytokines released from macrophages. Peritoneal macrophages purified from 3-wk-old mice untreated (−) or treated with (+) LPS and IFN-γ for 72 h. The cytokine levels are indicated by mean values ± SD.

Article Snippet: Purified T cells (5 × 105 /ml) were incubated with or without coated anti-CD3 antibody or coated anti-CD3 plus anti-CD28 antibodies for 24 h. Purified peritoneal macrophages (5 × 105 /ml) were incubated in the presence or absence of 1 μg/ml of LPS and 100 U/ml of IFN-γ (Genzyme Corp., Cambridge, MA) for 72 h. Cytokine levels for IL-1β, IL-4, IL-6, IL-10, GM-CSF, and TNF-α in supernatants were measured by ELISA (R & D Systems, Inc.); levels of IFN-γ and IL-2 were also determined by ELISA (BIOSOURCE, Camarillo, CA).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Mouse Assay