intracellular cytokine staining Search Results


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  • 99
    Thermo Fisher intracellular cytokine staining
    Immune responses against mutated ligands in PBMC of patient Mel15. Clinical course and retrieval of patient material ( a ). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 ( b ). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation ( c ). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 10 4 cells ( d ). Intracellular <t>cytokine</t> staining (ICS) of an expanded NCAPG2 P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h ( e ). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4 S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells ( f ). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining ( g ) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) ( h ). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.
    Intracellular Cytokine Staining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson intracellular cytokine staining
    <t>Cytokine</t> production kinetics in T cells. ( A ) T cells that were rested for 3 d in the presence of IL-7 ( Top ), IL-2 ( Center ), or IL-15 ( Bottom ) were activated with 100 nM SIINFEKL (N4) or SIITFEKL (T4) OVA peptide for indicated time points ( n = 3 mice). Brefeldin A (BrfA) was added during the last 2 h of culture. ( B ) Representative dot plots for TNF-α and IFN-γ ( Upper ) and IFN-γ and IL-2 ( Lower ) production of T cells that were activated for indicated time with 100 nM OVA 257–264 peptide (N4) in the presence of BrfA during the last 2 h of stimulation. Numbers indicate percentage of cytokine-producing T cells. ( C ) mRNA levels of Tnfa , Ifng , and Il2 of T cells that were rested for 3 d in the presence of IL-7, IL-2, or IL-15.
    Intracellular Cytokine Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 4775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen intracellular cytokine staining
    HLA-A*0201-restricted epitope-specific cellular immunogenicity of various smallpox vaccines. ( A and B ) Peptide-specific intracellular <t>cytokine</t> release of splenocytes from vaccinated HHD mice. Mice were immunized i.p. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 IU of MVA ( A ) or i.m. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 , 10 6 , 10 5 , and 10 4 IU of MVA ( B ). #1 and #2 refer to individual mice analyzed. Splenocyte preparations were analyzed for the presence of VP35#1 peptide-specific, activated (CD62L low ) CD8 + T cells. The magnitude of the induced T cell response is depicted as percentages of cytokine-secreting CD8 + T cells within the live CD8 + cell population. ( C ) Specific lysis of peptide-pulsed human target cells by VP35#1-specific CTLs from vaccinated HHD mice. Effector CTLs were generated by single i.p. immunization of HHD mice with MVA (■ and □), CVA (● and ○), or Wyeth (▴ and ▵). CTLs were assayed for cytotoxicity at the indicated E/T ratio against T2 targets pulsed with either VP35#1 (filled symbols) or influenza M1 peptide (open symbols). ( D ) Processing and presentation of peptide epitope VP35#1 after heterologous expression of the vaccinia virus H3L gene. Human A375/H3L (■ and □) or control A375 (● and ○) cells were tested against A*0201-restricted murine CTLs specific for VP35#1 (filled symbols) or human tyrosinase 369–377 epitopes (open symbols) as effector cells at the indicated E/T ratio.
    Intracellular Cytokine Staining, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cytokine staining
    HLA-A*0201-restricted epitope-specific cellular immunogenicity of various smallpox vaccines. ( A and B ) Peptide-specific intracellular <t>cytokine</t> release of splenocytes from vaccinated HHD mice. Mice were immunized i.p. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 IU of MVA ( A ) or i.m. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 , 10 6 , 10 5 , and 10 4 IU of MVA ( B ). #1 and #2 refer to individual mice analyzed. Splenocyte preparations were analyzed for the presence of VP35#1 peptide-specific, activated (CD62L low ) CD8 + T cells. The magnitude of the induced T cell response is depicted as percentages of cytokine-secreting CD8 + T cells within the live CD8 + cell population. ( C ) Specific lysis of peptide-pulsed human target cells by VP35#1-specific CTLs from vaccinated HHD mice. Effector CTLs were generated by single i.p. immunization of HHD mice with MVA (■ and □), CVA (● and ○), or Wyeth (▴ and ▵). CTLs were assayed for cytotoxicity at the indicated E/T ratio against T2 targets pulsed with either VP35#1 (filled symbols) or influenza M1 peptide (open symbols). ( D ) Processing and presentation of peptide epitope VP35#1 after heterologous expression of the vaccinia virus H3L gene. Human A375/H3L (■ and □) or control A375 (● and ○) cells were tested against A*0201-restricted murine CTLs specific for VP35#1 (filled symbols) or human tyrosinase 369–377 epitopes (open symbols) as effector cells at the indicated E/T ratio.
    Cytokine Staining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2931 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson intracellular cytokine staining kit
    Transgenic IL-15 in in vivo model. (A) CLL-1 expression of AML PDX model. (B) Schematic figure of AML PDX model. in the PDX model, mice received PDX cells intravenously (1.5×10 6 cells per mouse) followed 18–21 days later by a single injection of CLL-1 CAR T cells (2.5×10 6 cells per mouse). (C) Residual tumor cells (top) and number of T cells (bottom) at day 5 after CLL-1 CAR (n=8) CLL-1 CAR+ IL15 (n=5) T cell infusion. P value was determined by unpaired Student’s t-test. (D) Kaplan-Meier curve showing the survival of mice in each experimental group. (E) Serum human <t>cytokine</t> levels (mean±SEM) (TNF alpha, IL15, IL-2, IFN-gamma, IL-1a, IL-6, IL-10) of representative mice at day 7 after CLL-1 CAR (n=3) and CLL-1 +IL15 CAR (n=3) T cell infusion. P value was determined by unpaired Student’s t-test. (F) Histopathological examination of lung (top) and liver (bottom) tissues (H E, ×100) in a mouse died at day 4 after CLL-1+ IL15 CAR infusion. Hemorrhage, edema is marked with green and apoptosis is marked with black in lung. The necrosis of liver is marked with green. P values were determined by log-rank Mantel-Cox test. CAR, chimeric antigen receptor; CLL, C-type lectin-like molecule 1; IFN, interferon; IL-15, interleukin 15; Iso Ctrl, isotype control; NS, not significant; NTR, non-transduced; PDX, patient derived xenografts; TNF, tumor necrosis factor.
    Intracellular Cytokine Staining Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson intracellular ifn γ cytokine staining
    Ginseng enhances <t>IFN-γ</t> cytokine-producing lung cells from BALB/c mice. (A) IL-4 ELISpot. (B) IFN-γ ELISpot. Cytokine (IL-4 and IFN-γ) producing lung cells isolated at day 5 postinfection ( n =5 BALB/c mice per group) were determined
    Intracellular Ifn γ Cytokine Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher intracellular cytokines
    Ginseng enhances <t>IFN-γ</t> cytokine-producing lung cells from BALB/c mice. (A) IL-4 ELISpot. (B) IFN-γ ELISpot. Cytokine (IL-4 and IFN-γ) producing lung cells isolated at day 5 postinfection ( n =5 BALB/c mice per group) were determined
    Intracellular Cytokines, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson intracellular cytokine stain
    Ginseng enhances <t>IFN-γ</t> cytokine-producing lung cells from BALB/c mice. (A) IL-4 ELISpot. (B) IFN-γ ELISpot. Cytokine (IL-4 and IFN-γ) producing lung cells isolated at day 5 postinfection ( n =5 BALB/c mice per group) were determined
    Intracellular Cytokine Stain, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson intracellular cytokine staining flow cytometry
    T cell subsets in spleen after immunization. A . Flow <t>cytometry</t> analysis strategies of T cell subsets in spleen, B and C . Levels of T cell subset are shown as percentages of <t>cytokine</t> producing cells (Interferon-γ, IL-17 and IL-4) per CD4+ T cells, and FoxP3 positive cells per CD4+ CD25+ T cells stimulated with AGE-LDL for each of the three groups (white bars = controls, bars with stripes = Alum adjuvant and black bars = AGE-LDL + Alum adjuvant, n = 10 per group). *indicates p
    Intracellular Cytokine Staining Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen intracellular cytokine staining kit
    The effects of dietary fatty acids on hepatic <t>cytokine</t> expression, inflammation signaling and serum adipokines. Wild-type C57BL6 mice were fed diets enriched with certain fatty acids (see ) for 12 weeks. A: Hepatic IFNγ and IL-4 expressions
    Intracellular Cytokine Staining Kit, supplied by Pharmingen, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson intracellular cytokine staining starter kit
    Enhanced efficacy of αDEC-205:OVA plus αCD40 relative to other immunization approaches. (A) C57BL/6 mice were immunized s.c. with several methods: Spleen DC pulsed ex vivo with 10 μg/ml each of αDEC-205:OVA and αCD40; 500 μg OVA in CFA; 50 μg OVA with 25 μg αCD40; 50 μg of SIINFEKL peptide with 25 μg αCD40; or 50 ng of OVA in αDEC-205:OVA with 25 μg of αCD40. 7 or 30 d later, lymph nodes were harvested and T cell expansion evaluated by K b -SIINFEKL–PE tetramer and CD62L staining. The gate for the y-axis was placed relative to the CD62L-negative tetramer binding cells in the right panel. Indicated percentages are percent of CD8 + lymphocytes. (B) As in A, but IFN-γ secretion evaluated by intracellular <t>cytokine</t> staining. Data are means of three experiments.
    Intracellular Cytokine Staining Starter Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend brilliant violet 605 anti human cd27
    Enhanced efficacy of αDEC-205:OVA plus αCD40 relative to other immunization approaches. (A) C57BL/6 mice were immunized s.c. with several methods: Spleen DC pulsed ex vivo with 10 μg/ml each of αDEC-205:OVA and αCD40; 500 μg OVA in CFA; 50 μg OVA with 25 μg αCD40; 50 μg of SIINFEKL peptide with 25 μg αCD40; or 50 ng of OVA in αDEC-205:OVA with 25 μg of αCD40. 7 or 30 d later, lymph nodes were harvested and T cell expansion evaluated by K b -SIINFEKL–PE tetramer and CD62L staining. The gate for the y-axis was placed relative to the CD62L-negative tetramer binding cells in the right panel. Indicated percentages are percent of CD8 + lymphocytes. (B) As in A, but IFN-γ secretion evaluated by intracellular <t>cytokine</t> staining. Data are means of three experiments.
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    Immune responses against mutated ligands in PBMC of patient Mel15. Clinical course and retrieval of patient material ( a ). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 ( b ). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation ( c ). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 10 4 cells ( d ). Intracellular cytokine staining (ICS) of an expanded NCAPG2 P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h ( e ). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4 S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells ( f ). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining ( g ) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) ( h ). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.

    Journal: Nature Communications

    Article Title: Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

    doi: 10.1038/ncomms13404

    Figure Lengend Snippet: Immune responses against mutated ligands in PBMC of patient Mel15. Clinical course and retrieval of patient material ( a ). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 ( b ). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation ( c ). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 10 4 cells ( d ). Intracellular cytokine staining (ICS) of an expanded NCAPG2 P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h ( e ). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4 S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells ( f ). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining ( g ) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) ( h ). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.

    Article Snippet: Intracellular cytokine staining was performed with IC staining kit (eBioscience).

    Techniques: Derivative Assay, In Vitro, Staining, Incubation, Co-Culture Assay

    Characterization of mutant-specific T-cell responses in HLA-matched healthy donors. T-cell responses of two different matched healthy donors against neoepitopes AKAP6 M1482I and NOP16 P169L . Effector cells were coincubated in duplicates with T2-A3 or T2-B7 pulsed either with the relevant peptide or control peptides with the same HLA restriction as the mutated ligands, results are shown as mean ( a ). Staining of T-cell line HD1-AKAP6 with the mutated or wt multimer ( b ). IFN-g release of the T-cell line on peptide titration of AKAP6 M1482I and its non-mutated counterpart using T2-A3 as targets (duplicates are depicted as mean) ( c ). IFN-g secretion (left Y-axis) and target-cell lysis (right Y -axis) after coincubation of the T-cell line HD1-AKAP6 with peptide-pulsed and minigene-transduced LCL1 cells performed in triplicates, data shown as mean±s.d. ( d ). Intracellular cytokine staining (IFN-g, TNF-a and IL-2) on co-culture of the T-cell line HD1-AKAP6 with LCL1 cells, either peptide-pulsed or minigene-transduced, determined by flow cytometry. Cells were gated on ethidium monoazide bromide-negative and CD8-positive events ( e ).

    Journal: Nature Communications

    Article Title: Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

    doi: 10.1038/ncomms13404

    Figure Lengend Snippet: Characterization of mutant-specific T-cell responses in HLA-matched healthy donors. T-cell responses of two different matched healthy donors against neoepitopes AKAP6 M1482I and NOP16 P169L . Effector cells were coincubated in duplicates with T2-A3 or T2-B7 pulsed either with the relevant peptide or control peptides with the same HLA restriction as the mutated ligands, results are shown as mean ( a ). Staining of T-cell line HD1-AKAP6 with the mutated or wt multimer ( b ). IFN-g release of the T-cell line on peptide titration of AKAP6 M1482I and its non-mutated counterpart using T2-A3 as targets (duplicates are depicted as mean) ( c ). IFN-g secretion (left Y-axis) and target-cell lysis (right Y -axis) after coincubation of the T-cell line HD1-AKAP6 with peptide-pulsed and minigene-transduced LCL1 cells performed in triplicates, data shown as mean±s.d. ( d ). Intracellular cytokine staining (IFN-g, TNF-a and IL-2) on co-culture of the T-cell line HD1-AKAP6 with LCL1 cells, either peptide-pulsed or minigene-transduced, determined by flow cytometry. Cells were gated on ethidium monoazide bromide-negative and CD8-positive events ( e ).

    Article Snippet: Intracellular cytokine staining was performed with IC staining kit (eBioscience).

    Techniques: Mutagenesis, Staining, Titration, Lysis, Co-Culture Assay, Flow Cytometry, Cytometry

    CX 3 CR1 + MNP-derived TL1A synergizes with IL-23 and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14 + MNPs and CD103 + DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3 − CD19 + ), CX 3 CR1 + MNPs, CD103 + DCs, and Ly6C + monocytes were sorted from the intestinal lamina propria of Cx3cr1 GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX 3 CR1 + MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: CX 3 CR1 + MNP-derived TL1A synergizes with IL-23 and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14 + MNPs and CD103 + DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3 − CD19 + ), CX 3 CR1 + MNPs, CD103 + DCs, and Ly6C + monocytes were sorted from the intestinal lamina propria of Cx3cr1 GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX 3 CR1 + MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.

    Article Snippet: Intracellular mouse cytokine staining was performed with IL-22 APC (IL22JOP), IFN-γ PECy7 (XMG1.2), IL17A FITC (eBio17B7), all from eBioscience.

    Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Two Tailed Test, Mouse Assay, Cell Culture, Staining, Transfection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from descending colon biopsies from patients with endoscopically mild to moderate Crohn’s’ disease ( n = 8, gray) or ulcerative colitis ( n = 6, black; Table S1 ), as well as age-matched non-IBD control patients undergoing routine screening colonoscopy ( n = 8, white), were stimulated ex vivo with PMA/ionomycin and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3 + or CD3 − fraction expressing IL-17 or IL-22 is indicated. *, P ≤ 0.05, two-tailed Student’s t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3 − IL-22 + (black lines) and CD3 − IL-22 − (gray) LPMCs. (C) Expression of RORγt by c-Kit + CD56 + LPMCs. Lin − cells (CD14/CD19/CD3/CD11b/CD11c/TCRγδ − ; Fig. S3 A ) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORγt. Lin − CD56 + c-Kit + ILC3 (black line) were compared with Lin − CD56 + c-Kit − NK cells (gray) for RORγt expression. (D) Surface staining of Lin − c-Kit + ILCs for the indicated markers (black line) compared with isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with control media (dotted line). Cells shown were gated on Lin − CD56 + c-Kit + . The isotype control is in gray. (F) CD11c + MHCII + human colonic APCs were electronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed (prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry. (left) Result from one representative donor. (right) Percentage of IL-22 + ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients. **, P ≤ 0.01, two-tailed Student’s t test. Black bars represent the geometric mean.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from descending colon biopsies from patients with endoscopically mild to moderate Crohn’s’ disease ( n = 8, gray) or ulcerative colitis ( n = 6, black; Table S1 ), as well as age-matched non-IBD control patients undergoing routine screening colonoscopy ( n = 8, white), were stimulated ex vivo with PMA/ionomycin and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3 + or CD3 − fraction expressing IL-17 or IL-22 is indicated. *, P ≤ 0.05, two-tailed Student’s t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3 − IL-22 + (black lines) and CD3 − IL-22 − (gray) LPMCs. (C) Expression of RORγt by c-Kit + CD56 + LPMCs. Lin − cells (CD14/CD19/CD3/CD11b/CD11c/TCRγδ − ; Fig. S3 A ) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORγt. Lin − CD56 + c-Kit + ILC3 (black line) were compared with Lin − CD56 + c-Kit − NK cells (gray) for RORγt expression. (D) Surface staining of Lin − c-Kit + ILCs for the indicated markers (black line) compared with isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with control media (dotted line). Cells shown were gated on Lin − CD56 + c-Kit + . The isotype control is in gray. (F) CD11c + MHCII + human colonic APCs were electronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed (prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry. (left) Result from one representative donor. (right) Percentage of IL-22 + ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients. **, P ≤ 0.01, two-tailed Student’s t test. Black bars represent the geometric mean.

    Article Snippet: Intracellular mouse cytokine staining was performed with IL-22 APC (IL22JOP), IFN-γ PECy7 (XMG1.2), IL17A FITC (eBio17B7), all from eBioscience.

    Techniques: Isolation, Ex Vivo, Staining, Expressing, Two Tailed Test, Cell Culture, Flow Cytometry, Cytometry

    TLR-stimulated CX 3 CR1 + MNPs are stronger inducers of ILC3 production of IL-22 than CD103 + CD11b + DCs and monocytes. (A–C) CD103 or CX 3 CR1 + MHCII + CD11c + CD11b + cells were isolated from the lamina propria of CX 3 CR1 GFP/+ mice (sort strategy shown in A and co-cultured with Lin − RORγt-GFP + ILCs with or without the indicated bacterial TLR ligands or IL-23. IL-22 was assessed by intracellular staining of CD90.2 + ILCs after 18 h. A representative flow cytometry plot is shown in B. (C) Percent IL-22 + ILCs is shown from seven independent experiments. **, P ≤ 0.01; *, P ≤ 0.05. One way ANOVA with Bonferroni’s correction. Error bars represent SEM. (D–F) Ly6C + MHCII lo (monocytes) and Ly6C − MHCII hi (MNPs) were isolated from CX 3 CR1 + CD11b + lamina propria cells (sort strategy is shown in D and co-cultured with intestinal ILCs with LPS or IL-23 as indicated. Intracellular cytokine staining for IL-22 is shown after 18 h (E). Supernatants were harvested after 18 h and IL-22 production quantitated by ELISA (F). Results are representative of two independent experiments performed in triplicate. ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: TLR-stimulated CX 3 CR1 + MNPs are stronger inducers of ILC3 production of IL-22 than CD103 + CD11b + DCs and monocytes. (A–C) CD103 or CX 3 CR1 + MHCII + CD11c + CD11b + cells were isolated from the lamina propria of CX 3 CR1 GFP/+ mice (sort strategy shown in A and co-cultured with Lin − RORγt-GFP + ILCs with or without the indicated bacterial TLR ligands or IL-23. IL-22 was assessed by intracellular staining of CD90.2 + ILCs after 18 h. A representative flow cytometry plot is shown in B. (C) Percent IL-22 + ILCs is shown from seven independent experiments. **, P ≤ 0.01; *, P ≤ 0.05. One way ANOVA with Bonferroni’s correction. Error bars represent SEM. (D–F) Ly6C + MHCII lo (monocytes) and Ly6C − MHCII hi (MNPs) were isolated from CX 3 CR1 + CD11b + lamina propria cells (sort strategy is shown in D and co-cultured with intestinal ILCs with LPS or IL-23 as indicated. Intracellular cytokine staining for IL-22 is shown after 18 h (E). Supernatants were harvested after 18 h and IL-22 production quantitated by ELISA (F). Results are representative of two independent experiments performed in triplicate. ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Article Snippet: Intracellular mouse cytokine staining was performed with IL-22 APC (IL22JOP), IFN-γ PECy7 (XMG1.2), IL17A FITC (eBio17B7), all from eBioscience.

    Techniques: Isolation, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    CX 3 CR1 + MNP-derived IL-23 and IL-1β activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1 STOP-DTR/GFP mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX 3 CR1 hi MNPs. (bottom) Expression of Ly6C and MHCII on CX 3 CR1 hi and CX 3 CR1 int populations. (B) Expression of IL-22 in Lin − CD90.2 + colonic ILCs from Cx3cr1 STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1 STOP-DTR/+ (Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days −2, −1, and 0 and every other day postinfection. One representative intracellular cytokine flow cytometry plot is shown on the left and a composite graph ( n = 6/group) on the right. *, P ≤ 0.05, two-tailed Student’s t test. Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures ( Fig. 3, B and C ) were harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX 3 CR1 + MNPs or CD103 + CD11b + DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1β by ELISA. Results are the mean of two independent experiments performed in duplicate and the SEM is shown. *, P ≤ 0.05; **, P ≤ 0.01. (E) Lin − CD90.2 hi ILCs from WT or Il1r −/− mice were co-cultured with sorted intestinal MNPs from WT or Il23p19 −/− mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after 18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P ≤ 0.05; ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: CX 3 CR1 + MNP-derived IL-23 and IL-1β activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1 STOP-DTR/GFP mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX 3 CR1 hi MNPs. (bottom) Expression of Ly6C and MHCII on CX 3 CR1 hi and CX 3 CR1 int populations. (B) Expression of IL-22 in Lin − CD90.2 + colonic ILCs from Cx3cr1 STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1 STOP-DTR/+ (Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days −2, −1, and 0 and every other day postinfection. One representative intracellular cytokine flow cytometry plot is shown on the left and a composite graph ( n = 6/group) on the right. *, P ≤ 0.05, two-tailed Student’s t test. Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures ( Fig. 3, B and C ) were harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX 3 CR1 + MNPs or CD103 + CD11b + DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1β by ELISA. Results are the mean of two independent experiments performed in duplicate and the SEM is shown. *, P ≤ 0.05; **, P ≤ 0.01. (E) Lin − CD90.2 hi ILCs from WT or Il1r −/− mice were co-cultured with sorted intestinal MNPs from WT or Il23p19 −/− mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after 18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P ≤ 0.05; ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Article Snippet: Intracellular mouse cytokine staining was performed with IL-22 APC (IL22JOP), IFN-γ PECy7 (XMG1.2), IL17A FITC (eBio17B7), all from eBioscience.

    Techniques: Derivative Assay, Mouse Assay, Injection, Expressing, Infection, Flow Cytometry, Cytometry, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

    Molecular and cellular mechanisms that contribute to tumour immunostimulatory reprogramming positive feedback loop a,b) . Quantitative RT-PCR analysis showing the effect of IFNγ and sCD40L on the mRNA levels of adhesion molecules, VEGFA (a) , and T cell attractant chemokines (b) . The experiments were repeated independently for three times (batches) with technical duplicates in each time. c) . Schematic of the experimental design and hypothetical model. d) . Tumours resected at Day12 – 13 post E0771 injection have similar size/weight, and the effect of Th1 adoptive transfer on vessel normalization as measured by the CD31 + endothelial cells to NG2 + pericytes ratio. e) . Flow cytometry quantification CD45.1 + adoptive transferred Th1 cells, and CD45.2 + host immune cells. f) . Characterization and quantification of CD45.2 + host immune cells showing that Th1-mediated immune infiltration is partially dependent on pericyte coverage. g) . Effect of Th1 adoptive transfer and pericyte depletion on CD11b + Ly6G + immune cells demonstrating different pattern with other tumour-infiltrating immune cells as from (f) . h) . Schematic summary of CD4 + -TL-mediated vessel normalization, and subsequent formation of positive feedback loop through cell-cell interaction, cytokine production and increased pericyte coverage. Checkpoint blockade therapy and antigen presentation enhance Th1-skewed CD4 + -TL activation and promote the vessel normalization/immunostimulatory reprogramming positive feedback loop. Data are presented as means ± s.e.m. Animal numbers used in (d–g) are denoted in (c) . P values were calculated using two-tailed unpaired Student’s t-test based on biological replicates ( a,b,d–g ). Technical replicates are averaged within each biological replicate ( a,b ).

    Journal: Nature

    Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

    doi: 10.1038/nature21724

    Figure Lengend Snippet: Molecular and cellular mechanisms that contribute to tumour immunostimulatory reprogramming positive feedback loop a,b) . Quantitative RT-PCR analysis showing the effect of IFNγ and sCD40L on the mRNA levels of adhesion molecules, VEGFA (a) , and T cell attractant chemokines (b) . The experiments were repeated independently for three times (batches) with technical duplicates in each time. c) . Schematic of the experimental design and hypothetical model. d) . Tumours resected at Day12 – 13 post E0771 injection have similar size/weight, and the effect of Th1 adoptive transfer on vessel normalization as measured by the CD31 + endothelial cells to NG2 + pericytes ratio. e) . Flow cytometry quantification CD45.1 + adoptive transferred Th1 cells, and CD45.2 + host immune cells. f) . Characterization and quantification of CD45.2 + host immune cells showing that Th1-mediated immune infiltration is partially dependent on pericyte coverage. g) . Effect of Th1 adoptive transfer and pericyte depletion on CD11b + Ly6G + immune cells demonstrating different pattern with other tumour-infiltrating immune cells as from (f) . h) . Schematic summary of CD4 + -TL-mediated vessel normalization, and subsequent formation of positive feedback loop through cell-cell interaction, cytokine production and increased pericyte coverage. Checkpoint blockade therapy and antigen presentation enhance Th1-skewed CD4 + -TL activation and promote the vessel normalization/immunostimulatory reprogramming positive feedback loop. Data are presented as means ± s.e.m. Animal numbers used in (d–g) are denoted in (c) . P values were calculated using two-tailed unpaired Student’s t-test based on biological replicates ( a,b,d–g ). Technical replicates are averaged within each biological replicate ( a,b ).

    Article Snippet: For cytokine intracellular staining, 0.5×106 enriched tumour-infiltrating immune cells were resuspened in 0.1 ml RPMI complete medium (RPMI-1640 medium containing 10% heat inactivated FBS, 100 IU·ml−1 penicillin/streptomycin, 55 μmol·L−1 β-Mercaptoethanol, 1× Protein Transport Inhibitor Cocktail (eBioscience)), and were incubated at 37°C with 5% CO2 for 5 hours.

    Techniques: Quantitative RT-PCR, Injection, Adoptive Transfer Assay, Flow Cytometry, Cytometry, Activation Assay, Two Tailed Test

    Cytokine production kinetics in T cells. ( A ) T cells that were rested for 3 d in the presence of IL-7 ( Top ), IL-2 ( Center ), or IL-15 ( Bottom ) were activated with 100 nM SIINFEKL (N4) or SIITFEKL (T4) OVA peptide for indicated time points ( n = 3 mice). Brefeldin A (BrfA) was added during the last 2 h of culture. ( B ) Representative dot plots for TNF-α and IFN-γ ( Upper ) and IFN-γ and IL-2 ( Lower ) production of T cells that were activated for indicated time with 100 nM OVA 257–264 peptide (N4) in the presence of BrfA during the last 2 h of stimulation. Numbers indicate percentage of cytokine-producing T cells. ( C ) mRNA levels of Tnfa , Ifng , and Il2 of T cells that were rested for 3 d in the presence of IL-7, IL-2, or IL-15.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Cytokine production kinetics in T cells. ( A ) T cells that were rested for 3 d in the presence of IL-7 ( Top ), IL-2 ( Center ), or IL-15 ( Bottom ) were activated with 100 nM SIINFEKL (N4) or SIITFEKL (T4) OVA peptide for indicated time points ( n = 3 mice). Brefeldin A (BrfA) was added during the last 2 h of culture. ( B ) Representative dot plots for TNF-α and IFN-γ ( Upper ) and IFN-γ and IL-2 ( Lower ) production of T cells that were activated for indicated time with 100 nM OVA 257–264 peptide (N4) in the presence of BrfA during the last 2 h of stimulation. Numbers indicate percentage of cytokine-producing T cells. ( C ) mRNA levels of Tnfa , Ifng , and Il2 of T cells that were rested for 3 d in the presence of IL-7, IL-2, or IL-15.

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Mouse Assay

    Preformed cytokine mRNA promotes early T cell responsiveness. ( A ) Tnfa , Ifng , and Il2 mRNA levels of resting T cells were measured by RT-PCR. ( B – D ) T cells were pretreated for 30 min with 1 μg/mL ActD or 10 μg/mL CHX and then stimulated for indicated time points with 100 nM OVA peptide in the presence of BrfA. Graphs depict the relative percentage of TNF-α ( B ), IFN-γ ( C ), and IL-2 ( D )-producing T cells compared with T cells activated in the absence of inhibitors. Data are presented as mean ± SD of three independently performed experiments. (One-way ANOVA corrected for multiple comparisons with a Tukey test; n = 7 mice; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Preformed cytokine mRNA promotes early T cell responsiveness. ( A ) Tnfa , Ifng , and Il2 mRNA levels of resting T cells were measured by RT-PCR. ( B – D ) T cells were pretreated for 30 min with 1 μg/mL ActD or 10 μg/mL CHX and then stimulated for indicated time points with 100 nM OVA peptide in the presence of BrfA. Graphs depict the relative percentage of TNF-α ( B ), IFN-γ ( C ), and IL-2 ( D )-producing T cells compared with T cells activated in the absence of inhibitors. Data are presented as mean ± SD of three independently performed experiments. (One-way ANOVA corrected for multiple comparisons with a Tukey test; n = 7 mice; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    Regulation of cytokine production through PKC and Ca 2+ -dependent signaling. ( A ) TNF-α and IFN-γ ( Upper ), and IL-2 and IFN-γ ( Lower ) production of CD44 hi memory-like T cells activated for 4 h with indicated stimuli. Unstimulated T cells were used as negative control. Representative of four mice from two independently performed experiments. ( B ) T cells were stimulated with PMA/ionomycin for indicated time points. The percentage of TNF-α, IFN-γ, and IL-2 protein producing T cells was determined by intracellular cytokine staining (mean ± SD; n = 7 mice). ( C and D ) IL-2 production of PMA/ionomycin-stimulated T cells ( C ), and IFN-γ and IL-2 production of OVA 257–264 -stimulated T cells ( D ) that were pretreated with the indicated inhibitor. Representative of six mice from three independently performed experiments. ( E ) Tnfa , Ifng , and Il2 mRNA levels (mean ± SD) of resting T cells that were stimulated for 2 h as indicated (one-way ANOVA with Tukey’s multiple comparison; n = 7 mice; ns, nonsignificant; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Regulation of cytokine production through PKC and Ca 2+ -dependent signaling. ( A ) TNF-α and IFN-γ ( Upper ), and IL-2 and IFN-γ ( Lower ) production of CD44 hi memory-like T cells activated for 4 h with indicated stimuli. Unstimulated T cells were used as negative control. Representative of four mice from two independently performed experiments. ( B ) T cells were stimulated with PMA/ionomycin for indicated time points. The percentage of TNF-α, IFN-γ, and IL-2 protein producing T cells was determined by intracellular cytokine staining (mean ± SD; n = 7 mice). ( C and D ) IL-2 production of PMA/ionomycin-stimulated T cells ( C ), and IFN-γ and IL-2 production of OVA 257–264 -stimulated T cells ( D ) that were pretreated with the indicated inhibitor. Representative of six mice from three independently performed experiments. ( E ) Tnfa , Ifng , and Il2 mRNA levels (mean ± SD) of resting T cells that were stimulated for 2 h as indicated (one-way ANOVA with Tukey’s multiple comparison; n = 7 mice; ns, nonsignificant; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Negative Control, Mouse Assay, Staining

    TNF-α, IFN-γ, and IL-2 follow individual production kinetics. ( A and B ) Cytokine production of resting CD8 + OT-I T cells stimulated with 100 nM OVA peptide. Brefeldin A (BrfA) was present throughout the stimulation ( A ) or added for the last 0.5, 1, or 2 h of activation ( B ). ( C ) Cytokine profile analysis of T cells activated for 2, 4, or 6 h from B . ( Upper ) Percentage of T cells that produce at least one cytokine. ( Lower ) Cytokine production of responding T cells. Pooled data from three independently performed experiments (mean ± SD; n = 7 mice). ( D ) FACS-sorted CD44 hi memory-like OT-I T cells activated with peptide-loaded bone marrow-derived dendritic cells. N4, SIINFEKL; T4, SIITFEKL ( n = 5 mice). ( E ) Effector memory (EM, Left ) and central memory (CM; Right ) T cells from LCMV-infected mice (d60) were reactivated with GP33 and NP396 peptide ( n = 5 mice).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: TNF-α, IFN-γ, and IL-2 follow individual production kinetics. ( A and B ) Cytokine production of resting CD8 + OT-I T cells stimulated with 100 nM OVA peptide. Brefeldin A (BrfA) was present throughout the stimulation ( A ) or added for the last 0.5, 1, or 2 h of activation ( B ). ( C ) Cytokine profile analysis of T cells activated for 2, 4, or 6 h from B . ( Upper ) Percentage of T cells that produce at least one cytokine. ( Lower ) Cytokine production of responding T cells. Pooled data from three independently performed experiments (mean ± SD; n = 7 mice). ( D ) FACS-sorted CD44 hi memory-like OT-I T cells activated with peptide-loaded bone marrow-derived dendritic cells. N4, SIINFEKL; T4, SIITFEKL ( n = 5 mice). ( E ) Effector memory (EM, Left ) and central memory (CM; Right ) T cells from LCMV-infected mice (d60) were reactivated with GP33 and NP396 peptide ( n = 5 mice).

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Activation Assay, Mouse Assay, FACS, Derivative Assay, Infection

    Role of PKC and Ca 2+ -dependent signaling in cytokine production. ( A ) Resting T cells were stimulated or not for 4 h with PMA, ionomycin, or with both (PMA/iono). Numbers in dot plots indicate percentage of TNF-α + and/or IFN-γ + T cells. Histogram depicts IL-2 production of the same sample. ( B and C ) T cells were pretreated or not with indicated inhibitor before activation with PMA/ionomycin ( B ), or with OVA peptide ( C ). Representative of three independently performed experiments. ( D – F ) TNF-α ( D ), IFN-γ ( E ), and IL-2 ( F ) protein levels ( Left ) and mRNA levels ( Right ) of CD44 hi memory-like T cells were determined after 4 h and 2 h of indicated stimulation, respectively ( n = 4 mice) (one-way ANOVA with Tukey’s comparison; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Role of PKC and Ca 2+ -dependent signaling in cytokine production. ( A ) Resting T cells were stimulated or not for 4 h with PMA, ionomycin, or with both (PMA/iono). Numbers in dot plots indicate percentage of TNF-α + and/or IFN-γ + T cells. Histogram depicts IL-2 production of the same sample. ( B and C ) T cells were pretreated or not with indicated inhibitor before activation with PMA/ionomycin ( B ), or with OVA peptide ( C ). Representative of three independently performed experiments. ( D – F ) TNF-α ( D ), IFN-γ ( E ), and IL-2 ( F ) protein levels ( Left ) and mRNA levels ( Right ) of CD44 hi memory-like T cells were determined after 4 h and 2 h of indicated stimulation, respectively ( n = 4 mice) (one-way ANOVA with Tukey’s comparison; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Activation Assay, Mouse Assay

    Signal strength determines cytokine specific pathway for production. ( A – C ) TNF-α ( A ), IFN-γ ( B ), and IL-2 ( C ) protein production upon 6 h of stimulation with 0.1 nM (low), 1 nM (intermediate), or 100 nM (high) OVA peptide. Representative of four independently performed experiments ( n = 6 mice). ( D – F , Left ) Tnfa ( D ), Ifng ( E ), and Il2 ( F ) mRNA levels after 2 h of stimulation. Mean ± SD of three ( D and F ) and four ( E ) independently performed experiments [ n = 5 ( D and F ); n = 6 ( E ) mice] (one-way ANOVA with Tukey’s comparison; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Signal strength determines cytokine specific pathway for production. ( A – C ) TNF-α ( A ), IFN-γ ( B ), and IL-2 ( C ) protein production upon 6 h of stimulation with 0.1 nM (low), 1 nM (intermediate), or 100 nM (high) OVA peptide. Representative of four independently performed experiments ( n = 6 mice). ( D – F , Left ) Tnfa ( D ), Ifng ( E ), and Il2 ( F ) mRNA levels after 2 h of stimulation. Mean ± SD of three ( D and F ) and four ( E ) independently performed experiments [ n = 5 ( D and F ); n = 6 ( E ) mice] (one-way ANOVA with Tukey’s comparison; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Mouse Assay

    Lung CD4 + T RM cells and heterotypic pulmonary protection imprint locally The left (L) and right (R) lobes of uninfected Het Imm (i.t.) and saline control mice were digested separately with collagenase. (A) Representative flow cytometry plots show lung T RM cells that were identified as CD3 + /CD4 + /CD11a hi /CD69 + /CD62L lo /CD44 hi . (B) T RM cells were quantified in the left (ipsilateral) and right (contralateral) lobes of Het Imm and saline mice. Data are displayed as % CD11a hi CD69 + of CD4 + cells. Bars represent means with SEM. Significance was determined by two-way ANOVA followed by Tukey’s post hoc test (n= 4–5 per group, experiment repeated three times). (C) Representative flow cytometry plot of CD44 and CD62L expression on CD11a hi CD69 + lung T RM cells (red) and lung CD4 + CD11a lo CD69- cells (blue). (D) Single cells from the left (ipsilateral) and right (contralateral) lobes of Het Imm (i.t.) and saline mice were stimulated with PMA/Ionomycin for 6h in the presence of a protein transport inhibitor. ICS and flow cytometry was performed to monitor cytokine production. Representative flow plots show IL-17A and IFN-γ-producing CD4 + cells from Het Imm and saline lungs. (E–F) IL-17A and IFN-γ-producing CD4 + cells were quantified in the left (ipsilateral) and right (contralateral) lobes of Het Imm and saline mice. Bars represent means with SEM. Significance was determined by two-way ANOVA followed by Tukey’s post hoc test (n= 5–6 per group). Three independent experiments were performed. (G) Mice on a mixed background were infected twice with saline, intranasal Sp19F (Het Imm i.n.), or intratracheal Sp19F (Het Imm i.t.). After 4 weeks mice from each group were challenged with 1 × 10 6 Sp3 CFU. Lungs were harvested after 24h and bacterial burden was determined. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Significance was determined by one-way ANOVA followed by Dunn’s post hoc test. (H) CD4 + lymphocytes from the lungs and spleens of uninfected Het Imm were isolated using FACS. Three minutes prior to euthanization intravital cells were fluorescently labeled with anti-CD45-APC antibody via i.v. injection. The cells for transfer were identified as live, i.v. label-negative CD45 + CD4 + cells. Naïve recipient mice were administered saline, lung-derived or spleen-derived CD4 + cells (1–4×10 6 per mouse) from Het Imm mice transferred via i.v. tail vein injection. Three days after the transfer, recipient mice were challenged with 1×10 6 Sp3 CFU. After 24h, left lung lobes were harvested, mRNA was extracted, and qRT-PCR for IL-17A was performed. Data were displayed as fold induction for each group normalized to uninfected lungs. Significance was determined by one-way ANOVA followed by Tukey’s post hoc test (n=3–5 per group). Bars represent means with SEM. (I) Ipsilateral vs contralateral heterotypic protection model. Het Imm (i.t.) mice received two Sp19F infections in their left lobes. After 4 weeks, half of the mice were challenged with 1×10 6 Sp3 CFU in their ipsilateral (left) lobe, and the other half challenged in their contralateral (right) lobes. Images represent the instillation (i.t.) of diluted colloidal carbon into C57BL/6 mouse lungs either in the left (L) or right (R) lobes. Lungs were immediately collected and the deposition of the dye was visualized. (J) Naïve C57BL/6 mice were infected with 1×10 6 Sp3 CFU selectively in either the left or right lobes. Lung bacterial burden was assessed after 24h. (K) The mice described in (I) were challenged with 1×10 6 Sp3 CFU in the specified location. After 24h whole lungs were collected and bacterial burden was assessed. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Significance was determined using a Mann-Whitney test. Two independent experiments were performed. * P

    Journal: Mucosal immunology

    Article Title: Regionally Compartmentalized Resident Memory T Cells Mediate Naturally Acquired Protection Against Pneumococcal Pneumonia

    doi: 10.1038/mi.2017.43

    Figure Lengend Snippet: Lung CD4 + T RM cells and heterotypic pulmonary protection imprint locally The left (L) and right (R) lobes of uninfected Het Imm (i.t.) and saline control mice were digested separately with collagenase. (A) Representative flow cytometry plots show lung T RM cells that were identified as CD3 + /CD4 + /CD11a hi /CD69 + /CD62L lo /CD44 hi . (B) T RM cells were quantified in the left (ipsilateral) and right (contralateral) lobes of Het Imm and saline mice. Data are displayed as % CD11a hi CD69 + of CD4 + cells. Bars represent means with SEM. Significance was determined by two-way ANOVA followed by Tukey’s post hoc test (n= 4–5 per group, experiment repeated three times). (C) Representative flow cytometry plot of CD44 and CD62L expression on CD11a hi CD69 + lung T RM cells (red) and lung CD4 + CD11a lo CD69- cells (blue). (D) Single cells from the left (ipsilateral) and right (contralateral) lobes of Het Imm (i.t.) and saline mice were stimulated with PMA/Ionomycin for 6h in the presence of a protein transport inhibitor. ICS and flow cytometry was performed to monitor cytokine production. Representative flow plots show IL-17A and IFN-γ-producing CD4 + cells from Het Imm and saline lungs. (E–F) IL-17A and IFN-γ-producing CD4 + cells were quantified in the left (ipsilateral) and right (contralateral) lobes of Het Imm and saline mice. Bars represent means with SEM. Significance was determined by two-way ANOVA followed by Tukey’s post hoc test (n= 5–6 per group). Three independent experiments were performed. (G) Mice on a mixed background were infected twice with saline, intranasal Sp19F (Het Imm i.n.), or intratracheal Sp19F (Het Imm i.t.). After 4 weeks mice from each group were challenged with 1 × 10 6 Sp3 CFU. Lungs were harvested after 24h and bacterial burden was determined. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Significance was determined by one-way ANOVA followed by Dunn’s post hoc test. (H) CD4 + lymphocytes from the lungs and spleens of uninfected Het Imm were isolated using FACS. Three minutes prior to euthanization intravital cells were fluorescently labeled with anti-CD45-APC antibody via i.v. injection. The cells for transfer were identified as live, i.v. label-negative CD45 + CD4 + cells. Naïve recipient mice were administered saline, lung-derived or spleen-derived CD4 + cells (1–4×10 6 per mouse) from Het Imm mice transferred via i.v. tail vein injection. Three days after the transfer, recipient mice were challenged with 1×10 6 Sp3 CFU. After 24h, left lung lobes were harvested, mRNA was extracted, and qRT-PCR for IL-17A was performed. Data were displayed as fold induction for each group normalized to uninfected lungs. Significance was determined by one-way ANOVA followed by Tukey’s post hoc test (n=3–5 per group). Bars represent means with SEM. (I) Ipsilateral vs contralateral heterotypic protection model. Het Imm (i.t.) mice received two Sp19F infections in their left lobes. After 4 weeks, half of the mice were challenged with 1×10 6 Sp3 CFU in their ipsilateral (left) lobe, and the other half challenged in their contralateral (right) lobes. Images represent the instillation (i.t.) of diluted colloidal carbon into C57BL/6 mouse lungs either in the left (L) or right (R) lobes. Lungs were immediately collected and the deposition of the dye was visualized. (J) Naïve C57BL/6 mice were infected with 1×10 6 Sp3 CFU selectively in either the left or right lobes. Lung bacterial burden was assessed after 24h. (K) The mice described in (I) were challenged with 1×10 6 Sp3 CFU in the specified location. After 24h whole lungs were collected and bacterial burden was assessed. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Significance was determined using a Mann-Whitney test. Two independent experiments were performed. * P

    Article Snippet: Intracellular cytokine staining (ICS) was performed using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences, San Jose, CA) and antibodies to label the following cell surface markers and cytokines were used: CD45 (clone 30-F11, Biolegend, San Diego, CA), CD3ε (clone 145-2C11, Biolegend), CD4 (clone GK1.5, eBioscience, San Diego, CA), IL-17A (clone eBio17B7, eBioscience), and IFN-γ (clone XMG1.2, eBioscience).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Infection, Isolation, FACS, Labeling, Injection, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY

    IL-17 and IFN-γ production in PBMC peaks 2 weeks post-vaccination. PBMC from vaccinated subjects were stimulated with Ag85A peptide pools with or without 100 uM ARL67156. No stimulation and phorbol 12-myristate 13-acetate with ionomycin were used as negative and positive controls. Percentages shown are unstimulated subtracted from Ag85A stimulation. Following staining, cells were gated as shown in (A): Lymphocytes were gated for on FSC vs. SSC. Singlets were then gated and dead cells, B cells and monocytes were gated out. CD3 + cells were selected for CD4 + CD8 − cells. Antigen-specific cytokine expression from these cells was evaluated. Cells expressing both IL-17 and IFN-γ were quantified and shown in (B). The effect of ARL67156 on cytokine expression was investigated by addition during ICS stimulation (C).

    Journal: PLoS ONE

    Article Title: Th1/Th17 Cell Induction and Corresponding Reduction in ATP Consumption following Vaccination with the Novel Mycobacterium tuberculosis Vaccine MVA85A

    doi: 10.1371/journal.pone.0023463

    Figure Lengend Snippet: IL-17 and IFN-γ production in PBMC peaks 2 weeks post-vaccination. PBMC from vaccinated subjects were stimulated with Ag85A peptide pools with or without 100 uM ARL67156. No stimulation and phorbol 12-myristate 13-acetate with ionomycin were used as negative and positive controls. Percentages shown are unstimulated subtracted from Ag85A stimulation. Following staining, cells were gated as shown in (A): Lymphocytes were gated for on FSC vs. SSC. Singlets were then gated and dead cells, B cells and monocytes were gated out. CD3 + cells were selected for CD4 + CD8 − cells. Antigen-specific cytokine expression from these cells was evaluated. Cells expressing both IL-17 and IFN-γ were quantified and shown in (B). The effect of ARL67156 on cytokine expression was investigated by addition during ICS stimulation (C).

    Article Snippet: Intracellular cytokine staining Cells were resuspended at 1×106 cells/mL and each sample divided into seven 1 mL aliquots in 5 mL polystyrene round bottom tubes (BD Falcon).

    Techniques: Staining, Expressing

    IL-17 and IFN-γ production in whole blood peaks 1 week post-vaccination. Whole blood (WB) from vaccinated subjects was stimulated for 6 h with a pool of 66 Ag85A peptides. Phytohaemagglutinin (PHA)-treated and untreated cells were used as positive and negative controls respectively. Lymphocytes were gated for and cytokine-expressing cells quantified as described above. Percentages of IL-17 + and IFN-γ + IL-17 + cells responding to the Ag85A peptide pool (with the percentages from unstimulated cells subtracted) are shown in (A) and (B). Differences in percentages of cells at the relevant peak time point in PBMC vs. WB is shown in (C). n = 7.

    Journal: PLoS ONE

    Article Title: Th1/Th17 Cell Induction and Corresponding Reduction in ATP Consumption following Vaccination with the Novel Mycobacterium tuberculosis Vaccine MVA85A

    doi: 10.1371/journal.pone.0023463

    Figure Lengend Snippet: IL-17 and IFN-γ production in whole blood peaks 1 week post-vaccination. Whole blood (WB) from vaccinated subjects was stimulated for 6 h with a pool of 66 Ag85A peptides. Phytohaemagglutinin (PHA)-treated and untreated cells were used as positive and negative controls respectively. Lymphocytes were gated for and cytokine-expressing cells quantified as described above. Percentages of IL-17 + and IFN-γ + IL-17 + cells responding to the Ag85A peptide pool (with the percentages from unstimulated cells subtracted) are shown in (A) and (B). Differences in percentages of cells at the relevant peak time point in PBMC vs. WB is shown in (C). n = 7.

    Article Snippet: Intracellular cytokine staining Cells were resuspended at 1×106 cells/mL and each sample divided into seven 1 mL aliquots in 5 mL polystyrene round bottom tubes (BD Falcon).

    Techniques: Western Blot, Expressing

    Recurrent GAS infection shifts the cytokine profile of CD4 + 2W:I-A b+ T cells to IL-17A + , IFN-γ + population in NALT. (A) B6 mice were inoculated intranasally with 2×10 8 CFU at weekly intervals for 12 weeks. CD4 + CD44 Hi 2W:I-A b+ T cells from Spleens, CLNs and NALT of infected mice were restimulated in vitro with PMA and ionomycin, stained and analyzed for IL-17A and IFN-γ and analyzed by flow cytometry. Representative result from one of two independent experiments is shown. (B) Pooled results of two independent experiments shown. (n = 4).

    Journal: PLoS Pathogens

    Article Title: Robust Antigen Specific Th17 T Cell Response to Group A Streptococcus Is Dependent on IL-6 and Intranasal Route of Infection

    doi: 10.1371/journal.ppat.1002252

    Figure Lengend Snippet: Recurrent GAS infection shifts the cytokine profile of CD4 + 2W:I-A b+ T cells to IL-17A + , IFN-γ + population in NALT. (A) B6 mice were inoculated intranasally with 2×10 8 CFU at weekly intervals for 12 weeks. CD4 + CD44 Hi 2W:I-A b+ T cells from Spleens, CLNs and NALT of infected mice were restimulated in vitro with PMA and ionomycin, stained and analyzed for IL-17A and IFN-γ and analyzed by flow cytometry. Representative result from one of two independent experiments is shown. (B) Pooled results of two independent experiments shown. (n = 4).

    Article Snippet: Intracellular cytokine staining Intracellular cytokine staining for IL-17A and IFN-γ was done using BD Cytofix/Cytoperm fixation-permeabilization solution and anti-IL-17A-PE and anti-IFN-γ-PE-Cy7 antibodies according to manufacturer's protocols.

    Techniques: Infection, Mouse Assay, In Vitro, Staining, Flow Cytometry, Cytometry

    HLA-A*0201-restricted epitope-specific cellular immunogenicity of various smallpox vaccines. ( A and B ) Peptide-specific intracellular cytokine release of splenocytes from vaccinated HHD mice. Mice were immunized i.p. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 IU of MVA ( A ) or i.m. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 , 10 6 , 10 5 , and 10 4 IU of MVA ( B ). #1 and #2 refer to individual mice analyzed. Splenocyte preparations were analyzed for the presence of VP35#1 peptide-specific, activated (CD62L low ) CD8 + T cells. The magnitude of the induced T cell response is depicted as percentages of cytokine-secreting CD8 + T cells within the live CD8 + cell population. ( C ) Specific lysis of peptide-pulsed human target cells by VP35#1-specific CTLs from vaccinated HHD mice. Effector CTLs were generated by single i.p. immunization of HHD mice with MVA (■ and □), CVA (● and ○), or Wyeth (▴ and ▵). CTLs were assayed for cytotoxicity at the indicated E/T ratio against T2 targets pulsed with either VP35#1 (filled symbols) or influenza M1 peptide (open symbols). ( D ) Processing and presentation of peptide epitope VP35#1 after heterologous expression of the vaccinia virus H3L gene. Human A375/H3L (■ and □) or control A375 (● and ○) cells were tested against A*0201-restricted murine CTLs specific for VP35#1 (filled symbols) or human tyrosinase 369–377 epitopes (open symbols) as effector cells at the indicated E/T ratio.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines

    doi: 10.1073/pnas.262668999

    Figure Lengend Snippet: HLA-A*0201-restricted epitope-specific cellular immunogenicity of various smallpox vaccines. ( A and B ) Peptide-specific intracellular cytokine release of splenocytes from vaccinated HHD mice. Mice were immunized i.p. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 IU of MVA ( A ) or i.m. with 2.5 × 10 5 PFU of CVA or Wyeth or 10 8 , 10 6 , 10 5 , and 10 4 IU of MVA ( B ). #1 and #2 refer to individual mice analyzed. Splenocyte preparations were analyzed for the presence of VP35#1 peptide-specific, activated (CD62L low ) CD8 + T cells. The magnitude of the induced T cell response is depicted as percentages of cytokine-secreting CD8 + T cells within the live CD8 + cell population. ( C ) Specific lysis of peptide-pulsed human target cells by VP35#1-specific CTLs from vaccinated HHD mice. Effector CTLs were generated by single i.p. immunization of HHD mice with MVA (■ and □), CVA (● and ○), or Wyeth (▴ and ▵). CTLs were assayed for cytotoxicity at the indicated E/T ratio against T2 targets pulsed with either VP35#1 (filled symbols) or influenza M1 peptide (open symbols). ( D ) Processing and presentation of peptide epitope VP35#1 after heterologous expression of the vaccinia virus H3L gene. Human A375/H3L (■ and □) or control A375 (● and ○) cells were tested against A*0201-restricted murine CTLs specific for VP35#1 (filled symbols) or human tyrosinase 369–377 epitopes (open symbols) as effector cells at the indicated E/T ratio.

    Article Snippet: Intracellular cytokine staining was performed for 30 min at 4°C by applying FITC-anti-IFN-γ (XMG1.2), FITC-anti-tumor necrosis factor α (MP6-XT22), or FITC-labeled IgG1 isotype control (R3-34) using Cytofix/Cytoperm (PharMingen/Becton Dickinson).

    Techniques: Mouse Assay, Lysis, Generated, Expressing

    Identification of HLA-A*0201-restricted peptide epitope VP35#1. ( A ) Vaccinia virus polypeptides selected for analysis of peptides with highest probability for HLA-A*0201 binding are shown. Incubation of splenocytes from vaccinated HHD mice with peptide VP35#1 results in specific secretion of IFN-γ. T cell response is depicted as percentage of cytokine-secreting CD8 + cells (%CD8+). An irrelevant HLA-A*0201-binding peptide (huTyr 369) was used as a negative control. The numbers marked by an asterisk refer to results from two independent experiments. ( B ) Alignment of VP35#1-peptide sequences derived from poxvirus gene products homologous to vaccinia virus H3L protein: vaccinia virus strains MVA (VV-MVA), Copenhagen (VV-Cop), WR (VV-WR), and Tian Tan (VV-Tan); monkeypox virus Zaire-96-I-16 (MPXV-ZRE); camelpox virus M-96 (CPXV-K/M96); ectromelia virus Naval (EV); variola major viruses Bangladesh-1975 (VAR-BSH) and India-1967 (VAR-IND); variola minor virus Garcia-1966 (VMN-GAR); yaba-like disease virus (YAB-YLD); molluscum contagiosum virus 1 (MC1); fowlpox virus (FPV-FCV); orf viruses NZ2 (Orf-NZ2), OV/20 (Orf-OV/20), and OV/7 (Orf-OV/7); myxoma virus Lausanne (MYX-LAU); lumpy skin disease virus Neethling 2490 (LSD-NEE); and swinepox virus 17077-99 (SPV-KAS).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines

    doi: 10.1073/pnas.262668999

    Figure Lengend Snippet: Identification of HLA-A*0201-restricted peptide epitope VP35#1. ( A ) Vaccinia virus polypeptides selected for analysis of peptides with highest probability for HLA-A*0201 binding are shown. Incubation of splenocytes from vaccinated HHD mice with peptide VP35#1 results in specific secretion of IFN-γ. T cell response is depicted as percentage of cytokine-secreting CD8 + cells (%CD8+). An irrelevant HLA-A*0201-binding peptide (huTyr 369) was used as a negative control. The numbers marked by an asterisk refer to results from two independent experiments. ( B ) Alignment of VP35#1-peptide sequences derived from poxvirus gene products homologous to vaccinia virus H3L protein: vaccinia virus strains MVA (VV-MVA), Copenhagen (VV-Cop), WR (VV-WR), and Tian Tan (VV-Tan); monkeypox virus Zaire-96-I-16 (MPXV-ZRE); camelpox virus M-96 (CPXV-K/M96); ectromelia virus Naval (EV); variola major viruses Bangladesh-1975 (VAR-BSH) and India-1967 (VAR-IND); variola minor virus Garcia-1966 (VMN-GAR); yaba-like disease virus (YAB-YLD); molluscum contagiosum virus 1 (MC1); fowlpox virus (FPV-FCV); orf viruses NZ2 (Orf-NZ2), OV/20 (Orf-OV/20), and OV/7 (Orf-OV/7); myxoma virus Lausanne (MYX-LAU); lumpy skin disease virus Neethling 2490 (LSD-NEE); and swinepox virus 17077-99 (SPV-KAS).

    Article Snippet: Intracellular cytokine staining was performed for 30 min at 4°C by applying FITC-anti-IFN-γ (XMG1.2), FITC-anti-tumor necrosis factor α (MP6-XT22), or FITC-labeled IgG1 isotype control (R3-34) using Cytofix/Cytoperm (PharMingen/Becton Dickinson).

    Techniques: Binding Assay, Incubation, Mouse Assay, Negative Control, Derivative Assay

    Analysis of VP35#1-peptide vaccine-induced immunity and protection in mouse challenge models. ( A ) Induction of HLA-A*0201-restricted VP35#1 peptide-specific CD8 + T cell responses after peptide immunization. HHD mice were immunized once with VP35#1 peptide-CpG-ODN. Splenocytes were stimulated with VP35#1 or influenza M1 peptide and stained for intracellular cytokines. Cells were analyzed by flow cytometry for the presence of VP35#1 peptide-specific, activated (CD62L low ) CD8 + T cells. The magnitude of the induced T cell response is depicted as percentages of cytokine-secreting CD8 + T cells within the live CD8 + cell population. ( B ) Protective efficacy of VP35#1/CpG-ODN vaccination in HLA-A*0201-transgenic mice. HHD mice ( n = 8) were immunized i.m. with 10 7 (■) IU of MVA or s.c. with 0.3 mg of VP35#1 peptide plus 10 nM CpG-ODN 1668 (●). Mock-vaccinated (□) mice served as control. Two (peptide) or 3 (MVA) weeks after vaccination animals were challenged with 2.5 × 10 6 PFU of WR and monitored daily for a 4-week period. The survival (%) of the animals was plotted. ( C and D ) Protective efficacy of vaccination in BALB/c mice. Groups of mice ( n = 10) were immunized i.m. with 10 4 (♦), 10 5 (■), 10 6 (▴), 10 7 (●), or 10 8 IU of MVA (−) ( C ) or 10 5 PFU of Wyeth (■) ( D ). Three weeks after vaccination animals were challenged intranasally with 1 × 10 6 PFU of WR. Individual animal weights were monitored daily and are expressed as means for each group. Mock-vaccinated (□) and mock-challenged (⋄) mice served as control.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines

    doi: 10.1073/pnas.262668999

    Figure Lengend Snippet: Analysis of VP35#1-peptide vaccine-induced immunity and protection in mouse challenge models. ( A ) Induction of HLA-A*0201-restricted VP35#1 peptide-specific CD8 + T cell responses after peptide immunization. HHD mice were immunized once with VP35#1 peptide-CpG-ODN. Splenocytes were stimulated with VP35#1 or influenza M1 peptide and stained for intracellular cytokines. Cells were analyzed by flow cytometry for the presence of VP35#1 peptide-specific, activated (CD62L low ) CD8 + T cells. The magnitude of the induced T cell response is depicted as percentages of cytokine-secreting CD8 + T cells within the live CD8 + cell population. ( B ) Protective efficacy of VP35#1/CpG-ODN vaccination in HLA-A*0201-transgenic mice. HHD mice ( n = 8) were immunized i.m. with 10 7 (■) IU of MVA or s.c. with 0.3 mg of VP35#1 peptide plus 10 nM CpG-ODN 1668 (●). Mock-vaccinated (□) mice served as control. Two (peptide) or 3 (MVA) weeks after vaccination animals were challenged with 2.5 × 10 6 PFU of WR and monitored daily for a 4-week period. The survival (%) of the animals was plotted. ( C and D ) Protective efficacy of vaccination in BALB/c mice. Groups of mice ( n = 10) were immunized i.m. with 10 4 (♦), 10 5 (■), 10 6 (▴), 10 7 (●), or 10 8 IU of MVA (−) ( C ) or 10 5 PFU of Wyeth (■) ( D ). Three weeks after vaccination animals were challenged intranasally with 1 × 10 6 PFU of WR. Individual animal weights were monitored daily and are expressed as means for each group. Mock-vaccinated (□) and mock-challenged (⋄) mice served as control.

    Article Snippet: Intracellular cytokine staining was performed for 30 min at 4°C by applying FITC-anti-IFN-γ (XMG1.2), FITC-anti-tumor necrosis factor α (MP6-XT22), or FITC-labeled IgG1 isotype control (R3-34) using Cytofix/Cytoperm (PharMingen/Becton Dickinson).

    Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry, Transgenic Assay

    Transgenic IL-15 in in vivo model. (A) CLL-1 expression of AML PDX model. (B) Schematic figure of AML PDX model. in the PDX model, mice received PDX cells intravenously (1.5×10 6 cells per mouse) followed 18–21 days later by a single injection of CLL-1 CAR T cells (2.5×10 6 cells per mouse). (C) Residual tumor cells (top) and number of T cells (bottom) at day 5 after CLL-1 CAR (n=8) CLL-1 CAR+ IL15 (n=5) T cell infusion. P value was determined by unpaired Student’s t-test. (D) Kaplan-Meier curve showing the survival of mice in each experimental group. (E) Serum human cytokine levels (mean±SEM) (TNF alpha, IL15, IL-2, IFN-gamma, IL-1a, IL-6, IL-10) of representative mice at day 7 after CLL-1 CAR (n=3) and CLL-1 +IL15 CAR (n=3) T cell infusion. P value was determined by unpaired Student’s t-test. (F) Histopathological examination of lung (top) and liver (bottom) tissues (H E, ×100) in a mouse died at day 4 after CLL-1+ IL15 CAR infusion. Hemorrhage, edema is marked with green and apoptosis is marked with black in lung. The necrosis of liver is marked with green. P values were determined by log-rank Mantel-Cox test. CAR, chimeric antigen receptor; CLL, C-type lectin-like molecule 1; IFN, interferon; IL-15, interleukin 15; Iso Ctrl, isotype control; NS, not significant; NTR, non-transduced; PDX, patient derived xenografts; TNF, tumor necrosis factor.

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Modulating TNFα activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia

    doi: 10.1136/jitc-2020-001229

    Figure Lengend Snippet: Transgenic IL-15 in in vivo model. (A) CLL-1 expression of AML PDX model. (B) Schematic figure of AML PDX model. in the PDX model, mice received PDX cells intravenously (1.5×10 6 cells per mouse) followed 18–21 days later by a single injection of CLL-1 CAR T cells (2.5×10 6 cells per mouse). (C) Residual tumor cells (top) and number of T cells (bottom) at day 5 after CLL-1 CAR (n=8) CLL-1 CAR+ IL15 (n=5) T cell infusion. P value was determined by unpaired Student’s t-test. (D) Kaplan-Meier curve showing the survival of mice in each experimental group. (E) Serum human cytokine levels (mean±SEM) (TNF alpha, IL15, IL-2, IFN-gamma, IL-1a, IL-6, IL-10) of representative mice at day 7 after CLL-1 CAR (n=3) and CLL-1 +IL15 CAR (n=3) T cell infusion. P value was determined by unpaired Student’s t-test. (F) Histopathological examination of lung (top) and liver (bottom) tissues (H E, ×100) in a mouse died at day 4 after CLL-1+ IL15 CAR infusion. Hemorrhage, edema is marked with green and apoptosis is marked with black in lung. The necrosis of liver is marked with green. P values were determined by log-rank Mantel-Cox test. CAR, chimeric antigen receptor; CLL, C-type lectin-like molecule 1; IFN, interferon; IL-15, interleukin 15; Iso Ctrl, isotype control; NS, not significant; NTR, non-transduced; PDX, patient derived xenografts; TNF, tumor necrosis factor.

    Article Snippet: Cytokine production by T-cells was measured by flow cytometry using Intracellular Cytokine Staining Kit (BD Biosciences).

    Techniques: Transgenic Assay, In Vivo, Expressing, Mouse Assay, Injection, Derivative Assay

    Ginseng enhances IFN-γ cytokine-producing lung cells from BALB/c mice. (A) IL-4 ELISpot. (B) IFN-γ ELISpot. Cytokine (IL-4 and IFN-γ) producing lung cells isolated at day 5 postinfection ( n =5 BALB/c mice per group) were determined

    Journal: Journal of Interferon & Cytokine Research

    Article Title: Ginseng Diminishes Lung Disease in Mice Immunized with Formalin-Inactivated Respiratory Syncytial Virus After Challenge by Modulating Host Immune Responses

    doi: 10.1089/jir.2013.0093

    Figure Lengend Snippet: Ginseng enhances IFN-γ cytokine-producing lung cells from BALB/c mice. (A) IL-4 ELISpot. (B) IFN-γ ELISpot. Cytokine (IL-4 and IFN-γ) producing lung cells isolated at day 5 postinfection ( n =5 BALB/c mice per group) were determined

    Article Snippet: Cells from BAL fluids were stimulated with phorbol myristate acetate (50 ng/mL) and ionomycin (500 ng/mL) for 4 h. After staining with surface antibodies (anti-CD3, CD45, CD4, CD8α, CD11b, CD11c, and Siglec F antibodies from eBiosciences), intracellular IFN-γ cytokine staining was followed by manufacturer's manuals (BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit).

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Isolation

    T cell subsets in spleen after immunization. A . Flow cytometry analysis strategies of T cell subsets in spleen, B and C . Levels of T cell subset are shown as percentages of cytokine producing cells (Interferon-γ, IL-17 and IL-4) per CD4+ T cells, and FoxP3 positive cells per CD4+ CD25+ T cells stimulated with AGE-LDL for each of the three groups (white bars = controls, bars with stripes = Alum adjuvant and black bars = AGE-LDL + Alum adjuvant, n = 10 per group). *indicates p

    Journal: Cardiovascular Diabetology

    Article Title: Immunization with advanced glycation end products modified low density lipoprotein inhibits atherosclerosis progression in diabetic apoE and LDLR null mice

    doi: 10.1186/s12933-014-0151-6

    Figure Lengend Snippet: T cell subsets in spleen after immunization. A . Flow cytometry analysis strategies of T cell subsets in spleen, B and C . Levels of T cell subset are shown as percentages of cytokine producing cells (Interferon-γ, IL-17 and IL-4) per CD4+ T cells, and FoxP3 positive cells per CD4+ CD25+ T cells stimulated with AGE-LDL for each of the three groups (white bars = controls, bars with stripes = Alum adjuvant and black bars = AGE-LDL + Alum adjuvant, n = 10 per group). *indicates p

    Article Snippet: Flow cytometry and intracellular cytokine staining Flow cytometry was performed on a FACS Calibur (BD, USA) after staining with appropriate antibodies; data were analyzed using Flowjo software (USA).

    Techniques: Flow Cytometry, Cytometry

    The effects of dietary fatty acids on hepatic cytokine expression, inflammation signaling and serum adipokines. Wild-type C57BL6 mice were fed diets enriched with certain fatty acids (see ) for 12 weeks. A: Hepatic IFNγ and IL-4 expressions

    Journal: Journal of Lipid Research

    Article Title: Dietary fatty acids modulate antigen presentation to hepatic NKT cells in nonalcoholic fatty liver disease [S]

    doi: 10.1194/jlr.M003004

    Figure Lengend Snippet: The effects of dietary fatty acids on hepatic cytokine expression, inflammation signaling and serum adipokines. Wild-type C57BL6 mice were fed diets enriched with certain fatty acids (see ) for 12 weeks. A: Hepatic IFNγ and IL-4 expressions

    Article Snippet: For intracellular staining of cytokines, we utilized an Intracellular Cytokine Staining Kit (Pharmingen).

    Techniques: Expressing, Mouse Assay

    SFA and MUFA diets selectively depleted hepatic NKT cells and alter NKT cell cytokine profile. Wild-type C57BL6 mice were fed diets enriched with certain fatty acids (see ) for 12 weeks. Hepatic mononuclear cells (HMNC) were isolated and NKT cells

    Journal: Journal of Lipid Research

    Article Title: Dietary fatty acids modulate antigen presentation to hepatic NKT cells in nonalcoholic fatty liver disease [S]

    doi: 10.1194/jlr.M003004

    Figure Lengend Snippet: SFA and MUFA diets selectively depleted hepatic NKT cells and alter NKT cell cytokine profile. Wild-type C57BL6 mice were fed diets enriched with certain fatty acids (see ) for 12 weeks. Hepatic mononuclear cells (HMNC) were isolated and NKT cells

    Article Snippet: For intracellular staining of cytokines, we utilized an Intracellular Cytokine Staining Kit (Pharmingen).

    Techniques: Mouse Assay, Isolation

    Enhanced efficacy of αDEC-205:OVA plus αCD40 relative to other immunization approaches. (A) C57BL/6 mice were immunized s.c. with several methods: Spleen DC pulsed ex vivo with 10 μg/ml each of αDEC-205:OVA and αCD40; 500 μg OVA in CFA; 50 μg OVA with 25 μg αCD40; 50 μg of SIINFEKL peptide with 25 μg αCD40; or 50 ng of OVA in αDEC-205:OVA with 25 μg of αCD40. 7 or 30 d later, lymph nodes were harvested and T cell expansion evaluated by K b -SIINFEKL–PE tetramer and CD62L staining. The gate for the y-axis was placed relative to the CD62L-negative tetramer binding cells in the right panel. Indicated percentages are percent of CD8 + lymphocytes. (B) As in A, but IFN-γ secretion evaluated by intracellular cytokine staining. Data are means of three experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: In Vivo Targeting of Antigens to Maturing Dendritic Cells via the DEC-205 Receptor Improves T Cell Vaccination

    doi: 10.1084/jem.20032220

    Figure Lengend Snippet: Enhanced efficacy of αDEC-205:OVA plus αCD40 relative to other immunization approaches. (A) C57BL/6 mice were immunized s.c. with several methods: Spleen DC pulsed ex vivo with 10 μg/ml each of αDEC-205:OVA and αCD40; 500 μg OVA in CFA; 50 μg OVA with 25 μg αCD40; 50 μg of SIINFEKL peptide with 25 μg αCD40; or 50 ng of OVA in αDEC-205:OVA with 25 μg of αCD40. 7 or 30 d later, lymph nodes were harvested and T cell expansion evaluated by K b -SIINFEKL–PE tetramer and CD62L staining. The gate for the y-axis was placed relative to the CD62L-negative tetramer binding cells in the right panel. Indicated percentages are percent of CD8 + lymphocytes. (B) As in A, but IFN-γ secretion evaluated by intracellular cytokine staining. Data are means of three experiments.

    Article Snippet: Cells were then harvested, stained for extracellular CD8, and then stained for cytokines with the BD intracellular cytokine staining starter kit (Becton Dickinson).

    Techniques: Mouse Assay, Ex Vivo, Staining, Binding Assay