15 kda recombinant granulysin  (R&D Systems)

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: Resting γδ T cells express granulysin. (a) Representative flow cytometry plots depicting the gating strategy used to identify cell populations of interest from peripheral blood mononuclear cells (PBMC) preparations. Lymphocytes were gated (G1) according to size (forward scatter; FSC) and granularity (side scatter; SSC). Within gate G1, CD8 + αβ T cells, natural killer (NK) cells and γδ T cells were further gated on using established lineage markers for these cells (G2–G4). (b) Following identification of each immune cell population, the percentage of cells expressing 15 000+ 9000 MW granulysin or 9000 MW granulysin was determined by flow cytometry (expression within γδ T‐cell populations depicted). (c–e) Percentage expression of 15 000+ 9000 MW granulysin, 9000 MW granulysin, and granzyme B within populations of CD8 + αβ T cells, NK cells and γδ T cells, as determined by flow cytometry. (f) Gating strategy used to differentiate between V δ 1 + γδ T cells and V δ 2 + γδ T cells. (g,h) Percentage expression of 15 000+ 9000 MW and 9000 MW granulysin within populations of V δ 1 + γδ T cells and V δ 2 + γδ T cells as determined by flow cytometry. Data shown are obtained from between six and ten independent experiments using PBMC from ten individual donors, with error bars (SD). Differences between groups were assessed by one‐way analysis of variance. * P < 0·05. *** P < 0·001 **** P < 0·0001.

Article Snippet: To test maturation, immature DC were treated for 24 hr with 100 ng/ml recombinant lipopolysaccharide (LPS) or 66 n m recombinant 15 000 MW granulysin (both R&D Systems).

Techniques: Flow Cytometry, Expressing

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: Stimulation of V δ 2 + γδ T cells with bacillus Calmette–Guérin (BCG) causes an increase in the intracellular expression of 9000 MW granulysin. (a–d) The percentage of V δ 2 + γδ T cells, natural killer (NK) cells and CD8 + T cells expressing early activation marker CD69, 15 000+ 9000 MW granulysin, granzyme B and 9000 MW granulysin following 24 hr of peripheral blood mononuclear cell (PBMC) stimulation with zoledronic acid (ZA), BCG or phorbol 12‐myristate 13‐acetate/ionomycin (PMA/I), as determined by flow cytometry. (e) The percentage of V δ 2 + γδ T cells expressing 9000 MW granulysin following either 24 hr of PBMC stimulation with BCG, or following isolation of this cell population from unstimulated PBMC, and subsequent stimulation of these isolated V δ 2 + γδ T cells for 24 hr. Data shown are mean values obtained from six independent experiments using PBMC from six individual donors, with error bars (SD). Statistics refer to the differences between treatment group and untreated group, and were assessed by one‐way analysis of variance. *** P < 0·001. **** P < 0.0001.

Article Snippet: To test maturation, immature DC were treated for 24 hr with 100 ng/ml recombinant lipopolysaccharide (LPS) or 66 n m recombinant 15 000 MW granulysin (both R&D Systems).

Techniques: Expressing, Activation Assay, Marker, Flow Cytometry, Isolation

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: V δ 2 + γδ T cells release granulysin in response to tumour. (a) The expression of exhaustion markers PD‐1 and Lag‐3 on, and the secretion of granulysin by, V δ 2 + γδ T cells during the 9‐day expansion process. (b) The percentage of V δ 2 + γδ T cells to express early activation marker CD69 following 24, 48 or 72 hr of culture with Daudi cells, Raji cells or Raji cells pre‐treated for 24 hr with 5 μ m zoledronic acid (ZA), as determined by flow cytometry. (c) The concentration of interferon‐ γ (IFN‐ γ ) found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (d) The percentage of V δ 2 + γδ T cells to express degranulation marker CD107a following 24, 48 or 72 hr of culture with Daudi cells, Raji cells or Raji cells pre‐treated for 24 hr with 5 μ m ZA, as determined by flow cytometry. (e) The concentration of granulysin found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (f) The concentration of granzyme B found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (g) Percentage killing of tumour cells by V δ 2 + γδ T cells following 24, 48 and 72 hr of culture, as determined by flow cytometry. Data shown are from six independent experiments, using V δ 2 + γδ T cells from six individual donors, with error bars (SD). Differences between groups were assessed by two‐way analysis of variance comparing negative control (V δ 2 + γδ T cells alone) with all other groups. * P < 0·05. ** P < 0·01. *** P < 0·001. **** P < 0·0001.

Article Snippet: To test maturation, immature DC were treated for 24 hr with 100 ng/ml recombinant lipopolysaccharide (LPS) or 66 n m recombinant 15 000 MW granulysin (both R&D Systems).

Techniques: Expressing, Activation Assay, Marker, Flow Cytometry, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: Granulysin can cause maturation of immature dendritic cells (DC). Changes in mean fluorescence intensity (MFI) of cell surface markers CD80, CCR7, CCR5 and HLA‐DR on monocyte‐derived immature DC following culture with 100 ng/ml lipopolysaccharide (LPS) or 66 n m recombinant 15 000 MW granulysin), as determined by flow cytometry. Treatment of cells with medium alone was included as a negative control. Data shown are the average taken from six individual donors. Differences between groups were assessed by two‐way analysis of variance comparing negative controls (pre‐maturation and medium alone) with all other groups. * P < 0·05. ** P < 0·01.

Article Snippet: To test maturation, immature DC were treated for 24 hr with 100 ng/ml recombinant lipopolysaccharide (LPS) or 66 n m recombinant 15 000 MW granulysin (both R&D Systems).

Techniques: Fluorescence, Derivative Assay, Recombinant, Flow Cytometry, Negative Control

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: Recombinant 15 000 MW granulysin causes differential migration of immature and mature dendritic cells (DC). (a) The migration of immature DC in response to 10 or 66 n m recombinant 15 000 MW granulysin, as determined by Ibidi μ‐migration assays. 500 ng/ml recombinant RANTES was used as a positive control of immature DC migration, while 2 ng/ml recombinant CCL19 was used as a negative control. (b) The migration of mature DC in response to 10 or 66 n m recombinant 15 000 MW granulysin, as determined by Ibidi μ‐migration assays. 500 ng/ml recombinant RANTES was used as a negative control of mature DC migration, whereas 2 ng/ml recombinant CCL19 was used as a positive control. Concentrations used for positive and negative controls were based on manufacturer’s recommendation. Data shown are from six independent experiments using immature and lipopolysaccharide‐matured DC differentiated from the monocytes of six individual donors, with error bars (SD). Differences between groups were assessed using one‐way analyses of variance. * P < 0·05. ** P < 0·01. *** P < 0·001. UN = untreated. GNLY = 15 000 MW granulysin.

Article Snippet: To test maturation, immature DC were treated for 24 hr with 100 ng/ml recombinant lipopolysaccharide (LPS) or 66 n m recombinant 15 000 MW granulysin (both R&D Systems).

Techniques: Recombinant, Migration, Positive Control, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis.

doi: 10.4049/jimmunol.1102563

Figure Lengend Snippet: FIGURE 4. Expression of cytotoxic granules’ components (mRNA and protein) by NK cells. (A–D) Analysis by qRT-PCR of the mRNA relative ex- pression for granzyme A (A), granzyme B (B), perforin (C), and granulysin (D) in NK cells isolated from peripheral blood of controls (n = 9) unstimulated (US), stimulated with IL-15 (5 ng/ml) or with P. brasiliensis yeast cells (strains Pb18 or Pb265; yeast cell/NK cell ratio 1:100) for 24 h. Results are expressed as mean 6 SEM of the relative expression determined as described in Materials and Methods. Statistical analysis: ANOVA for repeated measures with Bonferroni posttest. *p # 0.05 (in relation to unstimulated cells), #p # 0.05 (in relation to IL-15–stimulated cells). (E–H) Flow cytometry analysis of the expression of granzyme A (E), granzyme B (F), perforin (G), and granulysin (H) in NK cells isolated from peripheral blood of controls (n = 10) and PCM patients (n = 7) unstimulated (US), stimulated with IL-15 (5 ng/ml) or with P. brasiliensis yeast cells (strains Pb18 or Pb265; yeast cell/NK cell ratio 1:100) for 24 h. Results are expressed as mean 6 SEM of the percentage of positive cells for each parameter. Statistical analysis: ANOVA for repeated measures with Bonferroni posttest. #p # 0.05 (in relation to unstimulated cells), *p # 0.05 (in relation to NK cells from healthy individuals). (I) Dosage of granulysin in supernatants of culture of NK cells from controls (C) or PCM patients supplemented with recombinant IL-15 (5 ng/ml). The values are expressed as micromolars of granulysin. Values represent the mean 6 SEM. Statistical analysis: paired t test. p values are shown in the graphics. (J) Analysis of the effect of recombinant granulysin on P. brasiliensis yeast cells (Pb18 or Pb265 strain). Yeast cells were incubated with the indicated concentrations of granulysin at 37˚C for 48 h. After incubation, the samples were diluted 1:100 with distilled water and spread on BHI agar plates, and after 5–10 d the number of CFUs was determined. The results shown are represented as mean 6 SEM of three independent experiments. (K) Representative image of Western blotting analysis of supernatants from IL-15–stimulated NK cells untreated (Ctrl) or depleted of granulysin by immunoprecipitation (GNL-D). Membranes were labeled with specific Abs against granulysin, perforin, granzyme B, and actin. (L) Number of CFU/ml of P. brasiliensis (strain Pb18) cultured for 48 h with supernatants from IL-15–stimulated NK cells untreated (Ctrl) or depleted of granulysin by immunoprecipitation (GNL-D) or RPMI. Results expressed as mean 6 SEM of the number of CFU/ml. Statistical analysis: ANOVA test with Bonferroni posttest. *p # 0.05 (in relation to the GNL-D supernatants or RPMI). (M–P) Immunohistochemical analysis of granulysin+ cells (M), granzyme B+ cells (N), and perforin+ cells (O) (brown-stained cells) in a representative biopsy lesion from a patient with PCM. Cells were stained using specific mAbs as described in the Materials and Methods. Note that positive cells were found surrounding P. brasiliensis yeast cells (arrows). (P) Representative result of a control slide (without the primary Ab). Original magnification (M–P) 31000.

Article Snippet: The dosage of granulysin in supernatants of NK cells cultured with P. brasiliensis yeast cells stimulated or not with rhIL-15 was performed by ELISA as previously described (18), and the results are expressed as micromolars of granulysin determined by comparison with a standard curve made of recombinant human granulysin (R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Recombinant, Incubation, Western Blot, Immunoprecipitation, Labeling, Cell Culture, Immunohistochemical staining, Staining, Control