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

Journal: PLoS Pathogens

Article Title: Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

doi: 10.1371/journal.ppat.1003501

Figure Lengend Snippet: (a). Representative flow cytometry histograms of IFNγ-producing (upper panels) and perforin-producing Vγ2Vδ2 T effector cells gated on CD3 at day 8 postinfection and graph data (right) of numbers of Vγ2Vδ2 T effector cells in BALF collected overtime from Picostim/IL-2-treated and control groups. Effector cells were measured by ICS after HMBPP stimulation. n = 9/group for IFNγ effectors; n = 7/group for perforin effectors. * and ** denote p<0.05 and p<0.01, respectively, when analyzed by ANOVA or student t test. Effector cells measured by direct ICS without HMBPP stimulation was shown in Fig. S3a in . (b) Representative in situ confocal microscopic images (63× NA) of Vγ2Vδ2 T effector cells producing perforin and granulysin in lung tissue sections. The bottom two panels show that Vγ2 TCR (green) appear to co-localize with perforin (purple) and granulysin (red) in the merge images(co-localization marked by arrows) in lung tissue sections, suggesting that Vγ2 cells co-produce perforin and granulysin. Images at the top and middle panels show that no Vγ2 or granulysin was detectable in the lung tissue sections from representative macaques treated with saline or IL-2 alone, although perforin was detected in sections from IL-2-treated macaques. 10 um bars are indicated in images. All tissues sections were prepared from right middle and caudal lung lobes. Vγ2 cells were temporarily interpreted as Vγ2Vδ2 T cells as our previous studies demonstrated that Picostim or HMBPP/IL-2 exclusively expanded Vγ2Vδ2 T cells in vivo and most γδ T cells in lungs during Mtb infection were Vγ2Vδ2 T cells. Control isotype IgG did not give rise to any immune staining in the lung TB tissue sections (data not shown). See Sup Fig. 3b for additional confocal microscopic images of other macaques.

Article Snippet: Sections were incubated for 1 hr in dark with FITC-conjugated anti-human TCR Vγ2 mAb (clone 7A5, Thermo), 1∶10 diluted biotinylated anti-human perforin mAb (clone pf-344, MABTECH AB) and PE-conjugated anti-human granulysin mAb (clone DH2, Novus Biologicals).

Techniques: Flow Cytometry, Control, In Situ, Saline, In Vivo, Infection, Staining

Journal: PLoS Pathogens

Article Title: Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

doi: 10.1371/journal.ppat.1003501

Figure Lengend Snippet: (a). Shown on left are real time quantitative PCR data for mRNA expression levels of perforin and granulysin expressed by macaque Vγ2Vδ2 T effector cells capable of restricting intracellular Mtb growth. The quantitation was done using the as we previously described . Data are means with SEM from phosphoantigen-activated Vγ2Vδ2 T effector cells from 3 Mtb-infected macaques in 2 independent experiments. Control B cells were purified using CD20 mAb and immunemagnetic beads . Right panel shows that co-culturing of Mtb-infected monocytes with Vγ2Vδ2 T effector cells expressing perforin/granulysin led to reduction in CFU bacterial counts. (b). Graph curves show that recombinant granulysin(Gnl), but not perforin(PC), killed extracellular Mtb (left); Gnl+PC inhibited intracellular Mtb growth(right). Data are the killing or inhibiting percentage of extracellular or intracellular Mtb CFUs relative to Mtb CFUs in medium-only control(see ). Data are means derived from Mtb-infected monocytes from three macaques in 3 experiments. ** p<0.001; ***p<0.0001; ****p<0.00001, by student t test.

Article Snippet: Sections were incubated for 1 hr in dark with FITC-conjugated anti-human TCR Vγ2 mAb (clone 7A5, Thermo), 1∶10 diluted biotinylated anti-human perforin mAb (clone pf-344, MABTECH AB) and PE-conjugated anti-human granulysin mAb (clone DH2, Novus Biologicals).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay, Infection, Control, Purification, Recombinant, Derivative Assay

Journal: bioRxiv

Article Title: Blinatumomab-driven T-cell activation in αβ and γδ T-cell subsets: Insights from in vitro assays§

doi: 10.1101/2025.11.17.688178

Figure Lengend Snippet: A: BCP-ALL cells in the presence (red) or absence (grey) of BLN for three days were co-cultured with freshly isolated γδ, CD4⁺ and CD8⁺ αβ T cells/ T- and B-cell frequencies in the coculture were assessed by staining for CD3 and CD19, as exemplary shown for CD8⁺ αβ T cells. B: The release of IFNγ, GrzB and Prf into supernatants of cocultures in the presence (red) or absence (grey) of BLN was assessed via ELISA after three days, and the release of Gnly was analyzed after seven days. Data includes 4 to 7 healthy donors and * indicates p value between 0.01-0.05, ** indicates p value between 0.01-0.001 (paired and unpaired T-test).

Article Snippet: Cell culture supernatants were analyzed for Interferon-γ (IFNγ; DIF50C), Granzyme B (GrzB; DGZB00), Perforin (QK8011), Granulysin (Gnly; DY3138), Perforin (Prf; QK8011) and soluble Fas-Ligand (sFAS-L; DFL00B) using sandwich ELISA kits from R&D Systems, Minneapolis, USA according to the manufacturer’s instructions.

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