Journal: Nature immunology

Article Title: γδ T cells suppress Plasmodium falciparum blood stage infection by direct killing and phagocytosis

doi: 10.1038/s41590-020-00847-4

Figure Lengend Snippet: a , Frequency of circulating lymphocytes from 8 healthy donors (HD) and 8 P. falciparum patients (Pf). CD3 + CD4 + (CD4), CD3 + CD8 + (CD8), CD3 + TCRδ2 + (γδ2), CD3 − CD56 + (NK). b , c , Circulating γδ2 T cell activation in 8 HD and 5 Pf was evaluated by CD69 and CD16 staining. d , Intracellular granulysin (GNLY), perforin (PFN) and granzyme B (GzmB) in 8 HD and 8 Pf. e , CFSE-stained iRBC were co-cultured with fresh purified γδ2 T cells from 3 HD and 3 Pf at indicated E:T ratios for 12 hr and iRBC lysis was assessed by flow cytometry analysis of the number of viable CFSE + cells relative to added counting beads. f , Plasma GNLY concentration in 16 HD and 22 Pf patients by GNLY ELISA. Statistical analysis was performed by two-tailed nonparametric unpaired t-test ( a,f ), two-way ANOVA with Sidak’s multiple comparisons test ( d ), and area under the curve, followed by two-tailed nonparametric paired t-test, followed by two-tailed nonparametric paired t-test ( e ). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01, ***<0.001, ****<0.0001.

Article Snippet: GNLY in plasma from HD and Pf patients was quantified using the Human Granulysin DuoSet ELISA (R&D Systems).

Techniques: Activation Assay, Staining, Cell Culture, Purification, Lysis, Flow Cytometry, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature immunology

Article Title: γδ T cells suppress Plasmodium falciparum blood stage infection by direct killing and phagocytosis

doi: 10.1038/s41590-020-00847-4

Figure Lengend Snippet: a , PBMCs were stained with the Live/Dead viability dye and antibodies to CD3, CD4, CD8, TCRδ2 and CD56. Single live cells were gated on SSC-A vs FSC-A and dead cells were excluded. Lymphocyte subpopulations were gated as: CD3 + CD4 + (CD4 + T cells), CD3 + CD8 + (CD8 + T cells), CD3 + TCRδ2 + (γδ2 T cells) and CD3 − CD56 + (NK). b, PBMCs co-stained for CD69, CD16 and cytotoxic effector protein expression (GNLY, PFN and GzmB) were gated on γδ2 T cells.

Article Snippet: GNLY in plasma from HD and Pf patients was quantified using the Human Granulysin DuoSet ELISA (R&D Systems).

Techniques: Staining, Expressing

Journal: Nature immunology

Article Title: γδ T cells suppress Plasmodium falciparum blood stage infection by direct killing and phagocytosis

doi: 10.1038/s41590-020-00847-4

Figure Lengend Snippet: a-c , PBMC from HD were evaluated ex vivo (day 0) and after co-culture with uRBCs or iRBCs for 7 days. Shown are representative flow cytometry histograms (left) and percent positive cells for 4 HD samples (right) after staining for cell surface CD3 + CD4 + (CD4), CD3 + CD8 + (CD8), CD3 + TCRδ2 + (γδ2), CD3 − CD56 + (NK) and intracellular GNLY ( a ), PFN ( b ) or GzmB ( c ). The vertical gating lines were drawn based on the unstained sample. d , e , γδ2 T cell activation before and during co-culture with uRBCs or iRBCs was evaluated by CD69 and CD16 staining, n=3 independent donor samples. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons test ( a-c ) and two-way ANOVA with Tukey’s multiple comparisons test ( d-e ). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01,***<0.001, ****<0.0001. Data shown are representative of three independent experiments.

Article Snippet: GNLY in plasma from HD and Pf patients was quantified using the Human Granulysin DuoSet ELISA (R&D Systems).

Techniques: Ex Vivo, Co-Culture Assay, Flow Cytometry, Staining, Activation Assay

Journal: Nature immunology

Article Title: γδ T cells suppress Plasmodium falciparum blood stage infection by direct killing and phagocytosis

doi: 10.1038/s41590-020-00847-4

Figure Lengend Snippet: a-c , RBCs, incubated with indicated combinations of cytotoxic effector proteins (n=3), were assayed for RBC lysis by LDH release ( a ), parasite reinvasion into fresh RBCs by flow cytometry ( b ), and parasite morphology by electron microscopy ( c,d ). ( c ) quantifies prominent morphological alterations in EM images - parasitophorous vacuole (PV) detachment (black arrows) and increased parasite vacuolization (red arrows) in 15 images from 3 sections. Parasite nucleus (N), food vacuole (FV), hemozoin (Hz) are labeled in untreated iRBCs. e, uRBCs or unsynchronized iRBCs, stained with 25-NBD or filipin as cholesterol probes and Hoechst for parasite DNA, were analyzed by imaging flow cytometry using DNA staining to gate for different parasite stages (R ring, T trophozoite, S schizont). BF, bright field, H, Hoechst dye. f , Representative histograms (top) and quantification of multiple samples (bottom) of AF647-GNLY binding to uRBCs and iRBCs (trophozoite stage) by flow cytometry (n=5). g,h , GzmB-AF488 internalization in the absence or presence of GNLY or PFN assessed by imaging flow cytometry (n=3). ( g ) shows GzmB-AF488 (Gzm) internalization in representative images and ( h ) shows the proportion of RBCs at different parasite stages that stained for GzmB in multiple experiments, n=3. i, Representative images (left) and quantification (right) of imaging flow cytometry showing RBC, uninfected or infected at indicated parasite stage, incubated with GzmB-AF488, GNLY-AF647 and unlabeled GNLY, n=3. M, merozoite; R, ring; T, trophozoite; S, schizont; H, Hoechst dye; BF, bright field; n, biological independent samples. Scale bars: 500 nm ( d ), 7 μm ( e,g,i ). Statistical analysis was by one-way ANOVA with Tukey’s multiple comparisons test ( a,b,c,h,i ) or two-tailed nonparametric unpaired t-test ( f ). Mean ± s.e.m. is shown. P value: *<0.05, **<0.01, ***<0.001, ****<0.0001. Data shown are representative of three independent experiments.

Article Snippet: GNLY in plasma from HD and Pf patients was quantified using the Human Granulysin DuoSet ELISA (R&D Systems).

Techniques: Incubation, Red Blood Cell Lysis, Flow Cytometry, Electron Microscopy, Labeling, Staining, Imaging, Binding Assay, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: γδ T cells suppress Plasmodium falciparum blood stage infection by direct killing and phagocytosis

doi: 10.1038/s41590-020-00847-4

Figure Lengend Snippet: a , Electron microscopy of a ring stage iRBC treated or not with GzmB, PFN and GNLY showing disruption of morphology after treatment (right). In ( a ), parasitophorous vacuole (PV) detachment is indicated by a black arrow and increased parasite vacuolization by a red arrow. Parasite nucleus (N), hemoglobin vacuole (Hb), hemozoin (H). b , Imaging flow cytometry gating strategy for parasite developmental stages based on DNA content by Hoechst staining. M merozoites, R rings, T trophozoites, S schizonts. c, GzmB-AF488 uptake in the presence or absence of GNLY. Scale bar: 500 nm ( a ). n, biological independent samples. Data shown are representative of at least three independent experiments.

Article Snippet: GNLY in plasma from HD and Pf patients was quantified using the Human Granulysin DuoSet ELISA (R&D Systems).

Techniques: Electron Microscopy, Disruption, Imaging, Flow Cytometry, Staining

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

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: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Establishment and Validation of the Ascore Prognostic Signature. A Multivariate Cox coefficients for four ARGs (CERCAM, EMP1, GNLY, PTPRR) in the prognostic signature. B Ascore distribution among BLCA patients, sorted from lowest to highest. C Survival status categorized by Ascore for each BLCA patient. D Heatmap displaying expression levels of four genes in different Ascore groups. E Sankey diagram correlating clusters, Ascore groups, and BLCA survival status. F Kaplan–Meier analysis comparing overall survival between high and low Ascore groups in BLCA ( P < 0.0001). G Receiver Operating Characteristic (ROC) curves depicting Ascore signature’s predictive performance for 1, 3, and 5-year overall survival in BLCA, with the Area Under the Curve (AUC) values of 0.709, 0.724, and 0.745, respectively. (H–K) Kaplan–Meier analysis and time-dependent ROC curves in two external validation sets: GSE32548 and GSE32894

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: Biomarker Discovery, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Single-Cell RNA Sequencing Analysis of Ascore Distribution and Biological Significance in Bladder Cancer. A t-SNE plot showing seven main cell types distribution in the integrated dataset, with doublets manually annotated. B Dot plot of marker genes' expression levels in each cell type. C Ascore and four genes (CERCAM, EMP1, GNLY, PTPRR) expression and distribution across cell types. D t-SNE plot showing Ascore expression levels and patterns in each cell type. E Left plot: Six main epithelial cell subgroups visualized by t-SNE dimensionality reduction. Right plot: Ascore distribution and expression in epithelial cells, highlighting Subgroup0 and Subgroup2. F GO analysis of biological function differences between Subgroup0 and Subgroup2

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: RNA Sequencing, Marker, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Ascore Predictive Capability for Anti-PD-1 Immunotherapy Response in Gulou-Cohort2. A Representative immunohistochemistry (IHC) images illustrating the expression of four key genes (CERCAM, EMP1, GNLY, PTPRR) in two patients from Gulou-Cohort2 (Scale bars = 100 μm). Patient 4, who responded to anti-PD-1 therapy, had a low Ascore, in contrast to non-responder Patient 7, who had a high Ascore. B Distribution of Ascores among different response groups (CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease; *** P < 0.001). C ROC curves comparing the predictive accuracy of Ascore (AUC = 0.913) versus PD-L1 expression in tumor-infiltrating immune cells (ICs) (AUC = 0.662). D Decision curve analysis (DCA) indicating the net benefit of using Ascore compared to evaluating ICs' PD-L1 expression. E Kaplan–Meier curves c showing a correlation between higher Ascore values in tissue samples and reduced survival rates ( P = 0.0194)

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: Immunohistochemistry, Expressing

Journal: Vaccine

Article Title: Ag85A-specific CD4 + T cell lines derived after boosting BCG-vaccinated cattle with Ad5-85A possess both mycobacterial growth inhibition and anti-inflammatory properties

doi: 10.1016/j.vaccine.2018.03.068

Figure Lengend Snippet: Evaluation of transcription of cytotoxic genes in BCG-activated Ag85A-specific CD4 + T cell lines derived from BCG-primed Ad5-85A boosted cattle. Ag85A-specific CD4 + T cell lines derived from BCG-vaccinated cattle, both pre- and post- boosting with Ad5-85A, were activated with BCG-infected CD14 + cells for six days as described in materials and methods. RNA was extracted from Ag85A-specific CD4 + T cells at day zero and day six after BCG-stimulation and analysed for bovine perforin, granulysin and granzyme A gene expression. Due to restrictions in the number of cells available for each cell line, it was possible to carry out transcription analysis for six pre-boost cell lines derived from two animals (two from one animal and four from the other) and for four post-boost cell lines derived from three animals (two from one animal and one from each of the remaining animals). Data shown are for transcription observed at day six relative to transcription observed at day zero.

Article Snippet: Granulysin , Bt03287460_g1.

Techniques: Derivative Assay, Infection, Gene Expression