Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: Representative immunohistochemical streptavidin-peroxidase staining in extranodal natural killer/T-cell lymphoma (upper row) and rhinitis tissues (lower row). The positive cases of (A and D) programmed death 1, (B and E) PD-L1 and (C and F) PD-L2 (magnification, ×200). PD-L, programmed death ligand.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Immunohistochemical staining, Staining

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: PD-1 expression in (A) CD4 + and (B) CD8 + T-cell subsets in 20 ENKL patients was significantly increased compared with that in 10 HVs (P<0.05). Representative PD-1 expression in (C) CD4 + and (D) CD8 + T-cell subsets in six ENKL patients was (E) downregulated with chemotherapy. (F) T-helper cell type 1 cytokine (IL-2 and IFN-γ) mean production levels in the serum of 20 ENKL patients were significantly lower than those in 10 HVs (P<0.05). PD1, programmed death 1; ENKL, extranodal natural killer/T-cell lymphoma; HVs, healthy volunteers; IL-2 interleukin 2; IFN-γ, interferon γ.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: (A) Purity of CD8 + T cells separated by magnetic-activated cell sorting was 99%. (B) Purity of CD8 + PD-1 + T cells was 96.2% following the stimulation of allogeneic CD8 + T cells with phytohemagglutinin for 48 h. (C) SNK-6 cells were used as the control group and, following the coculture of SNK-6 cells and CD8 + T cells for 72 h, a significant inhibitory effect of PD-L1 on allogeneic CD8 + T-helper type 1 cytokine (IL-2 and IFN-γ) secretion was observed; (A and B) P<0.05. (D) CD8 + T-cell apoptosis in groups A and B was not altered significantly compared with activated CD8 + T cells at 72 h (P>0.05). (E) SNK-6 cells were used as the control group and cells harvested at 0, 24, 48 and 72 h were analyzed by flow cytometry gating CFSE + events. The proliferation index was not significantly different among the groups (P>0.05). PD-1, programme death 1; PD-L. programmed death ligand; IL-2 interleukin 2; IFN-γ, interferon γ; CFSE, carboxy-fluorescein succinimidyl ester.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: FACS, Flow Cytometry

Journal:

Article Title: Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

doi: 10.1093/emboj/cdg092

Figure Lengend Snippet: Fig. 3. Identification of gG as the vCKBP encoded by alphaherpesviruses. (A) Cross-linking with EGS of [125I]CXCL8 to supernatants from insect cell cultures infected with recombinant baculoviruses expressing the indicated ORF from EHV-1 strain AB4. The baculovirus clone number is shown in parenthesis. (B) Cross-linking with EGS of [125I]CXCL8 or [125I]CCL19 to supernatants from insect cell cultures infected with the indicated recombinant baculoviruses expressing full-length or secreted versions (S) of gG encoded by EHV-1, BHV-1, BHV-5, HSV-1 and HSV-2. Samples were analysed by SDS–PAGE and autoradiography. Molecular masses are in kilodaltons and the positions of 125I-labelled chemokine (CK) and vCKBP-chemokine complexes (asterisks) are indicated.

Article Snippet: Recombinant His-tagged human GITRL and FasL were from R&D Systems.

Techniques: Infection, Recombinant, Expressing, SDS Page, Autoradiography

Journal:

Article Title: Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

doi: 10.1093/emboj/cdg092

Figure Lengend Snippet: Fig. 6. Secreted gG from EHV-1 and BHV-1 expressed in the baculovirus system inhibits chemokine binding to cells in a dose-dependent manner. Binding assay of [125I]CCL3 and [125I]CXCL8 to U937 cells in the absence (solid triangles) or presence of increasing amounts of supernatants from Sf21 cells infected with recombinant baculovirus expressing full-length gG from either EHV-1 (B and D) or BHV-1 (A and C) (solid squares), or with control baculovirus (AcNPV) (open squares). The dose of supernatant is expressed as cell equivalents. Binding specificity was determined in the presence of a 500-fold excess of unlabelled CCL3 or CXCL8 (open circles). Purified M3 protein was used as a positive control (open triangles). Binding of chemokines is expressed as the mean ± SD of triplicate assays.

Article Snippet: Recombinant His-tagged human GITRL and FasL were from R&D Systems.

Techniques: Binding Assay, Infection, Recombinant, Expressing, Purification, Positive Control

Journal:

Article Title: Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

doi: 10.1093/emboj/cdg092

Figure Lengend Snippet: Fig. 7. Inhibition of chemokine biological activity by gG. (A) Chemokine-induced calcium flux. The indicated amount of purified BHV-1 gG.His was pre-incubated with 75 ng of CXCL1 before addition to Indo-1-loaded U937 cells. Similarly, purified BHV-5 gG. His was pre-incubated with 75 ng of CCL3 before addition to Indo-1-loaded HeLa cells transfected with CCR5 and CXCR4. Intracellular calcium mobilization was measured by FACS analysis. (B) Chemokine-induced chemotaxis in a Transwell migration assay. The top panel shows the migration of human neutrophils in response to 100 ng/ml CXCL8 in the presence of supernatants, expressed as cell equivalents, from insect cells infected with a recombinant baculovirus expressing full-length EHV-1 gG. The middle panel shows chemotaxis of human neutrophils induced by 100 ng/ml CXCL1 in the presence of increasing doses of purified BHV-1 gG.His. The bottom panel shows migration of U937 cells in response to 100 ng/ml CCL3 in the presence of purified BHV-5 gG.His. Supernatants from AcNPV-infected insect cells or an irrelevant purified His-tagged VV protein (A40R.His) were used as negative controls. Leukocytes migrating in duplicate assays are expressed as the percentage (mean ± SD) of the total cells added or as the number of cells counted (mean ± SD) in four high powered fields.

Article Snippet: Recombinant His-tagged human GITRL and FasL were from R&D Systems.

Techniques: Inhibition, Activity Assay, Purification, Incubation, Transfection, Chemotaxis Assay, Transwell Migration Assay, Migration, Infection, Recombinant, Expressing

Journal:

Article Title: Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

doi: 10.1093/emboj/cdg092

Figure Lengend Snippet: Fig. 8. Inhibition of chemokine–heparin interactions by purified gG. (A and B) FlashPlates precoated with heparin–BSA were incubated with [125I]CXCL8 in the absence or presence of purified gG from EHV-1 (EHV-1gG.His), BHV-1 (BHV-1gG.His) or BHV-5 (BHV-5gG.His), and the bound radio-activity (mean ± SD of duplicate samples) was determined. FlashPlates coated with BSA alone (BSA) were also tested to determine background binding. Increasing doses (10, 50 and 150 ng per well) of murine TNF (mTNF), human IgG, His-tagged Fas ligand (FasL) or His-tagged glucocorticoid-induced TNFR-superfamily-related protein ligand (GITRL) were also tested. (C and D) FlashPlates precoated with heparin–BSA were pre-incubated with [125I]CXCL1 or [125I]CCL2. Purified recombinant EHV-1 gG (300 ng per well) or medium was added to the wells and the bound radiolabelled chemokine (mean ± SD of triplicate samples) was determined at the indicated times. Background binding to BSA alone was subtracted.

Article Snippet: Recombinant His-tagged human GITRL and FasL were from R&D Systems.

Techniques: Inhibition, Purification, Incubation, Activity Assay, Binding Assay, Recombinant

Journal:

Article Title: Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

doi: 10.1093/emboj/cdg092

Figure Lengend Snippet: Fig. 9. Chemokine binding activity expressed at the surface of recombinant baculovirus-infected insect cells. Sf21 insect cells were infected with AcEHV-1gG or AcEHV-1gGs for 24 h and incubated for 30 min at room temperature with the indicated chemokines in suspension. The bound chemokine was determined. The binding difference between AcEHV-1gG and AcEHV-1gGs was observed three times for CXCL8 and twice for CXCL1.

Article Snippet: Recombinant His-tagged human GITRL and FasL were from R&D Systems.

Techniques: Binding Assay, Activity Assay, Recombinant, Infection, Incubation, Suspension