Journal: Cancer immunology research

Article Title: Mouse PVRIG Has CD8 + T Cell–Specific Coinhibitory Functions and Dampens Antitumor Immunity

doi: 10.1158/2326-6066.CIR-18-0460

Figure Lengend Snippet: PVRIG-deficient mice have increased T-cell function. A, RNA expression of PVRIG was assessed by qRT-PCR from purified mouse immune-cell subsets. Relative expression of the housekeeping gene, RPL18, was determined by the ΔCT method. One of two experiments performed is represented here. B, Pmel-1 CD8+ TCR transgenic T cells were activated with gp100 (25–33) peptide. PVRIG and TIGIT RNA transcripts were analyzed by qRT-PCR at the indicated time points. The graph shows mean ± SEM of results from five different experiments. C, Spleens were harvested from PVRIG−/− and wild-type littermates and analyzed by FACS for expression of PVRIG on CD8+ T and NK cells ex vivo (“resting” cells). In addition, CD3+ splenic T cells were activated for 11 days with anti-CD3/anti-CD28 beads. Following the activation, PVRIG expression on CD4+ and CD8+ T cells (“activated” cells) was analyzed by flow cytometry. Cells were gated based on viability-dye exclusion followed by gating on CD3−/CD49b+, CD3+/CD4+, and CD3+/CD8+ for acquisition of NK, CD4+ T, and CD8+ T cells. Each dot represents cells derived from an individual mouse. D, Wild-type and PVRIG−/− splenocytes were labeled with Cell Proliferation Dye eFluor450 and cultured in the presence of control Fc (mouse IgG2a) or with mouse PVRL2-Fc. After 4 days of culture, cell division was analyzed by flow cytometry. Representative FACS plots from one experiment (left) and the summary of percentage inhibition by PVRL2 Fc (% inhibition of proliferation control Fc - PVRIG Fc) from three independent experiments (right) are presented. *, P < 0.05, paired Student t test for the change in proliferation in the presence of PVRL2-Fc relative to proliferation in the presence of protein control in wild-type versus PVRIG−/− T cells. E, Pmel-1 CD8+ T cells derived from Pmel-1 PVRIG−/− or Pmel-1 wild-type mice were activated for 11 days with their cognate peptide and IL2. Activated Pmel-1 CD8+ T cells were then cocultured with B16-Db/gp100 cells for 18 hours and evaluated for CD107 expression and for cytokine production. Four independent experiments are presented as indicated by each paired dot. *, P < 0.05, Student t test comparing PVRIG−/− versus wild-type.

Article Snippet: For ELISA experiments, recombinant mouse PVRL2 HIS protein (Sino Biological) was diluted to 2 μg/mL and coated onto an EIA/RIA plate (Costar) overnight at 4°C.

Techniques: Cell Function Assay, RNA Expression, Quantitative RT-PCR, Purification, Expressing, Transgenic Assay, Ex Vivo, Activation Assay, Flow Cytometry, Derivative Assay, Labeling, Cell Culture, Inhibition

Journal: Translational Oncology

Article Title: Novel IL-15 dendritic cells have a potent immunomodulatory effect in immunotherapy of multiple myeloma

doi: 10.1016/j.tranon.2022.101413

Figure Lengend Snippet: Antibodies used in Flow cytometry analysis

Article Snippet: CD112-PE , R2.525 , Miltenyi Biotec (CA,USA).

Techniques: Flow Cytometry, Control, Staining

Journal: Translational Oncology

Article Title: Novel IL-15 dendritic cells have a potent immunomodulatory effect in immunotherapy of multiple myeloma

doi: 10.1016/j.tranon.2022.101413

Figure Lengend Snippet: IL-15 mDCs showed the enhanced cytotoxicity by generation of potent autologous cytotoxic T lymphocytes, CIK cells, and NK cells against primary autologous myeloma cells (paired). (A) We analyzed CD138 + primary myeloma cells isolated from bone marrow aspiration samples of newly diagnosed multiple myeloma patients to assess the expression levels of MHC class I, MICA, MICB, ULBP1, ULBP2/5/6, HLA-E, CD38 (n = 12), and CD112, CD155, ICAM-1 and PD-L1 (n = 5) using flow cytometry. ( B) We measured the expression of the degranulation marker CD107a in CTLs, CIK cells, and NK cells generated by mDCs against primary autologous myeloma cells. ( C) The killing capacity of activated lymphocytes by mDCs against primary autologous myeloma cells was investigated using an LDH-release cytotoxicity assay without or with anti-MHC class I mAb (n = 3). The data indicate that CLTs, CIK cells, and NK cells generated by the IL-15 mDCs (6 days) and IL-15 mDCs (8 days) groups exhibited stronger cytotoxicity than those of the conventional mDCs group. Data are representative from three independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 (One-way ANOVA, multiple comparisons test: Tukey for B. Multiple t tests – one per row for C).

Article Snippet: CD112-PE , R2.525 , Miltenyi Biotec (CA,USA).

Techniques: Isolation, Expressing, Flow Cytometry, Marker, Generated, Cytotoxicity Assay

Journal: Cell Death & Disease

Article Title: Deciphering the tumor immune microenvironment of imatinib-resistance in advanced gastrointestinal stromal tumors at single-cell resolution

doi: 10.1038/s41419-024-06571-3

Figure Lengend Snippet: a Cell communication analysis on TIGIT-NECTIN2 and BTLA-TNFRSF14 pair between different cell types in imatinib resistant and sensitive patients respectively. Expression of NECTIN2 ( b ), BTLA ( c ) and TNFRSF14 ( d ) in each cell type in imatinib resistant and sensitive patients respectively. e IHC analysis of NECTIN2, BTLA and TNFRSF14 between imatinib resistant (upper) and sensitive (bottom) patients. f Schematic diagram of the unique tumor-immune microenvironment of imatinib-resistance in advanced GIST.

Article Snippet: Finally, two experienced pathologists independently evaluated staining results for Foxp3 (Servicebio, GB11093), BTLA (Boster, A03149), TNFRSF14 (Affinity, AB2838686) and NECTIN2 (Boster, A08081-2).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Characterization of a novel anti-PVRIG antibody with Fc-competent function that exerts strong antitumor effects via NK activation in preclinical models

doi: 10.1007/s00262-024-03671-z

Figure Lengend Snippet: Binding affinity and ligand blocking activity of IBI352g4a. a Affinity of BMK and IBI352g4a for human PVRIG and cynoPVRIG assayed using FortéBio. b Human PVRIG cell-based binding was conducted for BMK and IBI352g4a using the GS-CHO/hPVRIG stable cell line. c CynoPVRIG cell-based binding was conducted for BMK and IBI352g4a using the GS-CHO/cynoPVRIG stable cell line. d ELISA-based human PVRIG/PVRL2 blocking assay. Biotinylated human PVRL2 cells were incubated with increasing amounts of IBI352g4a or BMK in precoated human PVRIG plates. After incubation and washing, biotinylated human PVRL2 was detected via streptavidin (HRP). e ELISA-based cynoPVRIG/PVRL2 blocking assay. CynoPVRL2 protein (his tag) was incubated with increasing amounts of IBI352g4a or BMK in precoated cynomolgus PVRIG plates. After incubation and washing, the expression of cynomolgus monkey PVRL2 was detected with an HRP-conjugated antibody

Article Snippet: After overnight incubation at 4 °C, the plates were blocked with PBS containing 1% BSA and 0.05% Tween-20 for 1 h. Serially diluted antibody and 2 μg/mL cynomolgus PVRL2 His protein (#90,206-C08H SinoBiological) were added to the plates and incubated for 3 h at room temperature.

Techniques: Binding Assay, Blocking Assay, Activity Assay, Stable Transfection, Enzyme-linked Immunosorbent Assay, Incubation, Expressing