Journal: Frontiers in Immunology

Article Title: IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer

doi: 10.3389/fimmu.2023.1163136

Figure Lengend Snippet: Induction of T cell activation against gastrointestinal cancer cell lines by CC-3. Monocyte-depleted PBMC of healthy donors were incubated with the indicated tumor cell lines (E:T 4:1) in the presence or absence of CC-3 or MOPCxCD3. If not stated otherwise, all constructs were used at 1 nM. (A) Activation of CD4+ and CD8+ T cells was determined by flow cytometric analysis for CD69 expression after 24 hours. Combined data obtained with PBMC of three independent donors are shown. (B) Degranulation of CD4+ and CD8+ T cells was determined by expression of CD107a after 4 h. Combined data obtained with PBMC of four independent donors are shown. (C) IL-2 and (D) IFNγ levels in culture supernatants were measured after 24 h using LEGENDplex assays. Combined data obtained with PBMC of four independent donors are shown. E:T, effector to target; PBMC, peripheral blood mononuclear cell.

Article Snippet: Where indicated, T cells within PBMC were isolated by using either Pan T cell Isolation Kit, human CD4 Micro Beads or human CD8 Micro Beads (Miltenyi Biotec).

Techniques: Activation Assay, Incubation, Construct, Expressing

Journal: Frontiers in Immunology

Article Title: IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer

doi: 10.3389/fimmu.2023.1163136

Figure Lengend Snippet: Induction of T cell proliferation and memory T cell populations by CC-3. (A) Monocyte-depleted PBMC of healthy donors (n=4) were labelled with CellTrace™ Violet cell dye and incubated with or without MOPCxCD3 or CC-3 (1 nM each) in the presence of PANC-1, Hep3B, or NCI-N87 cells (E:T 4:1). After 72 h, PBMC were reexposed to fresh target cells and the respective treatment for additional 72 h. On day 6, proliferation was determined by flow cytometry. (B, C) PBMC of healthy donors (n=5) were incubated with or without MOPCxCD3 or CC-3 (1 nM each) in the presence of PANC-1, Hep3B, or NCI-N87 cells (E:T 1:1). After 72 h, cells were reexposed to fresh target cells and the respective treatment. On day 6, subpopulations of CD4 + and CD8 + T cells were determined by flow cytometric analysis. Effector T cells were defined as CD62L - CD45ro - , naive T cells as CD62L + CD45ro - , effector memory T cells as CD62L - CD45ro + and central memory T cells as CD62L + CD45ro + . (B) representative t-distributed stochastic neighbor embedding (tSNE) plots and (C) pooled data are shown. E:T, effector to target. PBMC, peripheral blood mononuclear cell.

Article Snippet: Where indicated, T cells within PBMC were isolated by using either Pan T cell Isolation Kit, human CD4 Micro Beads or human CD8 Micro Beads (Miltenyi Biotec).

Techniques: Incubation, Flow Cytometry

Journal: Oncology Letters

Article Title: Breast cancer stem cell RNA-pulsed dendritic cells enhance tumor cell killing by effector T cells

doi: 10.3892/ol.2020.11338

Figure Lengend Snippet: Analysis of activated DCs and lymphocyte phenotypes. (A) Profiles of the specific markers CD14, CD11c, CD40, HLA-DR, CD83 and CD86 on monocytes and mDCs. (B) Percentage distribution of lymphocyte subpopulations before and after activation and following the isolation of CD8 + T cells. (C) The percentage of IFN-γ-positive CD8 + T lymphocytes after co-culture with unpulsed or RNA-pulsed DCs from one donor. (D) Level of IFN-γ in the culture media of effector lymphocytes from three donors. Bars represent mean ± SD of three independent experiments. *P<0.05. CD, cluster of differentiation; DCs, dendritic cells; mDC, mature dendritic cell; HLA-DR, human leukocyte antigen DR; IFN, interferon; L, lymphocyte; MFI, mean fluorescence intensity; NK, natural killer.

Article Snippet: The cell suspension was mixed and incubated with 10 µl of a CD8 + T cell biotin-antibody cocktail (Miltenyi Biotec, Inc.) for 5 min, followed by 20 µl of a CD8 + T cell micro-bead cocktail (Miltenyi Biotec, Inc.) for 10 min at room temperature.

Techniques: Activation Assay, Isolation, Co-Culture Assay, Fluorescence

Journal: Oncology Letters

Article Title: Breast cancer stem cell RNA-pulsed dendritic cells enhance tumor cell killing by effector T cells

doi: 10.3892/ol.2020.11338

Figure Lengend Snippet: Tumor cell apoptosis caused by CSC RNA-pulsed DC-activated T cells. Cancer cell apoptosis was assessed by an immune cell killing assay using the IncuCyte ® live cell analysis system. (A) Representative field images of cell-mediated cancer cell apoptosis at effector T cells at E:T ratios of 5:1, 10:1, and 20:1. (B and C) Time course of apoptotic cell counts of cancer cells exposed to different E:T ratios of total effector T lymphocytes to targets (B) BCA55-121-WP (C) BCA55-121-CSC and (D and E) Time course of apoptotic cell counts of cancer cells exposed to different ratios of CD8 + T cells in (D) BCA55-121-WP and (E) BCA55-121-CSC. The y-axis represents the numbers of apoptotic cells compared with those in the control at the starting point of each treatment condition. Results shown are from triplicate wells from two independent experiments. *P<0.05 vs. T cells from the unpulsed DCs. CSC, cancer stem cell; DCs, dendritic cells; WP, whole population; E, effector T cells; T, target cells.

Article Snippet: The cell suspension was mixed and incubated with 10 µl of a CD8 + T cell biotin-antibody cocktail (Miltenyi Biotec, Inc.) for 5 min, followed by 20 µl of a CD8 + T cell micro-bead cocktail (Miltenyi Biotec, Inc.) for 10 min at room temperature.

Techniques: