Journal: The Journal of Clinical Investigation

Article Title: An EBV-related CD4 TCR immunotherapy inhibits tumor growth in an HLA-DP5 + nasopharyngeal cancer mouse model

doi: 10.1172/JCI172092

Figure Lengend Snippet: ( A ) TCR135-transduced JK4NF cells were cocultured overnight with DCs pulsed with 10 μM or 1 μM GST–C-terminus EBNA1 recombinant protein (rEBNA1), 1 μM EBNA1 564–583 peptide, or PBS (NC). The frequency of ZsGreen-expressing cells was measured by flow cytometry. ( B ) SNU-719-EBNA1 tumor cells were cocultured with DCs pulsed with rEBNA1 protein (DC + rEBNA1) or PBS (DC only) and TCR135-transduced T cells (TCR135-T) or non-transduced T cells (TCRneg-T) for 72 hours, and then the living tumor cells were analyzed by Celigo Image Cytometer fluorescence photography. The representative fluorescent images and statistical results are shown. 2-way ANOVA and Šidák’s multiple comparisons, **** P < 0.0001. ( C ) DCs pulsed with rEBNA1 protein were cocultured with TCR135-T cells or TCRneg-T cells for 24 hours. Afterward, the supernatant was collected, and the cytotoxic effectors were detected using RayPlex Human Cytotoxic T Cell Array Kit 1. 2-tailed unpaired Student’s t test, ** P < 0.01, *** P < 0.001. ND, not detected. ( D ) DCs pulsed with rEBNA1 protein were cocultured with TCR135-T cells for 48 hours. Then, the supernatant was added to SNU-719-EBNA1 tumor cells and cultured for 72 hours, with or without neutralizing antibodies against TNF-α, IFN-γ, and FasL. The analysis of living tumor cells was carried out using Celigo Image Cytometer fluorescence photography. 1-way ANOVA and Dunnett’s multiple comparisons, **** P < 0.0001.

Article Snippet: The supernatant was collected, and the concentrations of 12 cytokines in the supernatant were quantitatively measured using RayPlex Human Cytotoxic T Cell Array Kit 1 (Ray Biotech) and flow cytometry.

Techniques: Recombinant, Expressing, Flow Cytometry, Cytometry, Fluorescence, Cell Culture