Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice
Fig 5
Transgenic TCRNLV expression, structural avidity, and cytolytic activity of TCR-transduced human T-cell subsets.
(A) Immunomagnetically selected human CD8 T cells (left panels) and CD4 T cells (right panels) were retrovirally transduced with TCRNLV (CD8-TCRNLV and CD4-TCRNLV, respectively) or empty vector (CD8 mock and CD4 mock, respectively) and were drug-selected. After in vitro expansion with anti-CD3/CD28 beads for a period of 10d, cells were analyzed cytofluorometrically for expression of CD4 and CD8, as well as of TCRNLV using HLA-A2.1/NLV tetramers. Shown are 2D dot plots with the percentages of labeled cells indicated. (B) and (C) Characterization of CD8-TCRNLV and CD4-TCRNLV cells respectively. (Left panel) Structural avidity (KD) of antigen binding was quantified by dose-dependent HLA-A2.1/NLV tetramer binding determined by flow cytometry (see the legend of Fig 2B). (Right panels) Cytolytic activities of TCRNLV-transduced and mock-transduced T cells (filled and open circles, respectively) determined by a standard [51Cr]-release assay at the indicated effector (i.e. T cell) to target (i.e. MEF of NSG/HHD mice) cell ratios (E:T). NSG/HHD MEF were pre-treated with IFN-γ for 48h and were either exogenously loaded with a high dose of synthetic NLV peptide as a positive control, or infected for 12h with mCMV-NLV and, as a non-antigenic control, with mCMV-NLVAla. Data represent means of duplicate assay cultures. Error bars indicate the range.