Figure 4.

Flow Cytometry Analyses of CD3+ T Cells Prior to Adoptive Transfer

Splenocytes of intact B10.D2(R101) mice were activated with ConA (3 μg/mL) and IL-2 (10 U/mL) for 24 h and transduced with SM1 TCRα or GFP. Flow cytometry analyses were performed on Day 3 post-transduction (6 days in vitro cultivation in total). Non-transduced cells (NTC), similarly cultured for 6 days were used as the control.

(A) Cells were gated as follows: lymphocytes were gated based on SSC-A vs. FSC-A followed by singlets gating based on FSC-H vs. FSC-A. Dead cells were excluded by staining with PI. The relative count of CD3+ cells were determined within the population of live singlets after staining with anti-CD3 monoclonal antibodies.

(B) The level of transduction measured by anti-Va2 staining for TCRα SM1 or by GFP expression (used as the reference) in the population of CD3+ live singlets.

(C) The relative number of CD4+ and CD8+ cells in the subset of SM1 transduced T cells. The relative count of Vα2⁺ cells were analyzed in CD4+ and CD8+ T cells subsets.

(D) The relative number of CD4+ and CD8+ cells in the subset of GFP transduced T cells. The relative count of GFP⁺ cells were analyzed in CD4+ and CD8+ T cells subsets. One of two representative experiments is shown (n = 2). Data are presented as mean ± SD.