Autologous fibroblasts infected with AD169 or RV798 can be used as stimulator cells to quantitate CMV-specific CD8⁺ T cells by cytokine flow cytometry. Fibroblasts were infected with AD169 or RV798 for 4, 16, 24, and 48 hours and then used to stimulate autologous PBMCs or whole blood in a CFC assay. CD8⁺ T cells that produced IFNγ were identified within the lymphocyte gate (based on forward/side scatter) by 2-color staining with anti-CD8 and anti–IFNγ monoclonal antibodies. The data shown are the results for donor 1 and are representative of the results obtained in 5 donors. (A) Frequency of CD8⁺ T cells induced to produce IFNγ after stimulation with mock-infected fibroblasts. (B) Frequency of CD8⁺ T cells induced to produce IFNγ after stimulation with fibroblasts infected with AD169 for various times. (C) Frequency of CD8⁺ T cells induced to produce IFNγ after stimulation with fibroblasts infected with RV798 for various times.