FIGURE 2.

CD8⁺ T-cell blast transformation and proliferation are inhibited by simultaneous exposure to HSV-1–infected corneal fibroblasts (cFb) and splenocytes (Spl). Eight days after HSV-1 corneal infection, DLN suspensions enriched for CD8⁺ T cells by CD4 depletion only (CD4⁻ DLN; A, B) or CD4 and MHC class II depletion (CD8⁺ DLN; C) were cultured with 1 × 10⁶ HSV-1–infected DiI-labeled (A, B) or unlabeled (C) Spls alone, with a monolayer of HSV-1–infected cFbs alone, or with Spls and cFbs. (A) Cells were then removed from the cultures after 48 hours, stained for surface CD45 and CD8α, and analyzed by flow cytometry. Cells undergoing blast transformation were identified based on forward angle and side scatter, and CD45 was used to distinguish bone marrow–derived cells including CD8⁺ T cells. Cells derived from the DLN and Spls were differentiated based on DiI staining, and blasting DiI⁻ CD8⁺ T cells were gated for further analysis. (B) The percentage of blasting DiI⁻ CD8⁺ T cells was multiplied by the total number of cells retrieved from each culture to obtain the mean ± SEM absolute number of blasting DLN-derived CD8⁺ T cells per culture. The graph contains combined data from duplicate cultures of two independent experiments. (C) ³H-Thymidine (³H-T) incorporation into cellular DNA was measured during the last 6 hours of a 72-hour culture as a measure of cellular proliferation. Data are recorded as the mean ± SEM ³H-T cpm in triplicate cultures of each group and are representative of three independent experiments with nearly identical results. *P < 0.001.