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. Author manuscript; available in PMC: 2008 Jun 2.
Published in final edited form as: Invest Ophthalmol Vis Sci. 2007 May;48(5):2194–2202. doi: 10.1167/iovs.06-1321

FIGURE 3.

FIGURE 3

Simultaneous exposure to HSV-1–infected corneal fibroblasts (cFb) and splenocytes (Spl) inhibits cytolytic arming but not degranulation of CTLps. Eight days after HSV-1 corneal infection, DLN suspensions enriched for CD8+ T cells by CD4 depletion only (CD4− DLN; A–C, E) or CD4 and MHC class II depletion (CD8+ DLN; D) were cultured for 48 hours with 1 × 106 HSV-1–infected DiI-labeled (A–C, E) or unlabeled (D) Spls alone, with a monolayer of HSV-1–infected cFbs alone, or with Spls and cFbs. (A) Cells were then removed from the cultures, stained for surface CD8α and intracellular GrB, and analyzed by flow cytometry. Cells undergoing blast transformation were identified based on forward angle and side scatter, and CD8+ T cells derived from the DLN and Spls were differentiated based on DiI staining. Dot plots are representative of duplicate cultures from two independent experiments. The percentage of blasting DiI− CD8+ GrB+ T cells (B) was multiplied by the total number of cells retrieved from each culture to obtain the mean ± SEM absolute number of blasting DLN-derived CD8+ GrB+ T cells per culture (C). Graphs contain combined data from duplicate cultures of two independent experiments. (D) Alternatively, the cells were removed from culture and resuspended in medium alone (spontaneous release) or medium containing PMA and ionomycin (experimental release) for 4 hours. BLT esterase (GrA) activity of culture supernatants was then measured spectrophotometrically. Values represent experimental release with spontaneous release subtracted. Data are presented as the mean ± SEM of triplicate samples and are representative of three independent experiments with nearly identical results. (E) Cells were removed from the cultures and exposed to HSV-1–infected cFb targets at an E:T ratio of 5:1 in the presence of protein transport inhibitor (GolgiPlug) and anti–CD107a mAb for 6 hours. Cells were then washed, stained for surface CD8α, and analyzed by flow cytometry as in (A). Dot plots are representative of two independent experiments.