FIGURE 5.

Inhibition of CTLp differentiation requires direct contact with HSV-1–infected corneal fibroblasts (cFb) and HSV-1 infection of spleen cells (Spl). (A) Eight days after HSV-1 corneal infection, DLN suspensions enriched for CD8⁺ T cells by CD4 and MHC class II depletion (CD8⁺ DLN) were cultured for 48 hours with different combinations of 1 × 10⁶ HSV-1–infected Spls and monolayers of HSV-1–infected cFbs in Transwell cultures containing cell-impermeable membranes between the top and bottom chambers. SEM was less than 2% for all culture conditions at all E:T ratios. (B) Cultures were prepared as in (A) except that the bottom chamber invariably contained CD8⁺ T cells and cFbs, whereas the top chamber contained no Spls, uninfected Spls, or HSV-1–infected Spls. CD8⁺ T cells were then removed from the cultures and exposed to ⁵¹Cr-loaded HSV-1–infected target cells. Cytotoxicity was measured in a 3.5-hour ⁵¹Cr release assay. Percentage of specific lysis against uninfected targets was less than 5% for all culture conditions at all E/T ratios. Data are representative of three independent experiments with nearly identical results.