Mice were immunized with gK, three times as described in Materials and Methods. Three weeks after the third immunization, splenocytes form immunized mice were isolated, and 2 ×10⁶ cells were stimulated with 10 μg of each peptide, 10 PFU/cell of UV-inactivated HSV-1 strain McKrae, 1 μg of ConA, or 10 μg of irrelevant peptide. Four hours before harvesting the cells for FACS analyses, BFA was added as described in Materials and Methods. CD4⁺ and CD8⁺ T cells expressing intracellular IFN-γ in response to stimulators were identified using appropriate Abs in a FACSCalibur. The frequency of CD4⁺ and CD8⁺ T cells expressing IFN-γ in one representative mouse from gK-immunized mice is shown. Experiments were repeated 2X.

To prepare target cells for detection of in vivo cytolytic activity, splenocytes from naive BALB/c mice were prepared as described in Materials and Methods. The cells were split into two populations. One population was pulsed with 10 μg of peptide #2, incubated at 37°C for 18 hr, and labeled with a high concentration of CFSE (2.5 μM) (CFSE^high cells). The second control target population was pulsed with a nonspecific control VSV peptide and labeled with a low concentration of CFSE (0.25 μM) (CFSE^low cells). For IV injection, an equal number of cells from each population were mixed together such that each mouse received one mouse’s worth of splenocytes in 100 μl of PBS. The target cells were then injected into mice that had previously been immunized with gK or KOS, or mock-immunized 3X as described in Materials and Methods. Four hours after IV injection of target cells, mice were sacrificed and their spleens were isolated. Cell suspensions were analyzed by FACS. Each population was detected by their differential CFSE fluorescence intensities. Representative histograms from various groups are shown. The specific lysis for each group was calculated as described in Materials and Methods using the intensity of low and high CFSE staining in relation to the unprimed control (panels G, H, and I). Panels: A) gK-immunized mice received lymphocytes primed with peptide #2; B) KOS-immunized mice received lymphocytes primed with peptide #2; C) Mock-immunized mice received lymphocytes primed with peptide #2; D) gK-immunized mice received lymphocytes primed with peptide #1; E) KOS-immunized mice received lymphocytes primed with peptide #1; F) Mock-immunized mice received lymphocytes primed with peptide #1; G) gK-immunized mice received unprimed lymphocytes; H) KOS-immunized mice received unprimed lymphocytes; and I) Mock-immunized mice received unprimed lymphocytes. Data are representative of FACS analyses from three mice per group.

Splenocytes from gK-immunized mice were stimulated with purified gK338, gK175, D2, or D3 in vitro for 72 hr as described in Materials and Methods. The concentrations of IFN-γ in the supernatants were measured by ELISA. Each point represents mean ± SEM for nine replicates from three mice.

Splenocytes from gK-immunized or KOS-immunized mice were separated into CD8⁺ or CD4⁺ T cells, and 2 × 10⁶ cells were stimulated with 10 μg of peptide #2 or control peptide #1 in vitro for 72 hr as described in Materials and Methods. The concentrations of IFN-γ in the supernatants were measured by ELISA. Each point represents mean ± SEM for three replicates from three mice.