Effective MCMV-specific CD8⁺ T-cell responses are elicited after inhibition of cross-presentation. (A) The cross-presenting capacity of DCs after CpG administration was assessed by measuring the proliferation of CL4 TCR transgenic HA-specific CD8⁺ T cells. BALB/c mice treated with saline (control) or 20 nmol CpG (CpG) received CFSE-labeled leukocytes from CL4 transgenic mice. Cell-associated HA was administered 18 hours later. Proliferation of CL4 CD8⁺ T cells was measured in the spleen 4 days after transfer. Results are representative of 3 experiments with at least 2 mice per group. (B) The cross-presenting capacity of DCs after CpG activation and MCMV infection was assessed as in panel A, except that mice were infected IP with MCMV at the time of injection of cell-associated HA. Results are representative of 3 experiments with at least 2 mice per group. (C) BALB/c mice received either saline or CpG 18 hours prior to infection with MCMV (5 × 10³ PFU per mouse) and T-cell responses were measured at various times PI. CD8⁺TCRβ⁺ (left), IE1-specific CD8⁺TCRβ⁺ (middle), and CD107a⁺CD8⁺TCRβ⁺ (right) cells were enumerated in the spleens of mice that were infected with MCMV with or without CpG pretreatment. Data are pooled from 2 to 4 independent experiments (where n = 5-12 mice per time point), except at day 1 PI (where n = 3) (*P < .05, **P < .01, ***P < .001). (D) The lytic activity of antiviral, IE1-specific CD8⁺ T cells was measured ex vivo by harvesting splenocytes on day 6 PI and culturing them with Cr⁵¹-labeled, IE1-pulsed P815 target cells for 4 hours at various E:T ratios. Specific lysis was determined as noted in supplemental Methods. Data are from 2 independent experiments (where n = 6 infected mice per group). (E) The lytic activity of antiviral, IE1-specific CD8⁺ T cells was measured using an in vivo CTL assay. Untreated or CpG pretreated mice were infected with MCMV (5 × 10³ PFU per mouse IP); at days 5 and 9 PI, the mice received a 1:1 mixture of CFSE^lo IE1 peptide-pulsed and CFSE^hi unpulsed splenocytes IV. Spleens were harvested on days 6 and 10 PI and IE1-specific CTL killing measured by flow cytometry as the loss of CFSE^lo IE1-pulsed targets compared with CFSE^hi unpulsed targets. Histograms are representative of 2 independent experiments (n = 4-6 mice per group per time point). Percentages shown are mean ± SEM. E:T, effector to target ratio.