Cross-presentation of ova to ova-specific CD8+ T cells in vivo by ova-loaded gp96 surface-expressing tumor cells. (A) Meth A-neo or Meth A-96tm cells were osmotically loaded with chicken ovalbumin. To quantify the loading efficiency, 2.5 x 10⁵ cells from each cell type were lysed in 20 µl PBS containing 0.5% NP-40. The supernatant, along with purified ovalbumin standard, were then subjected to 10% SDS-PAGE, followed by immunoblotting with ova-specific Ab. In this experiment, approximately 100 ng of ovalbumin was loaded into 5 x 10⁵ cells. The loading efficiency of Meth A-96tm and Meth A-neo cells appeared to be equivalent. (B) 2 x 10⁶ purified CD8+Valpha2+ OT-I cells were labeled with CFSE and adoptively transferred into C57BL/6 (H-2^b) mice by tail vein injections. Two days later, the mice were immunized i.p. with PBS, 1 mg ova, 5 x 10⁵ Meth A-neo or Meth A-96tm cells mixed with 100 ng ova, 5 x 10⁵ Meth A-neo or Meth A-96tm cells loaded with 100 ng ova intracellularly. Three days after immunization, splenocytes from each group of mice were analyzed by FACS after staining with propidium iodine (PI) and PE-labeled anti-Valpha2 mAb (Pharmingen). Cells were gated on PI-Valpha2+ populations. Shown is a representative result from three separate experiments.