Figure 4.

Vesicular stomatitis virus (VSV)-δ51 loading activates OT-I. (a) A total of 1 × 10⁶ OT-I, or VSV-δ51-loaded OT1 (OT-I(VSV)), were plated in six-well plates. Culture supernatants were harvested at 16 and 40 h postinfection (p.i.), and were assayed by enzyme-linked immunosorbent assay (ELISA) for interferon (IFN)-α. (b, c) OT-I cells were loaded at an MOI of 1 with VSV-δ51 or left unloaded. After 24 h, cells were analyzed by flow cytometry for expression the T-cell activation markers CD44 (b) or CD25 (c) along with CD8. (d) Three-day-activated OT-I, or OT-I cells loaded with VSV-δ51 (MOI 1) were used as effectors against B16 or B16ova targets loaded with Cr⁵¹. OT-I cells (effectors) were incubated for only short periods of time with their targets (B16 or B16ova) at the ratios shown, supernatants were harvested and analyzed for Cr⁵¹ release. Levels of lysis were relatively low in these assays because of the short period of time over which the assay needed to be conducted to exclude tumor cell lysis as a result of infection by VSV-δ51 released from the loaded OT-I T cells. Thus, control experiments demonstrated that cytotoxicity to B16 or B16ova tumor cells as a result of infection with VSV-δ51 is only significant after 6 h of virus incubation (not shown). Data shown are representative of three separate experiments; bars, s.d. *P<0.05 (VSV/OT1+B16 versus OT1+B16). (e) Three-day activated OT-I cells were loaded at an MOI of 1 with VSV-δ51 or left unloaded. The OT-I cells were grown for a further 24 h in the absence of added SIINFEKL peptide and were analyzed by flow cytometry for intracellular expression of IFN-γ along with CD8.