The sites of cross-presentation and induction of cross-tolerance by intravenous and intragastric administration of soluble OVA. (A) CFSE-labeled OT-I T cells were transferred into their syngenic C57BL/6 mice, which then received OVA via intravenous (IV; 5 mg) or intragastric (IG; 25 mg) routes, respectively (filled curve), or PBS as a control (open curve). Forty-eight hours later, lymphoid cells from the indicated secondary lymphoid organs were obtained and analyzed after gating with Vα2⁺ cells. (B-D) Groups of C57BL/6 mice were given 5 mg intravenous OVA or 25 mg intragastric OVA or PBS. Seven days later, the mice were primed with OVA-loaded splenocytes. (B) Cr-51 release assay was performed at 7 days after priming. The lytic unit was calculated as described in “Materials and methods,” and the mean value is expressed as a bar. (C) ELISPOT assay was performed to assess IFN-γ secretion by antigen-specific cells in response to 1 μM OVA peptide 7 days after priming. Values represent the mean ± SE. (D) Intracellular IFN-γ accumulation was assessed 7 days after priming as described in “Materials and methods.” i.v. indicates intravenous; i.g., intragastric. Means ± SEs from 3 mice per group are shown.