Allogeneic CD8α⁺ DC immunization preserves T-cell responses to third-party antigens. B6 donor mice were immunized with diluent or recipient BALB/c BM-derived CD8α⁺ DC or CD8α⁻ DCs as described in “Vaccination protocol.” T cells were harvested and used for in vitro and in vivo studies with C3H/HeJ recipients. (A) B6 splenic CD90⁺ T cells were stimulated with irradiated (3000 cGy) splenocytes (10⁵/well) from C3H/HeJ. During the final 6 hours of a 72-hour culture, cells were pulsed with ³H thymidine and assayed for proliferation. (B) Supernatants were collected at 66 hours and assayed by ELISA for IL-2, IFN-γ, and IL-10. Error bars represent SE. P = not significant between diluent control and BALB/c-derived CD8α⁺ DC groups. Each graph represents one of 3 similar experiments. (C) C3H/HeJ mice were lethally irradiated with 9000 cGy TBI and injected with 5 × 10⁶ TCD BM and 10⁶ CD90⁺ T cells from syngeneic (black straight line, n = 3), allogeneic diluent-treated (circles, dotted line, n = 7), or BALB/c-derived CD8⁺ DC-vaccinated (triangles, n = 6) or CD8⁻ DC-vaccinated (diamonds, n = 6) B6 animals and evaluated for survival. P = not significant between diluent control and the other groups. Data represent one of 2 similar experiments. (D) B6 OT-II transgenic mice were immunized with diluent or BALB/c BM-derived CD8α⁺ DCs or CD8α⁻ DCs as above. Responder T cells were harvested from the OT-II B6 mice and cultured for 48 hours with B6 syngeneic splenocytes that were either not pulsed or pulsed with 100nM or 500nM OVA_323-339 peptide. During the final 6 hours of a 48-hour culture, cells were pulsed with [³H] thymidine and assayed for proliferation. P = not significant between diluent control and the other immunized groups. Data are representative of one of 2 similar experiments.