OVA/IC–mediated antigen presentation in vitro induces Th1 CD4 and CD8 cellular proliferation and effector differentiation of naive transgenic T cells. (a) Anti-OVA T cell responses. IC-mediated antigen presentation efficiently drives OT-I and OT-II proliferative responses in vitro. 2 × 105 CD4 OT-II and 4 × 105 CD8 OT-I cells were cultured with 104 or 2 × 104 antigen-pulsed BMDCs in 96-well culture plates. 3H-thymidine incorporation was measured between 36 and 48 hours later and expressed in cpm. OVA (10 μg/ml), OVA-containing ICs (10 μg/ml OVA and 50 μg/ml anti-OVA), or OVA (10 μg/ml) plus “irrelevant” peroxidase/antiperoxidase (PAP) complexes (50 μg/ml) were added to DCs for 48 hours prior to incubation with T cells. The results are representative of five separate experiments. (b) Cytokine production. IC-loaded BMDCs induce IFN-γ production by naive OT-I and OT-II transgenic cells. CD4 OT-II or CD8 OT-I transgenic T cells were cultured at 106/ml with 5 × 104/ml pulsed DCs in 24-well plates. IL-4 and IFN-γ production was determined by ELISA after 3–5 days of culture. The results are from two independent experiments. The sensitivity of the ELISAs were 1 pg/ml. Culture of OT-I or OT-II transgenic T cells with DCs alone (i.e., without OVA peptides or soluble OVA) did not elaborate detectable IFN-γ or IL-4.