SWAP-70^−/− DCs are impaired in generating CD4⁺ T-cell responses in vitro and in vivo. (A,B) Activation of T-cell hybridomas. Magnetic-activated cell sorting (MACS)–isolated CD11c⁺ BMDCs (A) or spleen DCs (B) were incubated with T-cell hybridomas BO9710 and 1H3.1, respectively, and different concentrations of peptides. IL-2 in the culture supernatants was measured by ELISA. Representative data of 3 independent experiments are shown. (C) ELISPOT results show IFN-γ–secreting CD4⁺ T cells specific for OVA_323-339 epitope. Results are expressed as the average number of IFN-γ–secreting T cells in the whole spleen. SWAP-70^+/+ mice were immunized with MACS-isolated CD11c⁺ BMDCs loaded with OVA_323-339 peptide, and ELISPOT was performed 10 days later. Representative data of 2 independent experiments are shown. White columns indicate wt and LPS; striped columns, wt untreated; white/black columns, SWAP-70^−/− untreated; and black columns, SWAP-70^−/− and LPS as indicated. *P < .001. (D) Retroviral infection with SWAP-70-GFP restores the capacity to stimulate CD4⁺ T cells of SWAP-70–deficient DCs. Seven-day CD11c⁺ BMDCs were transfected with SWAP-70-IRES-GFP (SWAP-70-GFP) or IRES-GFP (empty vector) by retroviral infection as in Figure 2. BMDCs were stimulated with LPS for 12 hours, fixed, and incubated with T-cell hybridomas BO9710 and 1 μg/mL OVA_329-332 peptide. IL-2 in the culture supernatants was measured by ELISA. Representative data of 2 independent experiments are shown. □ indicates wt; ■, ko. (E) BMDCs were incubated for 2 hours with 2 mg/mL OVA protein (left panel) or E coli–expressing OVA or Eα proteins at a ratio of 100:1 (bacteria/DCs) (right panel). Cells were washed, fixed, and mixed with T-cell hybridoma cells. IL-2 in the culture supernatants was measured by ELISA. Representative data of 2 independent experiments are shown. □ indicates wt; ■, ko. *P < .001.