T-bet expression rescued IgG2a production in Ets-1^−/− B cells. B cells from Ets-1^−/− chimeras were stimulated in vitro with 25 μg/mL of LPS and 50 ng/mL of IFN-γ and simultaneously infected with GFP or GFP-T-bet retroviruses. Seventy-two hours after stimulation, cells were analyzed by FACS and RNA was harvested. (A) FACS analysis of IgG2a and IgG1 expression on wild-type (WT) and Ets1^−/− (KO) cells transduced with GFP alone (GFP) or GFP-T-bet (T-bet). The percentages of cells falling in the gates are indicated. Results are representative of 3 experiments. (B) The bar graphs show the mean percentages of IgG2a-expressing cells in 3 independent experiments as in panel A. The statistical significance was calculated with the Student t test and the P values are shown. (C) Top panel, semiquantitative RT-PCR of 10-fold serial dilutions of template cDNA from cells infected with the indicated viruses. Transcription of T-bet, γ2a germline (GL), and post-switch (PS) were analyzed. The Hprt gene was used as a control. Bottom panel, Western blot analysis of Ets-1^−/− B cells transduced with the empty (GFP) or T-bet–expressing viruses (T-bet) in the same experiment as in panel A. Whole-cell lysates were subjected to Western analysis with Abs recognizing T-bet or actin as loading control. (B) B cells from the spleens of Ets-1^−/− (KO) or wild-type (WT) chimeras were left untreated (NA) or stimulated with 25 μg/mL of LPS and 50 ng/mL of IFN-γ and infected with retrovirus expressing GFP alone, Ets-1-GFP (Ets-1), or T-bet-GFP (T-bet). Secretion of IgG2a was measured by ELISA after 5 days in culture. Results are representative of 3 independent experiments.