Fig. 1.

Ubc12 is required for TCR-induced proliferation and activation. (A) Immunoblot analysis of Ubc12 was performed in sorted GFP⁺ control (ctl) or GFP⁺ shUbc12 CD4⁺ T cells (Inset). Peripheral naïve CD4⁺ T cells sorted from shRNA expressing chimeric mice were stimulated with various concentration of anti-CD3 and anti-CD28 mAb for 3 d and then pulsed for an additional 8 h with ³H-thymidine. Cell proliferation was measured by ³H-thymidine uptake. (B) Cell cycle analysis of control (Left) and shUbc12 CD4⁺ (Right) T cells. The cells were activated for 23 h with anti-CD3/CD28 mAbs, then pulsed for 1 h with EdU and analyzed by flow cytometry after staining for EdU and 7-AAD. (C) Control (Left) and shUbc12 CD4⁺ (Right) violet-labeled CD4⁺ T cells were stimulated with anti-CD3/CD28 mAbs for 2 d, after which cell division was analyzed by flow cytometry. (D) Flow cytometry for cell surface expression of CD69 (Left) and CD25 (Right) in sorted control and shUbc12 CD4⁺ T cells stimulated for 16 h with anti-CD3/CD28 mAbs. The results are representative of three repeated experiments. (E) IL-2 (Left) and IFN-γ (Right) production in control and shUbc12 CD4⁺ T cells stimulated for 48 h with anti-CD3 and anti-CD28, measured by ELISA. (F) Control (Left) and Ubc12 shRNA-expressing (Right) OT-II TCR transgenic mice were generated by reconstitution of retrovirally transduced bone marrow cells from OT-II mice into lethally irradiated recipient mice (n = 3). After 8 w, isolated CD4⁺ T cells from OT-II control or OT-II Ubc12 knockdown chimeric mice were adoptively transferred into C57BL/6J recipient mice, followed by immunization with OVA plus CFA. At 5 d after immunization, splenocytes and lymph node cells were stimulated with OVA_323–339 peptide for 24 h and analyzed by flow cytometry using anti–IL-2 and anti–IFN-γ antibodies. (G) Sorted naïve CD4⁺ T cells were stimulated with anti-CD3/CD28 mAbs and differentiated in vitro under Th1-inducing (Upper) or Th2-inducing (Lower) conditions. Results are from three repeated experiments.