Figure 4.

Aberrant production of IL-21 by DEF6-deficient T cells plays a key role in the humoral abnormalities observed in mice lacking DEF6. (A) Purified CD4⁺ T cells from 8–10-wk-old WT, S70^−/−, Def6^tr/tr, and DKO female mice were subjected to a primary stimulation, and then restimulated with αCD3 and αCD28 for 48 h. Supernatants were then collected and assayed for IL-17 (left), IL-21 (middle), and IFN-γ (right) production by ELISA. The data are representative of three independent experiments. The error bars represent mean ± SD. (B) Splenocytes from 24-wk-old WT, S70^−/−, Def6^tr/tr, and DKO female mice were stimulated with PMA (50 µg/ml) and Ionomycin (1 µM) for 5 h. After stimulation, cells were subjected to intracellular staining for IL-17 and IL-21, and then analyzed for cytokine production by flow cytometry. The data are representative of three independent experiments. (C–F) 8–10-wk-old female WT, Def6^tr/tr, IL-21^−/−, and Def6^tr/trIL-21^−/− mice were immunized with SRBC (n = 4–7/group). Unimmunized control mice (not depicted) received PBS only. At day 7 after immunization, splenocytes were analyzed for numbers of B220⁺PNA⁺ cells (C), B220^loCD138⁺ cells (D), CD4⁺CD62L^loCD44^hi cells (E), and CD4⁺ICOS⁺ cells (F). Each dot represents individual mice and horizontal bars indicate mean for each group. The data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005.