![Fig. 4. IKK2dn DC blocks CD40L-induced NF-κB activation and cytokine production in immature DCs. / Immature DCs were left uninfected (white) or were infected with Adβ-gal (gray), AdNIKdn (cross-hatched), AdIKK2dn (checkered), or AdIκBα (black), and cultured for 2 additional days. (A) Graded doses of uninfected cells were cultured with 105 allogeneic T cells in the presence of a neutralizing CD40L antibody or its isotype-matched control. Proliferation was determined on day 6 using the [3H]thymidine uptake assay. (B-D) Cells were stimulated for 24 hours with irradiated 1 × 105 mock control– or CD40L-transfected mouse fibroblasts, and supernatants were collected and assayed for the presence of TNFα, IL-6, and IL-8 by ELISA. Mean values ± SDs of 1 experiment representative of 4 independent experiments are shown. (E) Cells were stimulated with 30 μg/mL of sCD40L for 45 minutes, cytosolic and nuclear extracts collected, and IκBα levels as well as NF-κB DNA-binding activity were determined by Western blotting and EMSA, respectively. (F) Immature DCs were left uninfected or infected with Adβ-gal, AdNIKdn, AdNIKwt, or combinations of them. An MOI of 100 was used for each virus except in the 2 coinfection conditions, where an MOI of 500 was used to provide a 5-fold excess of Adβ-gal or AdNIKdn virus. After further stimulation for 24 hours, supernatants were collected and assayed for the presence of TNFα by ELISA. Mean values ± SDs of 1 experiment representative of 3 independent experiments performed on samples from unrelated donors are shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/3/10.1182_blood-2002-06-1835/4/h80333721004.jpeg?Expires=1722765310&Signature=J8buYMBhnYRMbGWK5usXvPx6WlEVJXSuh6i-BbDOGsu1XjDjEUYiMGadvT2ZmmrOddbYM9HZExHtTWW0eTdPqakt8UU0ews0eTiHzGdczyJhx-BMlAaQarKiSs3bwkREDyXRHciocmm2~GwzFIyn4JX2X2H9VfUlYqaG4r4xq2HMfod-QuG7iVu7GcEylbLAFvwnl8QOGOWzb48ABhHfaWg1jdi-~dCYtEH7tIsdpInmTX8dOxqcbtuuQNob7c~H~WY5F1Wu0qUiYGUUtBFWJ7pM907GiczPk-DG~0O0k~gmtF9qFN3pSp0tLEwQ5mvvp5QPaODnn-w~T-l5bgfR-A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
IKK2dn DC blocks CD40L-induced NF-κB activation and cytokine production in immature DCs.
Immature DCs were left uninfected (white) or were infected with Adβ-gal (gray), AdNIKdn (cross-hatched), AdIKK2dn (checkered), or AdIκBα (black), and cultured for 2 additional days. (A) Graded doses of uninfected cells were cultured with 105 allogeneic T cells in the presence of a neutralizing CD40L antibody or its isotype-matched control. Proliferation was determined on day 6 using the [3H]thymidine uptake assay. (B-D) Cells were stimulated for 24 hours with irradiated 1 × 105 mock control– or CD40L-transfected mouse fibroblasts, and supernatants were collected and assayed for the presence of TNFα, IL-6, and IL-8 by ELISA. Mean values ± SDs of 1 experiment representative of 4 independent experiments are shown. (E) Cells were stimulated with 30 μg/mL of sCD40L for 45 minutes, cytosolic and nuclear extracts collected, and IκBα levels as well as NF-κB DNA-binding activity were determined by Western blotting and EMSA, respectively. (F) Immature DCs were left uninfected or infected with Adβ-gal, AdNIKdn, AdNIKwt, or combinations of them. An MOI of 100 was used for each virus except in the 2 coinfection conditions, where an MOI of 500 was used to provide a 5-fold excess of Adβ-gal or AdNIKdn virus. After further stimulation for 24 hours, supernatants were collected and assayed for the presence of TNFα by ELISA. Mean values ± SDs of 1 experiment representative of 3 independent experiments performed on samples from unrelated donors are shown.
