Impaired cell cycle entry and survival after TCR stimulation of p50^−/− cRel^−/− T cells. (A) WT and p50^−/− cRel^−/− CD4⁺ T cells were activated with plate-bound αCD3, αCD3+IL-2, αCD3+αCD28, or αCD3+ αCD28+IL-2 for 2 d before DNA content staining and FACS^® were performed. The percentages show the sub-G₀ population, which represents apoptotic cells and cells in different phases of the cell cycle. Typical results of several independent experiments are shown. (B) RT-PCR was performed to determine expression of IL-2, c-Myc, Bcl2, and Bcl-X_L in WT and p50^−/− cRel^−/− CD4⁺ T cells after 6 h of activation.

High susceptibility of activated p50^−/− cRel^−/− T cells to cell death can be rescued by Bcl-2. (A) WT and p50^−/− cRel^−/− CD4⁺ T cells were activated by plate-coated αCD3+αCD28 for 3 d after which dead cells were removed on a Ficoll gradient. These cells were either stained with PE-αCD25 and SYTOX immediately (day 0) or cultured in T cell medium without αCD3 or IL-2 for 24 h before PE-αCD25 and SYTOX staining (day 1). Apoptosis rate represent the percentage of CD25⁺ SYTOX⁺ cells (apoptotic) in the total CD25⁺ population. (B) p50^−/− cRel^−/− CD4⁺ T cells were infected with MIG retrovirus during a 4-d stimulation in the presence of αCD3+ αCD28. On day 4, viable cells were stained by αCD25 before FACS^® analysis. (C) MIG and Bcl2 retroviral-infected WT and p50^−/− cRel^−/− CD4⁺ T cells were obtained as described in B, and either stained with PI and analyzed by FACS^® immediately (day 0) or cultured in T cell medium without αCD3 or IL-2 for 2 d before PI staining and FACS^® (day 2). The viable infected cells were GFP⁺ PI⁻. The percentage was calculated based on the percentage of GFP⁺ PI⁻ cells in the total cell population. The increase in the percentage of Bcl-2–infected cells after 2 d in this experiment is likely due to the decrease in the number of uninfected cells because of cell death, rather than an increase in the absolute number of Bcl-2–infected cells.

NF-κB activation is sufficient to promote T cell survival. (A) αCD3+αCD28+IL-2–activated WT CD4⁺ T cells were either used to make nuclear extract immediately or cultured in T cell medium without stimulation for 12 h before nuclear extract was made. EMSA was performed with an H2 site probe. The two complexes are described in the text. (B) MIG and CA-IKKβ retrovirus-infected WT CD4⁺ T cells were used immediately (0 h) or cultured in T cell medium without stimulation for 12 h. Nuclear extracts were made and EMSA was performed with the H2 site probe. (C and D) MIG, CA-IKKβ, and Bcl2 retrovirus-infected WT T cells were cultured in T cell medium without stimulation for 0, 1, 2, or 3 d, after which cells were stained with PI and analyzed by FACS^®. FACS^® data on days 0 and 3 are shown in C. The percentage indicates the proportion of GFP⁺ PI⁻ cells in total cell population. The percentage survival of infected T cells from days 0 to 3 are shown in D. (E) MIG, CA-IKKβ, and Bcl2 retroviral-infected WT T cells were cultured in T cell medium without αCD3 or IL-2 for 12 h. RNA was extracted before and after culturing. Bcl2, Bcl-XL, and β actin expression was examined by Northern blotting. 1, endogenously expressed Bcl2; 2, ectopically expressed Bcl2. (F) MIG, CA-IKKβ retrovirus-infected OT-II×IL-2^+/+ and OT-II×IL-2^−/− T cells were cultured in T cell medium without stimulation for 0–3 d. Survival rate of the infected cells was determined by PI staining and FACS^® analysis as in C and D.

Impaired antigen-induced responses and effector/memory and regulatory T cell generation in WT and p50^−/− cRel^−/− mice. (A) WT and p50^−/− cRel^−/− mice were either uninjected (day 0) or injected with SEB. Vβ8⁺ and Vβ6⁺ T cell populations were determined 3 and 6 d after injection. Two mice of each genotype were used per condition. (B) Naive and memory T cell populations were determined using 3- and 8-wk-old WT and p50^−/− cRel^−/− mice. Splenocytes from these mice were stained with CD4, CD44, and CD62L antibodies. FACS^® analysis was performed on gated CD4⁺ T cells. Naive T cells were CD44^low CD62L⁺ and memory T cells were CD44^high CD62L⁻. (C) Splenocytes from 2-mo-old WT and p50^−/− cRel^−/− mice were stained with CD4 and CD25. FACS^® analysis was performed on gated CD4⁺ T cells.

Combined absence of p50 and cRel NF-κB subunits does not affect T cell development. (A) FACS^® analysis of CD4 and CD8 expression in WT and p50^−/− cRel^−/− thymocytes. (B) FACS^® analysis of B220, CD3, CD4, and CD8 expression in WT and p50^−/− cRel^−/− splenocytes. (C) CD3 expression level in WT and p50^−/− cRel^−/− CD4⁺ T cells. FACS^® was performed by gating on CD4⁺ cells. (D) EMSAs were performed with nuclear extracts from naive CD4⁺ T cells either untreated or activated with 1 μg/ml plate-bound αCD3 or 1 μg/ml plate-bound αCD3 plus 1 μg/ml αCD28 for 6 h. NF-κB binding sites used were from H-2K^band IL-Rα (CD25). Complexes 1 and 2 are described in the text. (E) EMSAs were performed with nuclear extracts from naive CD4⁺ T cells activated with 1 μg/ml plate-bound αCD3 plus 1 μg/ml αCD28 for 6 h. NF-κB binding site was from H-2K^b. The addition of antibodies to RelA and p100/p52 is indicated.

Impaired cell division of p50^−/− cRel^−/− CD4⁺ T cells after activation. (A and B) WT and p50^−/− cRel^−/− CD4⁺ T cells were CFSE labeled and activated under different conditions as shown for 1–3 d. FACS^® was performed on viable cells by gating on the forward and side scatter characteristics. As shown in the bottom right of B, peaks/shoulders represent the number of times cells underwent division. The percentage indicates cell population that has divided at least once. (C) FACS^® analysis of CD25 (IL-2Rα) expression on WT and p50^−/− cRel^−/− CD4⁺ T cells after 3 d of activation by plate-bound αCD3+αCD28 and IL-2.

IL-2–induced survival pathway does not require p50+cRel. αCD3+αCD28–activated WT and p50^−/− cRel^−/− CD4⁺ T cells were cultured in T cell medium alone or in the presence of 20 ng/ml IL-2 for 1 or 2 d. Cell death was determined by DNA content staining and sub-G₀ quantification.

Lack of involvement of p50+cRel in Akt-induced T cell survival. MIG, myr-AKT, and CA-IKKβ retrovirus-infected WT and p50^−/− cRel^−/− CD4⁺ T cells were cultured in T cell medium without αCD3 or IL-2 for 1 or 2 d. Survival rate of the infected cells was determined by PI staining and FACS^® analysis as in Fig. 6, C and D.