Volume 13, Issue 5, Pages 677-689 (November 2000) NF-κB Activation by the Pre-T Cell Receptor Serves as a Selective Survival Signal in T Lymphocyte Development  Reinhard E Voll, Eijiro Jimi, Roderick J Phillips, Domingo F Barber, Mercedes Rincon, Adrian C Hayday, Richard A Flavell, Sankar Ghosh  Immunity  Volume 13, Issue 5, Pages 677-689 (November 2000) DOI: 10.1016/S1074-7613(00)00067-4

Figure 1 NF-κB Activity Peaks in CD44−CD25+ L Cells and CD8+ Thymocytes (A) Schematic depiction of thymocyte development based on expression of different cell surface markers (Hoffman et al. 1996). (B) Nuclear NF-κB DNA binding activity in tissues. Nuclear extracts from spleen, bone marrow, brain, liver, kidney, and thymus were analyzed for NF-κB DNA binding activity by electromobility shift assay (EMSA). (C) Luciferase assay of tissue extracts from NF-κB luciferase reporter mice. Spleen, bone marrow, brain, liver, kidney, and thymus extracts of NF-κB luciferase transgenic mice were analyzed for luciferase activity. Light units per 100 μg of tissue extract are shown. (D) NF-κB nuclear DNA binding activity in thymocyte subsets. Nuclear extracts were prepared from sorted thymocyte subsets of wild-type mice. NF-κB DNA binding activity of 1.5 μg nuclear extract was analyzed by electromobility shift assay. (E) Peak of NF-κB DNA binding activity in stage III L cells, which express the pre-T cell receptor. Nuclear extracts of E, L, and CD4+/CD8+ thymocytes from wild-type mice were analyzed for NF-κB DNA binding capacity by electromobility shift assay. (F) Supershift analysis of nuclear extracts from L cells reveals predominantly p50-p65 heterodimers. Nuclear extracts from L cells were incubated with antibodies against p50, p52, p65, RelB, c-Rel, and c-myc before electromobility shift assay. (G) Peak of luciferase activity in stage III and stage IV cells, coinciding with expression of the pre-T cell receptor (middle panel). After depletion of CD4- and CD8-positive cells, stage III thymocytes of NF-κB luciferase reporter mice were further separated, according to size, into small E cells and large “β-selected” L cells (right panel) (see flow chart on the left-hand side of the figure). The TCRαβ+, CD4, or CD8 SP cells were sorted as described in Experimental Procedures. Equal numbers of sorted cells were used to determine the luciferase activity. Error bars represent the standard deviations. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)

Figure 2 Absence of NF-κB Activation in Stage III Thymocytes of TCR β–Deficient and RAG1-Deficient Mice (A) Decreased DNA binding activity in nuclear extracts from TCR β–deficient and RAG1-deficient mice. Stage III thymocytes from TCR β–deficient, RAG1-deficient, and wild-type mice were sorted. In addition, stage III cells were obtained from wild-type mice. Nuclear extracts were prepared and analyzed by electromobility shift assay for NF-κB DNA binding activity. (B) Decreased NF-κB transcriptional activity in stage III and stage IV thymocytes from TCR β–deficient mice. TCR β–deficient mice were crossed with NF-κB luciferase reporter mice. Thymocytes of NF-κB-luc+/TCR βo/o and NF-κB-luc+/TCR βwt/wt littermates were sorted into stage III and stage IV subsets, and equal numbers of sorted cells were used to determine luciferase activity. Results shown are means of three independent experiments, normalized to cell number. Error bars represent standard deviations. (C) Activation of NF-κB in stage III cells of RAG1-deficient mice by anti-CD3 treatment. RAG1-deficient mice were intraperitoneally injected with 100 μg of the anti-CD3ε antibody 145-2C11. After 12 hr, thymocytes were prepared, and stage III cells were sorted. Equal amounts of nuclear extracts from the untreated and treated mice were analyzed by electromobility shift assay for NF-κB DNA binding activity. (D) The T cell line 4G4, which lacks either a TCR or pre-TCR, was stably transfected with TCR β, either alone or in combination with one of the pre-TCR isoforms pre-Tαa or pre-Tαb. The stable cell lines have been previously described (Barber et al. 1998). Nuclear extracts (5 μg) were prepared from unstimulated cells and were analyzed by electromobility shift assay using labeled κB and Oct-1 probe. Representative results from at least three independent experiments are shown. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)

Figure 3 Generation of Transgenic Mice Expressing a Constitutively Inhibiting IκBα in Thymocytes (A) The IκBα superinhibitor transgene. IκBα was mutated at the positions indicated, and the coding sequence of the hemagglutinin tag was attached at the 5′ end. This superinhibitor of NF-κB was cloned behind the proximal lck promoter, which is active in thymocytes. For increased expression, a part of the human growth hormone sequence containing introns and a polyadenylation signal was cloned 3′ of the mutated IκBα. (B) Expression levels of the mutated IκBα in transgenic mouse lines. Thymocytes of transgenic mouse lines were tested for expression of the transgenic IκBα by immunoblot. Staining with anti-IκBα visualizes both the slower migrating transgenic and the endogenous IκBα. (C) Reduced NF-κB nuclear DNA binding activity in thymocytes of transgenic mice expressing a mutant IκBα. The transgenic lines #12 and #28 were crossed, and nuclear extracts from thymocytes of 4-week-old male littermates, which were either nontransgenic, single transgenic (#28), or double transgenic (#12 × #28), were analyzed by electromobility shift analysis. (D) Decrease of CD8+ thymocytes in transgenic mice expressing a mutated IκBα. CD4 and CD8 expression on thymocytes of nontransgenic littermates, single-transgenic mice and double-transgenic mice of the lines (#12 × #28) were analyzed by flow cytometry. (E) Decreased numbers of stage III and stage IV subsets in mice expresssing a mutated IκBα in thymocytes. Thymocytes were stained with anti-CD4-Quantumred, anti-CD8-Quantumred, anti-HSA-PE, anti-CD44-biotin/Avidin-Texas red, and anti-CD25-FITC and analyzed by flow cytometry. In the upper panel, a gate was set on HSA+ cells negative for CD4 and CD8 (DN thymocytes). These DN cells were further analyzed according to their expression of CD44 and CD25, as shown in the lower panel. (F) The average number of cells in the indicated fractions from the DN population from transgenic mice (hatched bars) expressing the IκBα superinhibitor compared to nontransgenic controls (solid bars) is shown. The results from the transgenic mice are the average from 14 mice (seven were #12 × #28 crosses, while seven were from #7 × #28 crosses). (G) Wild-type or mutated IκBα transgenic mice received 4 i.p. injections of 1 mg BrdU. Thymocytes were stained with anti-CD4-APC, anti-CD8-Quantumred, anti-CD25-PE, and anti-BrdU. The percentage of BrdU-positive cells was measured in CD25+ or CD25− cells by flow cytometry, after electronically gating on CD4−CD8− cells. The results are from four independent experiments. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)

Figure 4 Expression of Constitutively Active IκB Kinase Leads to Increase in Numbers of Stage IV Cells (A) The constitutively active IKKβ transgene. FLAG-tagged IKKβ was mutated at the positions indicated and cloned behind the thymocyte-specific proximal lck promoter. (B) IκB kinase activity (left panel) and NF-κB DNA binding activity (right panel) in thymocytes from transgenic mice expressing the dominant-active form of IKKβ. (C) Increased numbers of DN stage IV subset in mice expressing the constitutively active form of IKKβ in thymocytes. Thymocytes were stained with anti-CD4-Quantumred, anti-CD8-Quantumred, anti-HSA-PE, anti-CD44-biotin/Avidin-APC, and anti-CD25-FITC and analyzed by flow cytometry, as described in Figure 3. The cell numbers of stage III E, L, and stage IV cells from 16 different transgenic mice from two different founders were tabulated, and the results are shown graphically. Solid bars are nontransgenic mice, while hatched bars represent IKKβ transgenic mice. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)

Figure 5 Expression of Constitutively Active IκB Kinase in RAG1−/− Mice Leads to Rescue of a Fraction of the Cells to the Double-Positive Stage (A) Immunoblot analysis of thymocytes from wild-type, RAG1−/−, and RAG1−/− crossed to dominant-active IKKβ trangenic mice. Total extracts (30 μg) were immunoblotted with antibodies to RAG1 and IKKβ. (B) Rescue of a fraction of DP cells in RAG1−/− mice expresssing a dominant-active IKKβ transgene in thymocytes. Thymocytes were stained with anti-CD4-FITC and anti-CD8-PE and analyzed by flow cytometry. (C) The average cell numbers of total and DP thymocytes from 24 different transgenic mice (12 RAG1−/−, 12 RAG1−/− × IKKβ) were tabulated, and the results are shown graphically. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)

Figure 6 Antiapoptotic Function of Bcl-2 Is Selectively Replaced by Activation of NF-κB during β Selection (A) Selective hypersensitivity to pharmacological inhibition of NF-κB in L cells compared to E cells. Stage III E and L cells, stage IV, DP, and CD4 SP cells were sorted and cultured in the presence of the NF-κB-inhibitor PDTC (pyrrolidine dithiocarbamate) (100 μM) for 12 hr. (B) Loss of Bcl-2 expression in stage III L cells and in the stage IV subset. Stage III E and L cells, stage IV, and DP thymocytes were sorted. Total cell extracts (25 μg/lane) were analyzed by immunoblot for expression of Bcl-2. Representative results of one of three independent experiments are shown. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)

Figure 7 Model of the Role of NF-κB during β Selection: Selective Rescue from Apoptotic Cell Death (A) Schematic depiction of the expression of Bcl-2 (immunoblot) and NF-κB (luciferase and EMSA) during T lymphocyte development. The expression of Bcl-2 is constitutive in early DN cells, whereas activation of NF-κB occurs only in the subset of cells expressing the pre-TCR. (B) A model showing the proposed role of pre-TCR-induced NF-κB as a selective survival signal in the development of α/β lineage T lymphocytes. Immunity 2000 13, 677-689DOI: (10.1016/S1074-7613(00)00067-4)