The Survival of Antigen-Stimulated T Cells Requires NFκB-Mediated Inhibition of p73 Expression  Yisong Y Wan, James DeGregori  Immunity  Volume 18, Issue 3, Pages 331-342 (March 2003) DOI: 10.1016/S1074-7613(03)00053-0

Figure 1 Adenoviral-Mediated mIκB Expression Blocks Endogenous NFκB Activity CAR Tg lymphocytes were transduced with Ad-GFP or Ad-mIκB (at a multiplicity of infection [MOI] of 5 for this and all subsequent experiments unless stated otherwise). 16 hr posttransduction, cells were activated by ConA. At 0, 24, or 30 hr postactivation, GFP- or mIκB-expressing T cells (GFP+ B220− cells in the lymphocyte size gate) were purified by FACS (greater than 97% purity in this and all subsequent experiments). (A) The expressions of mIκB and endogenous IκB were detected by immunoblotting. The HA-tagged mIκB migrates slower than endogenous IκB during gel electrophoresis. (B) The mRNA expression of Bfl-1/A1 relative to 18S rRNA was determined by real time RT-PCR. At 30 hr, there was a significant difference between Ad-GFP and Ad-mIκB transduced T cells in Bfl-1 expression. For all figures, * indicates P < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 using paired Student's t test, and standard deviation is indicated. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)

Figure 2 NFκB Is Required for T Cell Survival following Activation In Vitro and In Vivo (A) CAR Tg T cells were transduced with Ad-GFP or Ad-mIκB, and 2 × 106 cells (total cells, including B cells, macrophages, etc.) were cultured for 16 hr in a 48-well plate and then cultured with or without ConA for 24, 30, or 40 hr. In the top panel, transduced T cells were cultured without activation, and the percentage of T cells (B220Neg) expressing GFP or GFP/mIκB is shown (i.e., transduction efficiency). In the bottom panel, at each time point the number of live T cells in the culture was determined by cell counting with trypan-blue exclusion using a hemocytometer and flow cytometry for Thy1.2 positive cells (that also exclude propidium iodide [PI]). The percentage of viable GFP+ T cells was multiplied by the counted number of total viable cells to obtain the number of GFP+ T cells in each culture. The average plated numbers of GFP+ T cells in each well postinfection (t = 0) were 143 (GFP) and 108 (mIκB) × 103 GFP+ T cells. (B) CAR Tg T cells were transduced with Ad-GFP or Ad-mIκB and then activated by ConA in the presence of 20 U of recombinant mouse IL-2 for 36 hr. To measure the survival rate of transduced T cells following ConA activation, the percentage of GFP+ T cells was multiplied by the percentage of cells in the live gate as determined by light scatter. This fraction was set at 1 for Ad-GFP transduced T cells without IL-2. (C) DO11.10/CAR Tg spleen and lymph node cells were transduced with Ad-GFP or Ad-mIκB ex vivo. Without culturing, transduced T cells (1 × 107 total cells including nontransduced T cells and other cell types) were immediately transferred into Balb/C recipient mice by subocular injection. 16 hr later, 0.5 mg OVA peptide was injected into each recipient intraperitoneally. At different time points post OVA injection, GFP- or mIκB-expressing T cells in peripheral blood were monitored by anti-CAR/anti-B220 staining and flow cytometry. The percentage of GFP+CAR+ T cells (B220Neg) in recipients is plotted. Similar relative differences were obtained following sacrifice of recipients at 3 days and analysis of spleen and lymph node lymphocytes (the total number of lymphocytes did not vary significantly between recipients). (D) CAR Tg T cells were transduced with Ad-GFP or Ad-mIκB at an MOI of 10 and then activated by ConA. At 30 and 36 hr postactivation, Annexin V-APC staining was detected on transduced T cells (B220Neg GFP+) to identify apoptotic cells. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)

Figure 3 NFκB Activation Is Dispensable for G1 to S Phase Progression CAR Tg T cells were either not transduced (mock) or transduced with Ad-GFP or Ad-mIκB for 16 hr. T cells were either not activated or activated by ConA and harvested at the indicated times postactivation. (A) Transduced T cells (B220Neg GFP+) were purified by FACS and c-Myc expression was detected by immunoblotting. (B) 24 hr postactivation (or not activated as the control), T cell size was monitored by increased forward scatter in GFP- or GFP/mIκB-expressing T cells using flow cytometry. (C) Rb expression levels and phosphorylation state in purified GFP+ T cells were determined by immunoblotting. (D) The mRNA levels of Cdk1, an E2F target gene, were detected by ribonuclease protection assays (RPA) in purified GFP+ T cells. CycD3 mRNA levels, which are not affected by antigen stimulation, served as an internal control for this assay. (E) 24 hr postactivation, BrdU was added to the culture medium. Under our T cell activation and culture conditions, S phase entry occurs between 24 and 30 hr post-ConA activation. 30 hr postactivation, viable GFP- or GFP/mIκB-expressing T cells were purified by FACS and stained with fluorescent labeled anti-BrdU, followed by PI staining as a measure of DNA content. BrdU incorporation and PI intensity were determined by flow cytometry. The percentage of GFP+ T cells that incorporated BrdU is indicated. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)

Figure 4 Cdk Activity Is Required for the Apoptosis of Antigen-Stimulated T Cells Lacking NFκB Activation (A) T cells were activated with ConA in the presence of Rosco (20 μM; Calbiochem), DMSO, or neither (mock). At 24 hr postactivation, T cells were purified by MACS. The expression and phosphorylation state of Rb was assessed by immunoblotting. (B) CAR Tg T cells (DO11.10 Tg for OVA activation) were transduced with Ad-GFP or Ad-mIκB and then activated in the presence of DMSO or Rosco (two experiments each using either ConA or OVA activation gave similar results and are combined). At different time points postactivation, GFP- or GFP/mIκB-expressing T cell survival rates were determined as described as in Figure 2B. (C) CAR Tg T cells were transduced with Ad-GFP or Ad-mIκB +/− Ad-p16, activated with ConA, and surviving T cell numbers at 30 and 40 hr (+/− ConA) were determined as in Figure 2A. An average of 133 (GFP + Con), 102 (mIκB + Con), 110 (GFP + p16) and 100 (mIκB + p16) ×103 GFP+ T cells were plated per well at t = 0. Differences between paired samples (indicated by arrows) are statistically significant. There were no significant differences between GFP and mIκB + Rosco samples and between GFP + p16 and mIκB + p16 samples. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)

Figure 5 NFκB Activation Is Required to Limit p73 Upregulation following Antigen Stimulation (A) CAR Tg T cells were transduced with Ad-GFP or Ad-p16, activated with ConA for 30 hr, and p73 and actin protein levels were assayed by immunoblotting. (B) Ad-GFP or Ad-mIκB transduced and ConA-activated T cells were FACS purified at different time points postactivation and p73 and actin protein levels determined. Note that the blot probed with anti-p73 was trimmed just above 50 kDa, preventing the detection of lower molecular weight immunoreactive species. (C) CAR Tg or CAR/DO11.10 double Tg lymphocytes were transduced with Ad-GFP or Ad-mIκB. 18 hr posttransduction, T cells were activated with either ConA or OVA in the presence or absence of 20 μM Rosco (as indicated). GFP- or GFP/mIκB-expressing T cells were purified by FACS. Total cellular protein was isolated using either the Trizol method (left panels) or by lysis in RIPA buffer (right panels). p73, Rb, and α-actin protein expression was assayed by immunoblotting (note that loading is variable in the left panel). p73−/− and p73+/+ T cells stimulated for 30 hr with ConA served as controls. (D) As in (B), except immunoblotted for p53 and actin expression. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)

Figure 6 The Inhibition of NFκB Activity Differentially Affects the mRNA Expression of p73 Isoforms and p53/p73 Target Genes CAR Tg T cells were transduced with Ad-GFP or Ad-mIκB. At different time points post ConA activation, GFP- and GFP/mIκB-expressing T cells were sorted by FACS, and total RNA was extracted and purified. TAp73 or total p73 mRNA levels were assayed by real time RT-PCR as described in Experimental Procedures. Expression levels were normalized to 18S in the Figure, but indistinguishable results were obtained by normalization to β-actin. The increased TAp73 expression in mIκB relative to GFP-expressing cells at 30 hr postactivation is statistically significant. Similar results were obtained using OVA activation. Similarly, the relative mRNA levels for p21 and PERP were assayed by real time RT-PCR. The differences in p21 and PERP mRNA levels between GFP and mIκB samples at 30 hr are significant. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)

Figure 7 p73 Is a Critical Effector of Apoptosis Resulting from Antigen Stimulation of IκB-Expressing T Cells (A) Lymphocytes from CAR Tg/p73+/+ or CAR Tg/p73−/− mice were transduced with Ad-GFP or Ad-mIκB, activated with ConA (or not), and surviving T cell numbers (+/− ConA) were determined as in Figure 2A. An average of 152 (p73+/+ with GFP), 136 (p73+/+ with mIκB), 110 (p73−/− with GFP), and 95 (p73−/− with mIκB) × 103 GFP+ T cells were plated per well at t = 0. The differences between paired samples (as indicated by arrows) are significant. (B) Lymphocytes from CAR Tg mice were transduced with the indicated combination of Ad-GFP, Ad-mIκB/GFP, and Ad-DNp53/GFP (MOI of 5 for each virus; combined MOI of 10), activated with ConA (or not), and surviving T cell numbers (+/− ConA) were determined as in Figure 2A. An average of 140 (GFP+GFP), 136 (mIκB+GFP), 147 (GPF+dnp53), and 122 (mIκB+dnp53) ×103 GFP+ T cells were plated per well at t = 0. Differences between mIκB + GFP-expressing and mIκB + DNp53-expressing CAR Tg T cells following stimulation are significant. (C) Model: NFκB-dependent inhibition of TAp73 expression prevents apoptosis and promotes proliferation by the Cdk-Rb-E2F pathway. Immunity 2003 18, 331-342DOI: (10.1016/S1074-7613(03)00053-0)