IκBα nuclear export enables 4–1BB induced cRel activation and IL-2 production to promote CD8 T cell immunity

Mice

Nfkbia^NES/NES mice were generated as previously described (25). Six- to twelve-week-old littermate Nfkbia^WT/WT (WT) and Nfkbia^NES/NES mice were age and sex matched for all experiments. Male host mice (B6.SJL-Ptprc^a Pepc^b/BoyJ, Ly5.1) utilized for mixed bone marrow were obtained from The Jackson Laboratory (Bar Harbor, ME). Rel^−/− mice were obtained from Dr. Jonathan Levenson (28) with permission from Dr. Hsiou-Chi Liou. The P14 LCMV GP33-specific TCR transgenic mice on the Ly5.1 background (provided by Dr. Rafi Ahmed, Emory University) were bred in University of Wisconsin–Madison. P14 mice were crossed with Nfkbia^NES/NES mice to generate P14/Nfkbia^WT/WT and P14/Nfkbia^NES/NES mice. All experiments were approved and conducted in accordance by the Institutional Animal Care and Use Committee of the University of Wisconsin–Madison. All mice were housed and bred in specific pathogen-free conditions at the University of Wisconsin–Madison.

In vitro T cell stimulation and ELISA

Naive T cells were isolated from spleens using negative selection with the Pan T cell Isolation Kit II (Miltenyi Biotec) in addition to anti-CD44 microbeads (Miltenyi Biotec). Naïve CD8 T cells were isolated from spleens using negative selection with the Naive CD8a+ T cell Isolation kit (Miltenyi Biotec) and purity was confirmed by flow cytometry to be >95% CD8 CD44^lo cells. Cells were cultured in RPMI medium supplemented with 10% FBS, 50 uM β-mercaptoethanol, 1X GlutaMAX (Life Technologies), 100 U/mL Penicillin and 100 ug/mL Streptomycin. Cell culture plates were coated with anti-CD3 (Bio X Cell, Clone 17A2) and anti-CD28 (Bio X Cell, Clone 37.51) antibodies overnight at 4°C or for 2 hours at 37°C. For 4–1BB and OX40 stimulation, soluble anti-4–1BB (Bio X Cell, Clone 3H3) or anti-OX40 (Bio X Cell, Clone OX86) antibodies were added at the start of stimulation with anti-CD3 and anti-CD28. For CD27 stimulation, soluble anti-CD27 antibody (eBioscience, Clone LG.3A10) was crosslinked with anti-Rat and anti-Hamster Ig κ Light Chain (BD, #550336) and added at the start of stimulation. For the IL-2 ELISA (eBioscience), cells were cultured in 96-well plates for 48 hours and supernatants were collected and frozen at −80°C until ready to assay. ELISA data are represented as the mean +/− SEM of at least three technical replicates and significance was determined using an unpaired Student’s t test. Generated p values are two-tailed and p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) was considered statistically significant. For proliferation assays, splenocytes were stained with 10 uM CFSE (Invitrogen) in RPMI with 10% FBS for 10 minutes at room temperature. Naive CD8 T cells were then isolated as described above. For cell survival assays, 1×10⁶ naive CD8 T cells were stimulated with plate bound 5 μg/mL each of anti-CD3 and anti-CD28 antibodies, and soluble anti-4–1BB antibody for 48 hours in 24-well plates. Following this initial 48 hours, viable cells were quantified by the MACSquant flow cytometer and 4×10⁵ cells were re-plated in 24 well plates in RPMI media supplemented with 10% FBS, 50 uM β-mercaptoethanol, 1X GlutaMAX (Life Technologies), 100 U/mL Penicillin and 100 ug/mL Streptomycin. Viable cells were quantified by the MACSquant flow cytometer 48 hours following re-plating. For cell survival studies, data are represented as the mean +/− SEM of at least three technical replicates and significance was determined using an unpaired Student’s t test. Generated p values are two-tailed and p < 0.001 (***) was considered statistically significant.

Electrophoretic mobility shift assay (EMSA) and supershift assay

T cells were lysed in Totex buffer (20 mM HEPES [pH 7.9], 350 mM NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT) with 1X HALT protease inhibitor cocktail (Thermo Fisher Scientific) and 1 uM DTT. Cell extracts (6–10 μg) were incubated with 1 μg of poly(dI-dC) and EMSA binding buffer (75 mM NaCl, 15 mM Tris HCl [pH 7.5], 1.5 mM EDTA, 1.5 mM DTT, 7.5% glycerol, 0.3% NP-40, 20 μg/mL BSA) in a total reaction volume of 9 uL for 20 minutes at 4°C. For supershift assays, 1 μL of each antibody (0.2 μg) for RelA (Santa Cruz, clone C-20), cRel (Santa Cruz, clone C) or RelB (Santa Cruz, clone C-19) was added to cell extracts, with the reaction volume not exceeding 10 μL. One μL of ³²P labeled double stranded oligonucleotides containing the κB site from the Igκ gene (5’-TCAACAGAGGGGACTTTCCGAGAGGCC-3’), the IL-2 gene (5’-GACCAAGAGGGATTTCACCTAAATCCA-3’), and the Oct-1 consensus site (Promega) was added to the reaction and incubated for 20 minutes at room temperature and spun down at 14,000 RPM prior to sample loading and separation on a 4% native polyacrylamide gel, which was then dried and exposed to film or a storage phosphor screen. A Typhoon Biomolecular Imager (GE Healthcare) and accompanying ImageQuant TL software were used to scan and quantify resulting EMSAs. NF-κB binding was first normalized to Oct-1 binding and fold change was determined. Data is represented as the mean +/− SEM of at least three biological replicates and significance was determined using a paired Student’s t test. Generated p values are two-tailed and p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) was considered statistically significant.

Immunoblotting and immunoprecipitation analyses

Samples were lysed in Totex buffer and normalized by total protein amount before being diluted 1:1 with 2X Laemmli sample buffer and boiled for 10 minutes. Protein extracts were separated on a 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore). Membranes were blocked in PBS containing 0.2% Tween 20 (PBST) and 5% milk for 1 hour. The membrane was then probed with the antibodies against RelA (Santa Cruz, clone C-20), RelB (Santa Cruz, clone C-19), cRel (Santa Cruz, clone C), p105/p50 (Santa Cruz, clone NLS), p100/p52 (Santa Cruz, clone C-5), IκBα (Santa Cruz, clone C-21), IκBβ (Santa Cruz, clone C-20) or tubulin (Calbiochem, clone DM1A) diluted in PBST overnight. The following day, membranes were washed 3 times in PBST and incubated with the secondary anti-rabbit IgG (GE, NA934V) or anti-goat IgG antibody (GE, NA931V) conjugated to horseradish peroxidase. Membranes were washed 3 times in PBST and developed using Amersham ECL Western Blotting Detection Reagents (GE Healthcare). To detect degradation of IκBα and IκBβ, cells were incubated with the protein synthesis inhibitor, cycloheximide, at 20 μg/mL for 30 minutes at 37°C prior to stimulation. For IκBα:NF-κB co-immunoprecipitation (IP) analyses, samples were lysed in IP buffer (20 mM Tris, 250 mM NaCl, 3 mM EDTA, 3mM EGTA, 0.5% NP-40, 1mM DTT, and 1X HALT) for 20 minutes at 4°C. Cells were then completely lysed by 3 cycles of freezing by incubating tubes containing cells in 70% ethanol in dry ice for 2 minutes and thawing in a 37°C water bath for 2 minutes. This was followed by an incubation at 4°C for 10 minutes and centrifugation at 14,000 RPM for 10 minutes at 4°C. Total protein concentration in lysates was determined and approximately 150–200 ug of protein was brought to a final volume of 500 μL in IP buffer. Samples were rotated at 4°C with 2 μg of anti-IκBα antibody (Santa Cruz, clone C-21) for 1 hour. In the meantime, protein A sepharose beads (GE Healthcare) were equilibrated with IP buffer. Approximately 50 μL of 1:1 equilibrated beads were added to each IP sample. Samples were rotated overnight at 4°C. Beads were washed with IP buffer 3 times and then resuspended in 100 μL Laemmli buffer, boiled for 10 minutes, and loaded onto a 10% SDS-PAGE gel for analysis by immunoblotting.

Immunofluorescence staining and ImageStream acquisition and analysis

To induce maximal RelA, cRel, and IκBα nuclear localization, naïve and activated CD8 T cells and B cells were incubated with 20 ng/mL Leptomycin B (LMB) (Cayman Chemical) for 40 minutes at 37°C. Activated CD8 T cells were stained with Fixable Viability Dye (FVD) eFluor 780 (eBioscience) prior to fixation. CD8 T cells and B cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.3% Triton X-100 for 30 minutes. Following permeabilization, cells were stained with anti-RelA (Cell Signaling #D14E12, 1:200), anti-cRel (Santa Cruz #SC-71, 1:150), or anti-IκBα (Santa Cruz #SC-371,1:200) antibodies with 1.5% normal goat serum overnight at 4°C. The following day, primary antibody was removed and cells were incubated with a goat anti-rabbit secondary antibody directly conjugated to Alexa Fluor 488 (Cell Signaling) in 1.5% normal goat serum for 1 hour at 4°C. Nuclear staining was achieved with 5 uM DRAQ5 (BD Biosciences) for 10 minutes at 37°C immediately prior to acquisition. Cells were collected at 60X magnification and the low speed/high sensitivity setting using an ImageStream flow cytometer (Amnis ImageStreamX Mk II, 2 lasers [488 nm and 642 nm] and 6 channels [457/45, 533/55, 577/35, 610/30, 702/85, 762/35]). Single, focused, and viable cells were collected based on Area and Aspect Ratio in the brightfield channel, Gradient RMS in the nuclear channel, and exclusion of FVD eFluor 780. Utilizing the IDEAS software package, the “similarity score” was calculated utilizing at least 1500–2000 cells based on positive staining for RelA, cRel, or IκBα and DRAQ5.

Gene expression analysis and RNA sequencing

Naïve WT and Nfkbia^NES/NES CD8 T cells stimulated with plate bound 5 μg/mL anti-CD3 and 5 μg/mL anti-CD28 antibodies with or without soluble anti-4–1BB antibody for 12 hours. Cells were lysed in TRIzol reagent (Invitrogen) and frozen at −80° C until total RNA was extracted with the Direct-zol RNA MiniPrep Plus (Zymo Research) kit. For RNA-seq, libraries were generated with the TruSeq Stranded mRNA Library Prep Kit for NeoPrep (Illumina) and sequencing was performed on an Illumina HiSeq 2000.

RNA-seq processing and analysis

Adapter-trimmed reads were aligned to the mouse (mm10) genome using STAR with default parameters (29). Only primary mapped reads with alignment score >30 were selected by using “samtools view” with “–F 2820” and “–q 30” and then input into featureCounts (30) to get counts per gene using GENCODE M6 (GRCm38.p4) annotation. The number of raw reads from sequencing is approximately 22 million per sample and the final number of reads after filtering was approximately 16 million reads per sample. The average Pearson’s correlation between biological replicates was approximately 0.995 using log2 CPM (counts per million reads).

To obtain the final “NES dependent” gene list, we performed two rounds of differential gene expression analysis by using R package EdgeR (glmFit and glmLRT functions) (31). First, we selected 4–1BB induced genes by testing between 12 hr 4–1BB stimulated samples (12hr+) against 12 hr unstimulated samples (12hr-) (N = 3 for each time point) in WT (Log2 fold change > 1 and FDR < 0.01). A second test between WT vs. NES at 12 hours of 4–1BB stimulation was performed to determine NES dependency (LFC < −0.5 and FDR < 0.05). Lastly, by intersecting the genes from this two-round analysis, we divided these genes into three categories: 1) induced in WT but induction is not NES dependent (“NES independent”); 2) induced in WT and induction is NES dependent (“NES dependent”); 3) not induced in WT and expression is reduced in NES mutant (“NES reduced”). Of these, NES-dependent genes’ Ensembl IDs were entered into DAVID 6.8 to perform functional enrichment analysis on KEGG_PATHWAY. The RPKM for these genes were normalized to [0,1] range for each gene and then used to produce an expression heatmap. The top 50 induced genes in WT were ranked based on induction fold change in WT and selected after filtering genes with average RPKM < 3 for WT 12hr-.

Motif enrichment analysis

To study the binding motif enrichment of RelA:p50, cRel:p50, and cRel:cRel (32), position weight matrixes (PWMs) (33) were inputted into HOMER motif discovery software (34) and their occurrence was scanned for in the promoter regions of “NES dependent” and “NES independent” genes (promoters defined as −1000bp to 300bp of TSS). Fisher’s exact test was performed to calculate the fold enrichment and p-value for each motif in “NES dependent” against “NES independent” genes.

Viral infections

Six- to eight- week old WT and Nfkbia^NES/NES mice were infected i.p. with 2×10⁵ PFU of the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) to induce an acute infection. For re-infection of mixed bone marrow chimeras, LCMV immunized mice were infected i.v. with 2×10⁶ PFU of the clone 13 strain of LCMV. Plaque assays on Vero cells were used for quantification of infectious LCMV, as previously described (27).

Flow cytometry and cell staining

Peripheral blood was collected in 4% sodium citrate and mononuclear cells were isolated following density centrifugation with Ficoll Histopaque (Sigma). Single cell suspensions of mononuclear cells from spleens were obtained using a standard procedure. Approximately 1×10⁶ cells were stained per sample. Cells were first stained with Fixable Viability Dye eFluor 780 or eFluor 450 (eBioscience), followed by staining with MHC Class I tetramers specific for the LCMV epitope H2-D^b NP_396–404 and/or H2-D^b GP_33–41 (NIH Tetramer Facility) and antibodies against cell surface markers, CD8 (BD Biosciencies, Clone 53–6.7), CD44 (BD Biosciencies, Clone IM7), IFN-γ (BD Biosciences, Clone XMG1.2), CD62L (BD Biosciences, Clone MEL-14), KLRG (BD Biosciences, Clone 2F1), Ly5.1 (BD Biosciences, Clone A20), Ly5.2 (BD Biosciences, Clone 45.2), Thy1.1 (BD Biosciences, Clone OX-7) and Thy1.2 (BD Biosciences, Clone 53–2.1). Cells were then fixed with 4% paraformaldehyde. For intracellular cytokine staining, splenocytes were stimulated ex vivo with LCMV epitope NP_396–404 (200 ng/mL), Golgi Plug (BD Biosciences), and IL-2 (2.5 ng/mL) for 5 hours at 37°C. Following incubation, cells were stained with cell surface markers first, fixed and permeabilized utilizing the Intracellular Fixation & Permeabilization Buffer Set (eBioscience) and then stained with anti-IFN-γ antibody. For LCMV studies, data are represented as the mean +/− SEM of biological replicates and significance was determined using an unpaired Student’s t test. Generated p values are two-tailed, and p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) was considered statistically significant.

Generation of mixed bone marrow chimeras

Bone marrow cells were obtained from the femurs and tibias of host strain male (B6.SJL-Ptprc^a Pepc^b/BoyJ, Ly5.1) mice, WT (Ly5.2) and Nfkbia^NES/NES mice (Ly5.2). An equal number of bone marrow cells from host strain mice (Ly5.1) was mixed with an equal number of cells from either WT (Ly5.2) or Nfkbia^NES/NES (Ly5.2) mice. A total of 8×10⁶ cells were adoptively transferred into lethally irradiated (450 rads x 2) male host mice (Ly5.1). Following reconstitution, host mice were given neomycin (25 μg/mL) and Polymyxin B (13 μg/mL) in drinking water every other day for 6 weeks. Reconstitution of the hematopoietic compartment was assessed 6 weeks following reconstitution by analysis of peripheral blood. Mice were infected with LCMV (Armstrong strain) one week later.

P14/Nfkbia^WT/WT and P14/Nfkbia^NES/NES adoptive cell transfer assay

For P14 adoptive transfer studies, naïve CD8 T cells were purified from P14/Nfkbia^WT/WT and P14/Nfkbia^NES/NES mice spleens and 10⁴ cells of the appropriate genotype were transferred into recipient mice (Jackson Laboratory, B6.PL-Thy1^a/CyJ). Mice were infected with LCMV (Armstrong strain) 24 hours later as above.

IκBα NES is not required for acute NF-κB activation and proliferation in CD3+CD28-stimulated CD8 T cells

To begin to analyze the activation and functional status of T cells in Nfkbia^NES/NES mice (25), we first investigated the responses of naïve splenic T cells to TCR and costimulatory CD28 signaling by stimulating them on culture plates coated with agonistic anti-CD3 and anti-CD28 antibodies (referred from here on as “CD3+CD28”). The amounts of antibodies used were based on preliminary titration studies (not shown). Electrophoretic mobility shift assay (EMSA) demonstrated that basal and CD3+CD28-induced NF-κB activities were partially reduced in Nfkbia^NES/NES T cells relative to Nfkbia^WT/WT (WT) littermate control cells (Supplemental Figure 1A). To specifically investigate the impact of mutant IκBα on CD8 T cells, naïve splenic CD8 T cells were analyzed as above. We evaluated the 2 hour time point to examine acute CD3+CD28 induced activation potential and further performed supershift assays to decipher which of the NF-κB subunits was involved. Basal NF-κB activity, primarily composed of RelA, was significantly reduced in Nfkbia^NES/NES CD8 cells relative to WT counterparts (Figure 1A). Reduced basal NF-κB activity suggested the possibility that expression of NF-κB-regulated NF-κB family members might be reduced in Nfkbia^NES/NES CD8 T cells as in the case with splenic mutant B cells (25), which was confirmed by western blot analysis showing reduced expression of cRel, RelB, p105/p50 and p100, but not RelA (Figure 1B). IκBα expression was slightly increased while IκBβ expression was slightly reduced in Nfkbia^NES/NES cells, as was the case in mutant splenic B cells (25). Despite these expression differences of NF-κB and IκB proteins, activation of both RelA and cRel complexes following CD3+CD28 stimulation was not significantly inhibited in the Nfkbia^NES/NES CD8 T cells (Figure 1A). Stimulation of Nfkbia^NES/NES and WT CD8 T cells with CD3+CD28 induced comparable levels of: (i) IL-2 receptor α chain (CD25) and TNFR family member (4–1BB, CD27, OX40) expression, as analyzed by flow cytometry (Figures 1C), (ii) IL-2 secretion, as assessed by ELISA with Rel^−/− CD8 T cells serving as a control (Figures 1D), and (iii) cell proliferation, as measured with a CFSE dilution assay (Figures 1E). Thus, contrary to B cells whose maturation, activation and proliferation were significantly defective in Nfkbia^NES/NES mice (25), the IκBα NES was not necessary for optimal acute CD3+CD28-induced NF-κB activation, activation marker expression, IL-2 secretion and proliferation in CD8 T cells.

IκBα NES mutation causes 4–1BB signaling defects and functional deficits in CD8 T cells

Proper T cell responses require signaling through not only TCR activation but also additional co-stimulatory signals (4). Because 4–1BB was expressed at low to undetectable levels in naïve cells, but was robustly induced relative to other TNFR family members in both WT and Nfkbia^NES/NES CD8 T cells (Figures 1C), we next focused our analyses on 4–1BB signaling. Stimulation of naïve CD8 T cells with an agonistic 4–1BB antibody elicited no measurable NF-κB signaling (not shown). Next, in order to measure the impact of the NES mutation on 4–1BB signaling, we stimulated naïve CD8 T cells with CD3+CD28 antibodies in the presence or absence of 4–1BB antibody. We reasoned that 4–1BB would be induced in CD3+CD28 activated CD8 T cells, which would then enable stimulation via the 4–1BB antibody, simulating the dynamics of naïve CD8 T cell stimulation by antigen presenting cells (35). Stimulation with CD3+CD28+4–1BB activated both cRel and RelA complexes in WT T cells (Figure 2A), but the dominant NF-κB complexes shifted from RelA in naïve cells activated by CD3+CD28 to cRel in CD3+CD28+4–1BB-stimulated CD8 T cells (Figure 2B). In striking contrast, activation of RelA and cRel in Nfkbia^NES/NES cells stimulated with 4–1BB was significantly reduced relative to WT controls and the impact of inhibition was greater for cRel than RelA (an average of 2.3-fold vs 1.5-fold, respectively) (Figure 2B). Similar differences were also evident at 12 hours of CD3+CD28+4–1BB stimulated T cells (Supplemental Figure 2A). Although certain members of the TNFR superfamily, such as CD40, can strongly activate non-canonical NF-κB (RelB:p52 complex) in B cells (Supplemental Figure 2B) (36), supershift analyses of 4–1BB-stimulated WT and Nfkbia^NES/NES CD8 T cells revealed the lack of detectable RelB complexes (Supplemental Figure 2C). In addition, while the processing of the precursor protein p100 to its active p52 form was nearly completely induced by CD40 engagement in WT and Nfkbia^NES/NES B cells (Supplemental Figure 2D), the majority of p100 remained unprocessed in 4–1BB-stimulated CD8 T cells of both genotypes (Supplemental Figure 2E). Thus, activation of only canonical NF-κB complexes was significantly inhibited in 4–1BB-stimulated Nfkbia^NES/NES CD8 T cells.

The change in cRel dominance of activation following 4–1BB stimulation in WT CD8 T cells (Figure 2B) correlated with significantly larger amounts of IL-2 production in response to CD3+CD28+4–1BB stimulation in comparison to CD3+CD28 alone (Figure 2C). 4–1BB-induced IL-2 production was significantly defective in Nfkbia^NES/NES CD8 T cells, approaching the level of the CD3+CD28 alone condition (Figure 2C). Furthermore, upregulation of CD25 (IL-2Rα) by CD3+CD28+4–1BB stimulation, which was again much higher than that induced by CD3+CD28, was also significantly reduced in Nfkbia^NES/NES CD8 T cells (Figure 2D). The level of phosphorylated STAT5A (pY694) downstream of CD25 signaling under CD3+CD28+4–1BB stimulation in WT T cells was reduced in Nfkbia^NES/NES cells, which was similar to that detected in similarly stimulated Rel^−/− cells (Figure 2E, right). Thus, the CD25 induction defect in the mutant cells likely stemmed from reduced feed forward signaling mediated by IL-2 (37). Moreover, exposure of naïve Nfkbia^NES/NES CD8 T cells to CD3+CD28 and agonistic antibodies to OX40 or CD27 also demonstrated defective IL-2 production (Figure 2F) and CD25 expression (for OX40 stimulation) (Figure 2G). These results suggested that Nfkbia^NES/NES CD8 T cell defects were not limited only to 4–1BB but were shared by other TNFR superfamily members.

Based on the above results, we expected that CD8 T cell proliferation in response to CD3+CD28+4–1BB would be significantly reduced in Nfkbia^NES/NES CD8 T cells relative to the WT cells. However, while both WT and mutant cells stimulated with CD3+CD28+4–1BB proliferated more than CD3+CD28 stimulation alone, Nfkbia^NES/NES CD8T cells proliferated similarly to the WT cells under the same conditions (Figure 2H). We therefore next considered the possibility of higher IL-2 secretion induced by 4–1BB stimulation in WT CD8 T cells relative to mutant cells affecting cell survival. To test this, CD8 T cells were first stimulated by CD3+CD28+4–1BB for 48 hours and then cultured for additional 48 hours without any stimuli (Supplemental Figure 2F for schematic). The recovery of Nfkbia^NES/NES CD8 T cells was less than 50% of WT cells (Figure 2I). When a CD25 blocking antibody was included during the first 48 hour activation phase to block IL-2 signaling, cell recovery was nearly completely eliminated for both WT and Nfkbia^NES/NES T cells (Figure 2I). Thus, IL-2 signaling was required for the accumulation of activated CD8 T cells upon withdrawal of activation signals. Based on these results, we propose that a major function of secreted IL-2 and CD25 signaling in response to 4–1BB stimulation was to promote survival and continued proliferation of activated CD8 T cells. Perhaps, this phenomenon reflects programmed proliferation and accumulation of activated CD8 T cells, following withdrawal of TCR signaling (38). This aspect of CD8 T cell biology was significantly defective in Nfkbia^NES/NES CD8 T cells.

IκBα NES is necessary for CD3+CD28 induced cytoplasmic localization of IκBα:cRel complexes to enable efficient 4–1BB signaling

Given that the NES of IκBα promotes its own cytoplasmic localization with its associated NF-κB proteins, in particular cRel (25), we next examined subcellular localization of IκBα, RelA and cRel in naïve versus CD3+CD28-stimulated Nfkbia^NES/NES and WT CD8 T cells. After fixation, permeabilization and staining with appropriate antibodies against these proteins (see Materials and Methods), we utilized ImageStream flow cytometry and the IDEAS software to calculate a log transformed Pearson correlation coefficient between the pixel values of two images to derive a “similarity score” (39). We used DRAQ5 nuclear stain along with NF-κB/IκBα staining to obtain similarity scores such that a higher similarity score denoted higher nuclear localization of the protein of interest. We further employed the nuclear export inhibitor, leptomycin B (LMB) (40), as a control to demonstrate maximal nuclear localization of NF-κB and IκBα proteins. We first tested the utility of this assay using splenic B cells isolated from WT and Nfkbia^NES/NES hosts. The similarity scores for IκBα and cRel were higher in Nfkbia^NES/NES B cells compared to WT cells, which were similar to those induced by LMB in WT cells (Supplemental Figure 3). However, RelA in both WT and mutant cells showed lower similarity scores than LMB conditions. These results are consistent with the NES-mutant IκBα causing nuclear localization of associated cRel, but not RelA, complexes, as reported previously (25).

Having validated the assay, we next evaluated subcellular localization of IκBα, RelA and cRel in naïve WT and Nfkbia^NES/NES CD8 T cells. Analysis of fluorescence imaging indicated that IκBα is largely cytoplasmic in T cells of both genotypes (Figure 3A, IκBα, upper). Correspondingly, the similarity scores for IκBα in both genotypes were similar and lower than that induced by LMB treatment in WT cells (Figure 3A, IκBα, lower). The similarity score for RelA in Nfkbia^NES/NES CD8 T cells was slightly higher than for WT cells but was still much lower than that induced by LMB (Figure 3A, RelA, lower). For cRel, the similarity score was slightly higher in the mutant cells than WT cells and similar to that increased with LMB treatment in WT cells (Figure 3A, cRel, lower). Following CD3+CD28 stimulation, expression of not only TNFR family members (Figure 1C) but also cRel and p50 increased in WT CD8 T cells (Figure 3B), which was accompanied by increased formation of IκBα/cRel complexes (Figure 3C). In addition, IκBα and RelA levels were slightly reduced in activated WT cells (Figure 3B). Now, LMB treatment of CD3+CD28 activated WT CD8 T cells demonstrated a greater increase in the similarity scores in both IκBα and cRel, similar to RelA, relative to those in naïve cells (Figure 3A and 3D, compare WT +/− LMB). In Nfkbia^NES/NES CD8 T cells, cRel and p50 levels also increased with a further reduction in RelA, but not IκBα (Figure 3B); IκBα and cRel, but not RelA, displayed high similarity scores that approached those scores induced by LMB in WT cells (Figure 3D, NES). These results suggested that newly formed IκBα/cRel complexes in activated T cells were mis-localized to the nucleus in Nfkbia^NES/NES CD8 T cells while RelA remained cytoplasmic. Accordingly, 4–1BB stimulation of CD3+CD28 activated CD8 T cells caused rapid IκBα degradation in WT cells but much less so in the mutant cells (Figure 3E). Degradation of IκBβ was barely detectable in both WT and mutant cells. This 4–1BB-induced resistance of IκBα to degradation (due to nuclear accumulation) could explain the significant cRel activation defects observed in Nfkbia^NES/NES CD8 T cells (Figure 2A–2B).

IκBα nuclear export is required for inducing a 4–1BB-mediated NF-κB transcriptional program in CD8 T cells

The biochemical and functional data thus far suggested that a 4–1BB-induced transcriptional program would likely be altered in Nfkbia^NES/NES CD8 T cells. To define a 4–1BB induced transcriptional program that requires proper IκBα nuclear export in CD8 T cells, we next performed RNA-seq analyses on WT and Nfkbia^NES/NES CD8 T cells stimulated with CD3+CD28 with or without 4–1BB stimulation (Figure 4A). We chose a 12-hour time point for analysis to capture early changes induced by 4–1BB signaling as 4–1BB mediated disruption of NF-κB signaling was already evident at this time point (Supplemental Figure 2A). Stimulation of WT CD8 T cells with CD3+CD28+4–1BB altered expression of a set of up- and down- regulated genes relative to CD3+CD28 alone (Figure 4B, left). Moreover, analysis of similarly stimulated Nfkbia^NES/NES CD8 T cells revealed 3 distinct groups of genes (Figure 4C); 1) “NES independent”: genes induced by 4–1BB in WT cells and similarly in Nfkbia^NES/NES (110 genes), 2) “NES dependent”: genes induced by 4–1BB in WT cells but less induced in Nfkbia^NES/NES (88 genes), and 3) “NES reduced”: genes not induced by 4–1BB in WT cells but reduced in Nfkbia^NES/NES (114 genes) (Figure 4C). Of these, the “NES dependent” category was of particular interest because these genes were candidates whose transcriptional programs were controlled by IκBα nuclear export in response to 4–1BB stimulation over CD3+CD28 alone. KEGG pathway analysis of the “NES dependent” category revealed an enrichment of pathways such as the TNF and Jak-STAT signaling pathways and processes such as cytokine signaling (Figure 4D). A ranking of the most upregulated genes by 4–1BB based on their fold induction in WT CD8 T cells revealed those relevant to 4–1BB biology, such as Icam1, Fas, and Jun (Figure 4E). This ranking also demonstrated that Il2 was the 2nd most highly induced gene. This corroborates our previous finding that IL-2 secretion was significantly reduced in 4–1BB-stimulated Nfkbia^NES/NES CD8 T cells (Figure 2C).

To assess whether “NES dependent” genes were under the control of the NF-κB pathway, in particular, cRel complexes, as our previous biochemical and imaging data suggest (Figures 2 and 3), promoter regions of these genes were tested for the enrichment of NF-κB consensus motifs for RelA:p50, cRel:p50, and cRel:cRel complexes (32, 33). We found the greatest enrichment of cRel:p50 motifs in “NES dependent” genes when compared to “NES independent” genes, along with less significant enrichment for RelA:p50 and cRel:cRel motifs (Figure 4F). Despite a cRel:p50 binding site was not detected in the IL-2 gene by the above bioinformatic analysis, we confirmed cRel binding (16.5-fold over basal) to an NF-κB consensus sequence from the IL-2 gene promoter in WT T cells stimulated with CD3+CD28+4–1BB for 12 hours, which was reduced (to 5.2-fold) in Nfkbia^NES/NES T cells (Supplemental Figure 2A). RelA binding also increased in CD3+CD28+4–1BB stimulated WT cells but was much less than cRel binding. These results suggested that 4–1BB stimulation induced a prominent cRel-dependent transcriptional program in CD3+CD28 activated CD8 T cells, which was enabled by IκBα NES-mediated proper subcellular distribution of cRel complexes.

The IκBα NES is necessary for cell-intrinsic anti-viral CD8 T cell responses in vivo

These results thus far demonstrated that CD8 T cells in Nfkbia^NES/NES mice have 4–1BB-mediated NF-κB signaling, transcriptomic and functional defects ex vivo. To test whether IκBα nuclear export was also necessary for activation and expansion of CD8 T cells in vivo, we utilized the Armstrong strain of lymphocytic choriomengitis virus (LCMV) that induces a well-defined virus-specific T cell response (26). At the peak of the anti-viral CD8 T cell response (i.e. day 8 after infection), the accumulation of activated (CD8 CD44^hi) and LCMV NP396-specific CD8 T cells in the spleen of Nfkbia^NES/NES mice was significantly lower than in WT mice (Figure 5A). This data suggested that loss of IκBα nuclear export reduced expansion of CD8 T cells during an acute viral infection. Our ex vivo results demonstrated that Nfkbia^NES/NES CD8 T cells produced significantly lower levels of Il2 transcripts (Figure 4E) and IL-2 protein (Figure 2C), and displayed defects in IL-2-dependent survival, following activation (Figure 2I). In order to test the hypothesis that a defective production of IL-2 was at least in part responsible for a defect in CD8 T cell accumulation in LCMV-infected Nfkbia^NES/NES mice in vivo, IL-2 was administered for 6 days following LCMV infection. Although IL-2 administration of LCMV-infected WT mice increased the percentage of CD8 T cells, it did not increase the percentages or the total number of LCMV NP396- or GP33-specific CD8 T cells in the spleen (Figures 5B & 5C). Strikingly, IL-2 administration completely restored the accumulation of LCMV-specific CD8 T cells in the spleens of Nfkbia^NES/NES mice (Figures 5B & 5C). These data suggested that exogenous IL-2 administration could overcome defective accumulation of CD8 T cells in LCMV-infected Nfkbia^NES/NES mice.

Although our data above demonstrated that virus-specific CD8 T cell responses were suboptimal in Nfkbia^NES/NES mice and could be restored by exogenous IL-2 administration, this defect could be cell extrinsic due to the presence of the IκBα NES mutation in the germline. Moreover, Nfkbia^NES/NES mice also harbor B cell and secondary tissue disruptions (25), which might have impacted anti-viral CD8 T cell responses. Thus, we further performed three different in vivo studies to address this question. First, to address whether defects in anti-viral CD8 T cell responses in Nfkbia^NES/NES mice originated from hematological cell populations, we generated mixed bone marrow chimeras. Lethally irradiated Ly5.1 host mice were reconstituted with a mix of bone marrow cells from congenically marked host strain (Ly5.1) and Ly5.2 Nfkbia^WT/WT or Nfkbia^NES/NES mice, generating control and experimental chimeras (CC and EC, respectively) (Supplemental Figure 3A). As expected, the Nfkbia^NES/NES compartment of CC and EC had normal T cell populations (Supplemental Figure 3B). At day 8 after infection, we quantified LCMV-specific CD8 T cell responses in control and experimental chimeras. The activation of WT (Ly5.1+) host CD8 T cell responses were comparable in control and experimental chimeras; percentages of WT NP396-specific CD8 T cells among Ly5.1+ CD8 T cells were comparable between control and experimental chimeras (Supplemental Figure 3C). CD8 T cells derived from Nfkbia^WT/WT (Ly5.2+) bone marrow cells showed strong activation in control chimeras; >10% of the CD8 T cells were specific to the immunodominant epitope NP_396–404 (Figure 6A). By contrast, the percentages of Nfkbia^NES/NES (Ly5.2) NP_396–404-specific CD8 T cells in experimental chimeras were lower, as compared to their Ly5.2 WT counterparts in control chimeras (Figure 6A). We also calculated the ratios of the percentages of Ly5.1+ to Ly5.2+ NP396-specific CD8 T cells in CCs and ECs. The Ly5.1:Ly5.2 ratio of NP396-specific CD8 T cells in CCs and ECs were 0.83 and 1.72, respectively and this was statistically significant (p=0.029). The significant reduction of NP_396–404 specific cells in the Nfkbia^NES/NES compartment persisted through the memory phase at 64 days post-infection to a re-challenge with LCMV clone 13 (Supplemental Figure 3D). These results support the conclusion that the anti-viral CD8 T cell defect in Nfkbia^NES/NES mice was of hematological origin and likely cell intrinsic.

Next, to specifically address whether the above Nfkbia^NES/NES mouse defects were CD8 T cell-intrinsic, we crossed Nfkbia^NES/NES mice with LCMV glycoprotein (GP) 33-specific TCR transgenic P14 mice to generate P14/Nfkbia^NES/NES mice. Thy1.1+ hosts were adoptively transferred with either P14/Nfkbia^WT/WT (Ly5.2+/Thy1.2+) or P14/Nfkbia^NES/NES (Ly5.2+/Thy1.2+) naïve CD8 T cells and then infected with LCMV to test the responses of donor mutant CD8 T cells (Supplemental Figure 3E, non-competitive environment). Analysis of mice 8 days post-infection revealed a significantly reduced expansion of P14/Nfkbia^NES/NES cells in comparison to P14/Nfkbia^WT/WT transgenic cells (Figure 6B). Thus, these results indicated that the anti-viral CD8 T cell expansion defect of Nfkbia^NES/NES mice was cell intrinsic. Finally, to further validate this, we performed the same experiment in a competitive environment in which an equal number of P14/Nfkbia^WT/WT (Ly5.1+/Thy1.2+) and P14/Nfkbia^NES/NES (Ly5.2+/Thy1.2+) naïve CD8 T cells were adoptively transferred into Thy1.1 mice and then infected with LCMV (Supplemental Figure 3F, competitive environment). Examination of mice after establishment of transferred cells prior to infection showed a slightly higher level of the Nfkbia^NES/NES CD8 T cells relative to Nfkbia^WT/WT cells in the hosts (Supplemental Figure 3F, right). However, at 8 days post-infection, P14/Nfkbia^NES/NES T cell expansion was significantly reduced in comparison to P14/Nfkbia^WT/WT cells (Figure 6C), demonstrating that the Nfkbia^NES/NES CD8 T cells were unable to effectively compete with Nfkbia^WT/WT cells in the identical host environment. Our data collectively supports the critical role of IκBα NES to promote proper cell-intrinsic anti-viral CD8 T cell responses in vivo.