Resting non-activated CD₄⁺ T lymphocytes were negatively isolated from human PBMCs by depletion of B cells, NK cells, monocytes, CD₈ ⁺ T cells and activated lymphocytes. Analysis by flow cytometry revealed they were CD₄⁺ CD₂₅ ^- CD₆₉ ^- HLA-DR^- with a purity >95%.

IκBα and p65/RelA shuttling between nucleus and cytosol was analyzed in resting blood CD₄⁺ T cells by using LMB, a specific inhibitor of the nuclear protein export. The subcellular distribution of IκBα and p65/RelA was first analyzed by immunofluorescence assays. Both IκBα and p65/RelA were localized in the cytosol of unstimulated CD₄⁺ T cells (Fig. 1), but after treatment with LMB, both IκBα and p65/RelA were retained in the nucleus. This nuclear translocation was observed in the absence of any stimulus and was not due to serum activation since similar results were observed in serum deprivation conditions (data not shown).

These results were confirmed using chimeric proteins formed by the enhanced yellow fluorescent protein (EYFP) fused to IκBα or p65/RelA. Resting CD₄⁺ T cells were transiently transfected with plasmids pEYFP-p65 and pEYFP-IκBα separately. Analysis was performed 24 hours after transfection by confocal microscopy. There was low quantity of both IκBα and p65/RelA in the nucleus of the resting T cells before LMB treatment (Fig. 2a) but after exposure to LMB, both EYFP-IκBα and EYFP-p65 fusion proteins were retained in the nucleus. Plasmid pEYFP-C1 containing the EYFP under the control of CMV promoter was used as control of non-specific intracellular distribution.

To exclude that nucleoporation could induce NF-κB activity, electrophoretic mobility shift assays (EMSA) were performed in nuclear extracts from CD₄⁺ T lymphocytes transfected with a control plasmid (pcDNA3.1) by two different methods: the Amaxa Nucleofector system and classical electroporation using an Equibio electroporator (Figure 2b).

In order to determine the dynamics of IκBα shuttling, resting CD₄⁺ T cells were transiently transfected with EYFP-IκBα vector, attached to fibronectine-coated slides and filmed in vivo by time-lapse confocal microscopy during treatment with LMB. Photographs were taken each minute after adding LMB and it was determined that less than 6 minutes were enough to saturate the nucleus with IκBα (Fig. 3 and additional file 1).

LMB toxicity was assessed by propidium iodide staining and flow cytometry in resting CD₄⁺ T cells treated up to 24 hours. Mortality due to LMB treatment (20 nM) was increased only 10% above controls after the longest incubation time (data not shown).

Because more than 10⁸ blood T lymphocytes for each experimental point were required to perform these experiments, T cells were expanded according to a protocol previously developed in our laboratory. PBMCs were cultured for 3 days with 5 μg/ml PHA and for the consecutive 9 days with 300 U/ml IL-2. These long-term cultures of PHA-treated T lymphocytes were maintained without supplemental IL-2 18 hours before the experiment to assure they were in a resting state concerning NF-κB activity. Following this protocol, it was proved that basal and induced NF-κB was similar as in resting T lymphocytes 7 (see Additional file 2).

Consequently, association between IκBα and p65/RelA was determined in the nucleus of long-term cultures of PHA-treated T lymphocytes. For this purpose, nuclear and cytosolic protein extracts were analyzed by immunoblotting assays. As previously shown for resting CD₄⁺ T lymphocytes (Fig. 1), both nuclear IκBα and p65/RelA levels increased in cells treated with LMB (Fig. 4a, Nucleus, lane 2). The accumulation of cytosolic proteins in the nucleus after LMB treatment has been ruled out by immunoblotting of cytosolic and nuclear extracts from PHA-treated T cells by using an antibody against both p105 and p50/NF-κB1 proteins (Fig. 4b). The p105 protein is the precursor of the p50 subunit and it presents exclusively a cytosolic location.

Immunoprecipitation assays with an antibody against p65/RelA showed the presence of NF-κB/IκBα complexes in the nucleus of T cells treated or not with LMB (Fig. 4a, Immunoprecipitation, Nucleus, lanes 1 and 2), whereas no association between p65/RelA and IκBα was observed in cells activated with PMA (Fig. 4a, Immunoprecipitation, Nucleus, lane 3).

Once it was confirmed that both p65/RelA and IκBα were able to shuttle between nucleus and cytosol in unstimulated T cells, NF-κB DNA binding activity was analyzed by EMSA. Despite the presence of p65/RelA in the nucleus, no binding was detected in unstimulated T cells treated or not with LMB (Fig. 4c, lanes 1 and 2). This correlated with the detection of NF-κB/IκBα complexes in the nucleus of these cells (Fig. 4a, Immunoprecipitation, Nucleus, lanes 1 and 2). As expected, NF-κB kept the binding activity to -κB motif in PMA-activated T cells (Fig. 4c, lane 3), due to the absence of NF-κB/IκBα complexes in the nucleus of these cells (Fig. 4a, Immunoprecipitation, Nucleus, lane 3).

Unstimulated T cells were incubated with CHX for 30 minutes before adding other stimulus in order to stop de novo protein synthesis. Then, LMB or PMA were added to the culture medium. Immunoblotting assays showed a decrease of IκBα levels in the nucleus of T cells incubated with both CHX and LMB (Fig. 4d, lane 2) or with CHX, LMB and PMA (Fig. 4d, lane 3), but not in those cells only incubated with LMB (Fig. 4d, lane 1). These data not only confirm previous results showing that nuclear translocation of IκBα is dependent on protein resynthesis 3 but also asserts that this de novo protein synthesis is carried out even in unstimulated T cells.

Resting blood CD₄⁺ T cells were transfected with a LTR-LUC vector alone or together with a Tat expression vector under the control of the CMV promoter in order to assess NF-κB-dependent transcriptional activity in these cells by measurement of luciferase activity (Fig. 5a and 5b). As expected, both Tat over-expression and PMA activation enhanced LTR-dependent transcription, as previously described 1116. However, when nuclear levels of IκBα were increased by LMB (Fig. 5a) or transient transfection of CMV-IκBα vector (Fig. 5b), a dramatic decrease in luciferase activity was observed, both in PMA-activated T cells and cells in which Tat was over-expressed. Interestingly, a basal NF-κB activity able to induce a low LTR transactivation was detected in unstimulated CD₄⁺ T cells. This low LTR transactivation was annulled when IκBα was over-expressed by both LMB or CMV-IκBα transfection, thus proving this basal LTR transactivation was due to a residual NF-κB activity in resting CD₄⁺ T cells.

To assess the role of basal NF-κB activity and IκBα over-expression on a model of HIV production in resting and activated CD₄⁺ T cells, highly purified CD₄⁺ CD₂₅ ^- CD₆₉ ^- DR^- T lymphocytes obtained from blood of different healthy donors were transfected with a full-length infectious HIV clone (NL4.3) together with a CMV-IκBα expression vector or pcDNA3 as negative control. Cells were maintained in culture up to 7 days either in the absence of activation or activated with two different stimuli, PHA and CD3 antibodies. HIV p24-gag was quantified 5 and 7 days after transfection. An intense HIV replication was detected in activated CD₄⁺ T cells after 7 days in culture (Fig. 6b). Besides, a discrete but significant HIV p24-gag production was assessed in resting CD₄⁺ T cells after 5 days of transfection (Fig. 6a). When IκBα was over-expressed in these cells, p24-gag production decreased as compared to cells transfected with a control plasmid and this difference was significant (p < 0.05) for resting and anti-CD₃ activated T cells. Although more than five-fold decrease was observed at day 7 for PHA-activated lymphocytes when IκBα was over-expressed, this result did not reach statistical significance (p = 0.081).

Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors by centrifugation through a Ficoll-Hypaque gradient (Pharmacia Corporation, North Peapack, NJ). Cells were collected in supplemented RPMI and maintained at a concentration of 2 × 10⁶ cells/ml. PHA-treated T lymphocytes were obtained from PBMCs incubated for 3 days with 5 μg/ml phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO) and for the consecutive 9 days with 300 U/ml IL-2 (Chiron, Emeryville, CA). These long-term cultures of PHA-treated T lymphocytes were maintained without supplemental IL-2 18 hours before the experiment. These PHA-treated T lymphocytes remained at a pre-activated status and expressed activation markers 35 although NF-κB did not show DNA-binding activity (Additional file 2).

Resting CD₄⁺ T lymphocytes were isolated from PBMCs by negative selection with CD₄ Negative Isolation Kit (T helper/inducer cells) (Dynal Biotech, Oslo, Norway), according to the manufacturer's instructions. Subsequently, isolated CD₄⁺ T cells were depleted of CD₂₅ ⁺ by positive selection with Dynabeads CD₂₅ (Dynal Biotech). Purity of isolated CD₄⁺ CD₂₅ ^- T cells was analyzed by flow cytometry with a FACScalibur flow cytometer (BD Biosciences, Erembodegem, Belgium). Cells were stained with monoclonal antibodies (mAb) against CD₄ and HLA-DR conjugated with fluorescein isothiocyanate (FITC), and anti-CD₂₅, -CD₆₉, and -CD₃ conjugated with phycoerythrin (PE), all provided by BD Biosciences. Analysis by flow cytometry revealed that the phenotype of isolated T lymphocytes was CD₄⁺ CD₂₅ ^- CD₆₉ ^- HLA-DR^- with a purity >95%.

Cells were incubated with 25 ng/ml of 5-phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 30 min-18 hours. Leptomycin B (LMB) was used at 20 nM (Sigma-Aldrich). Cells treated with 10 μg/ml of cycloheximide (CHX) (Sigma-Aldrich) were incubated with this reagent 30 minutes before adding other stimuli. Primary antibodies against p65/RelA, p105/p50 and IκBα were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies conjugated to horseradish peroxidase were purchased from GE Healthcare (Uppsala, Sweden). Secondary antibodies conjugated to Alexa 488 or Texas Red were purchased from Molecular Probes (Eugene, OR).

Luciferase reporter gene under the control of the U3+R regions of the HIV-long terminal repeat (LTR) (LAI strain) was previously reported 36. pSV-β-Galactosidase vector (Promega, Madison, WI) was used to cotransfect the cells as an internal control reporter. IκBα gene cloned in pcDNA3.1(+) vector under the control of CMV promoter (CMV-IκBα) was described previously 37. Viral Tat gene under the control of the CMV promoter (CMV-Tat) was also described previously 38. pcDNA3.1(+) vector was used as negative control (Invitrogen, Carlsbad, CA). The vector pNL4.3 that contained the HIV complete genome and induced an infectious progeny after transfection in several cell lines was kindly provided by Dr M.A. Martin [39; National Institute of health AIDS Research and Reference Reagent Program #3418]. Dr. Johannes Schmid kindly provided the constructions of p65/RelA and IκBα genes in the enhanced yellow fluorescent protein vector (pEYFP-p65 and pEYFP-IκBα, respectively) 4041. Expression vector pEYFP-C1 (Clontech, BD Biosciences) that contains the yellow fluorescent protein gene under CMV promoter control was used as negative control. All plasmids were purified using Qiagen Plasmid Maxi Kit (Qiagen, CA), following the manufacturer's instructions.

CD₄⁺ T cells (5 × 10⁶) were transiently transfected with 2 μg of plasmid DNA under U-14 electroporation program conditions by nucleoporation with an Amaxa Nucleofector (Amaxa, Cologne, Germany) according to the manufacturer's instructions. Alternatively, CD₄⁺ T cells were also transfected by electroporation with an Easyjet Plus Electroporator (Equibio, Middlesex, UK). In brief, 10 × 10⁶ cells were resuspended in 350 μl of RPMI without supplements and mixed with 1 μg of plasmid DNA per 10⁶ cells in a 4 mm electroporation cuvette (Equibio). Cells were transfected at 320 V, 1500 μF and maximum resistance. After transfection, cells were incubated in supplemented RPMI at 37°C for 24 hours before analysis. Luciferase and β-Galactosidase activities were assayed using Luciferase Assay System and β-Galactosidase Enzyme Assay System, respectively, according to manufacture's instructions (Promega).

Cytosolic and nuclear protein extracts were obtained as described previously 7. Ten micrograms of nuclear extracts were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond-ECL nitrocellulose paper (GE Healthcare). After blocking and incubation with primary and secondary antibodies, proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Cytosolic and nuclear protein extracts were subjected to immunoprecipitation with agarose-conjugated antibody against p65/RelA (Santa Cruz Biotechnology). In brief, nuclear or cytosolic proteins (100 μg) were incubated overnight at 4°C with 10 μg of specific agarose-conjugated antibody in RIPA buffer (PBS 1×, 0.1% SDS, 1% NP-40) and 0.5% sodium deoxycholate (DOC). Immunoprecipitate was collected by centrifugation at 4°C, 2.500 rpm for 5 minutes and washed four times with RIPA/DOC buffer. Finally, the agarose pellet was denatured at 95°C for 2 minutes and analyzed by SDS-PAGE, followed by immunoblotting with the specific antibodies.

Nuclear protein extracts (3 μg) were analyzed using the [α-³²P]-dCTP-labeled double-stranded synthetic wild-type HIV enhancer oligonucleotide 5'-AGCTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA-3' containing both κB consensus motifs. The nucleoprotein-oligonucleotide complexes were analyzed by electrophoresis on a non-denaturing 6% polyacrylamide gel.

Resting CD₄⁺ T cells were transfected with pNL4.3 vector alone or with either CMV-IκBα or pcDNA3.1(+) vectors and further activated with 1 μg/ml anti-CD₃ (BD Biosciences) plus 300 IU/ml IL-2. Viral replication was assessed by quantification of HIV p24 gag antigen in culture supernatants every 48 hours using an enzyme-like immunoassay (Innotest™ HIV Ag mAb, Innogenetics, Barcelona, Spain).

For immunofluorescence assays, cells were immobilized in PolyPrep slides (Sigma-Aldrich) for 15 minutes and then fixed with 2% paraformaldehyde-0.025% glutaraldehyde in PBS for 10 minutes at room temperature. After washing twice with 0.1% glycine/PBS, cells were permeabilized with 0.1% Triton ×-100/PBS for 10 minutes. After washing, cells were treated with 1 mg/ml NaBH₄ for 10 minutes. Incubation for 1 hour at room temperature with each primary and secondary antibodies and subsequent washes were performed with PBS/2% bovine serum albumin (BSA)/0.05% saponine buffer. Coverslips were immobilized with 70% glycerol/PBS. Images were obtained with a Radiance 2100 confocal microscope (BioRad, Hercules, CA). For time-lapse fluorescence confocal microscopy, coverslips were coated with fibronectin (20 μg/ml) for 2 h at 37°C and blocked with PBS containing BSA 0.1%. Then, coverslips were washed with 1× Hanks Balanced Salt Solution (HBSS) and mounted in Attofluor open chambers (Molecular Probes). Cells were allowed to adhere on these chambers for 30 min. Confocal images were acquired using a Leica TCS-SP Confocal Laser Scanning Unit (Leica, Heidelberg, Germany) equipped with Ar and He-Ne laser beams and attached to a Leica DMIRBE Inverted Epi-Fluorescence Microscope. Images were processed and assembled into movies using Leica confocal software.

Differences in HIV replication in the presence of IκBα over-expression were assessed by Mann-Whitney test using Statistical Product and Service Solutions (SPSS) software v14 (Addlink Software Científico, Madrid, Spain).