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The Journal of Immunology, 1998, 161: 649-658.
Copyright © 1998 by The American Association of Immunologists

Inhibition of CD28/CD3-Mediated Costimulation of Naive and Memory Human T Lymphocytes by Intracellular Incorporation of Polyclonal Antibodies Specific for the Activator Protein-1 Transcriptional Complex1

Darren G. Woodside and Bradley W. McIntyre2

Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A number of indirect methods have been utilized in demonstrating activator protein-1 transcription factor function in IL-2 promoter activity. However, there has been no direct demonstration that activator protein-1 is involved in CD28-dependent costimulation of IL-2 gene transcription in freshly isolated naive and memory human T lymphocytes. To address this issue, the method of scrape loading was applied to purified peripheral blood T lymphocytes. Since scrape loading relies on adherent cells, peripheral blood human T (PB-T) cells were immobilized on the nonspecific cell attachment factor poly-L-lysine. Cells scraped off poly-L-lysine in the presence of Ig FITC efficiently incorporated Ig, with relatively uniform fluorescence. T cells retained their physical parameters as measured by forward and side light scatter, and functional activity as measured by costimulation of proliferation and IL-2 production after being scraped off this substrate. CD28/CD3-costimulated T cells produced intracellular IL-2 from all subsets measured (CD4+, CD4-, CD45RO+, and CD45RO-). IL-2 production and intracellular accumulation in nonscraped PB-T cells activated with CD28/CD3 coligation were skewed favoring CD45RO+ and CD4+ subsets, as was IL-2 production in scraped PB-T cells. The intracellular incorporation of Abs specific for c-Fos and c-Jun family members by scrape loading inhibited the production and intracellular accumulation of IL-2 within 6 h of costimulation with PMA/ionomycin, or costimulation by CD28 and CD3 ligation. Scrape loading thus provides an efficient mechanism for intracellular incorporation of macromolecules, and the first direct evidence that c-Fos and c-Jun are involved in transcription of the IL-2 gene within its correct chromosomal context, in resting human T lymphocyte subpopulations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tlymphocyte activation requires complex synergism between the signals generated through TCR/MHC interactions and signals provided by costimulatory molecules (reviewed in 1 . This provides a mechanism whereby T cell clonal expansion can take place in the correct antigenic context, effectively lowering TCR-signaling thresholds required for specific immune responses (2). The interdependency of both of these signals for functional T lymphocyte coactivation is emphasized by the observation that triggering the TCR alone results in a state of Ag unresponsiveness (3). IL-2 is a key player in the regulation of anergic states, and costimulation through the CD28 cell surface molecule dramatically induces its expression (4).

There are two mechanisms controlling IL-2 expression initiated by CD28 costimulation. One mechanism involves stabilization of IL-2 message (5). The other, and perhaps the main mechanism in resting T cells seems to be regulation at the transcriptional level (6). Indirect evidence demonstrating transcription factor involvement in the IL-2 promoter has been provided mainly by a number of molecular approaches. These include in vitro and in vivo DNA footprinting (7, 8), electrophoretic mobility shift (9) and supershift assays (10) of nuclear protein extracts, and transcriptional reporter assays involving extrachromosomal promoters transfected into T cells (11). The current view is that IL-2 transcription involves the coordinate regulation of disparate transcription factors, in which maximal promoter activity relies on the presence of all factors (8). One of the major components of the IL-2 promoter is the activator protein-1 (AP-1)3 (7). This transcriptional complex is composed of c-Jun and c-Fos proto-oncogene families arranged in hetero- and homodimers, and can associate with different transcription factors, such as octamer-binding protein (Oct) and the nuclear factor of activated T cells (NF-AT) in the IL-2 promoter. This allows the possibility of five different interaction sites in the IL-2 promoter, besides its own consensus binding site (12). Because of this, AP-1 is thought of as a key regulator in IL-2 gene expression (12). In fact, AP-1 activity is proposed to be involved in the regulation of c-rel, which is a transcription factor of the NF-{kappa}B family implicated in IL-2 promoter activity (13). There has been, however, no direct demonstration that AP-1 is necessary in the costimulation of IL-2 production in freshly isolated human naive or memory peripheral blood T lymphocytes.

Genetic approaches have not provided conclusive results. Whole populations of T cells isolated from mice deficient in c-Fos or c-Jun genes have no noticeable differences in development, cytokine induction, or proliferation in response to TCR stimulation (14, 15). Therefore, molecular genetic approaches have not demonstrated the role played by these transcription factors in the control of IL-2 gene expression. Nor have basic molecular approaches addressed the question of function or necessity of the identified transcription factors in the costimulation of IL-2 production in resting peripheral blood T cells in an in vivo, intact cell model, in which the IL-2 promoter is in its correct original chromosomal context.

To address this issue, we have developed a method independent of genetic manipulation for determining transcription factor activity in the induction of IL-2 in human peripheral blood T (PB-T) lymphocytes. This method involves scrape loading Abs specific for the AP-1 transcription factor into freshly isolated T lymphocytes. By this method, T lymphocytes adherent to plastic coated with the nonspecific cell attachment factor poly-L-lysine are physically scraped off the plastic, resulting in a transient perforation or tear in the cell surface membrane. Since the medium in which the scrape loading occurs has been spiked with anti-transcription factor Abs, when the membranes quickly reseal, intracellular incorporation of the Abs occurs. This procedure maintains functional integrity of the scraped cells, while incorporating sufficient quantities of Ig for the subsequent analysis of effects on specific T lymphocyte activation pathways. Coupling this procedure with methods involving multicolor FACS analysis of cell surface molecules and intracellular cytokines, we demonstrate that within 6 h of coactivation, intracellular production of IL-2 can be inhibited by Abs to components of the AP-1 transcriptional complex in the initial activation of resting human naive and memory T lymphocyte subpopulations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PMA, ionomycin, BSA, and poly-L-lysine were purchased from Sigma (St. Louis, MO). mAbs rat anti-human IL-2 PE (IgG2a), mouse anti-human CD45RO FITC (IgG2a), mouse anti-human CD4 Cychrome (IgG1), control mouse IgG2a FITC, control rat IgG2a PE, and control mouse IgG1 Cychrome were all purchased from PharMingen (San Diego, CA). Mouse anti-human CD28 mAb Leu28 was purchased from Becton Dickinson (Lincoln Park, NJ). The mouse anti-human CD3 mAb OKT3 hybridoma was obtained from American Type Culture Collection (Manassas, VA), and Ig was purified from ascites. Mouse anti-human ß1 integrin mAb 33B6 was generated in this laboratory and has been previously described (16). Goat anti-rabbit (GAR)-FITC was purchased from PharMingen. Polyclonal transcription factor Abs used were rabbit anti-c-Fos (Oncogene Science, Uniondale, NY) and rabbit anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA). Rabbit anti-mouse Ig was purchased from Cappel (Durham, NC). The extracellular matrix component fibronectin (FN) was affinity purified from 200 ml of human plasma (Gulf Coast Regional Blood Center, Houston, TX), as follows. Briefly, gelatin-Sepharose columns were prewashed with PBS containing 2 mM EDTA and 0.1 mM PMSF. Plasma containing 2 mM EDTA and 0.1 mM PMSF was passed over the column twice and then washed with 4 vol PBS/EDTA/PMSF. Elution of FN from the columns was performed with 4 M urea in 0.15 M NaCl, 2 mM EDTA, 0.1 mM PMSF, and 20 mM Tris-HCl, pH 8. Collected fractions were dialyzed into PBS. All procedures were conducted at room temperature. Purity was determined by SDS-PAGE.

Cells

PB-T cells were isolated by negative selection, as previously described (17). Briefly, mononuclear cells were isolated from buffy coats (Gulf Coast Regional Blood Center) of healthy donors by density-dependent cell separation on Ficoll (1.077 g/ml; Pharmacia, Uppsala, Sweden). Monocytes were removed by several rounds of plastic adherence on tissue culture dishes (Costar, Cambridge, MA) in complete media (RPMI 1640, supplemented with 10% v/v FBS, 1 mM L-glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin, all from Life Technologies, Grand Island, NY). Further density-dependent cell separation was performed on discontinuous Percoll (295 mOsm; Sigma) gradients (44, 48, and 60% v/v Percoll in RPMI 1640; Life Technologies). Cells at the interface of the 48/60% layer were carefully collected and washed in complete media, and the B cells were removed by adherence to nylon wool (Polysciences, Warrington, PA). The lymphocyte population obtained this way was routinely >95% CD3+, as determined by flow-cytometric analysis (Epics Profile, Coulter, Miami, FL). Purified T cells were maintained in complete media at 37°C and 5% CO2 and were used within 24 h of isolation.

Protein immobilization

For costimulation of proliferation, 50 µl of 1 µg/ml anti-CD3 mAb OKT3 was added to 96-well plates (Corning Easy Wash ELISA plates, Corning, NY) in 0.1 M NaHCO3 (pH 8). Then 50 µl of costimulatory mAb Leu28 (anti-CD28), or 33B6 (anti-ß1 integrin), or the extracellular matrix component FN (all at 4 µg/ml in 0.1 M NaHCO3, pH 8) was added. This was incubated for at least 2 h at 37°C. BSA (2% w/v) was then added and incubated for 1 h at room temperature. For costimulation of cytokine production, 35-mm bacteriologic petri dishes (Becton Dickinson) were precoated with 1 µg/ml OKT3 and 4 µg/ml Leu28 in 0.05 M Tris-HCl (pH 9.5) in a final volume of 1.5 ml for 2 h at 37°C. Plates were then blocked with 2% w/v BSA for 1 h. Plates and dishes were washed extensively in PBS before use.

T cell costimulation of proliferation

PB-T cells were plated at a density of 5 x 105 cells/ml (2 x 105 cells/well) in triplicate, in 96-well plates that were precoated with appropriate costimulatory reagents. Approximately 2 to 3 days after initial plating, cells were pulsed with 0.5 µCi [3H]thymidine (Amersham, Arlington Heights, IL) in 50 µl of complete medium. After a 24-h incubation period, cells were harvested onto glass fiber filter mats (Whatman, Maidstone, U.K.) using a PHD cell harvester (Cambridge Technology, Cambridge, MA). [3H]Thymidine incorporation was measured via standard liquid scintillation counting (Beckman LS2800; Beckman Instruments, Fullerton, CA).

Intracellular cytokine measurements

Cells were resuspended in complete media containing 4 µM monensin (Sigma), then plated in 12-well tissue culture plates for PMA/ionomycin activation or 35-mm bacteriologic petri dishes that had been precoated with the appropriate costimulatory mAbs. After 6 h of activation, cells were harvested and intracellular cytokine production was measured. Briefly, cells to be stained were washed once in PBS containing 1% (w/v) FBS and 0.1% NaN3 (stain/wash buffer). When three-color cell staining was performed, directly conjugated Abs were added, incubated for 30 min at 4°C, and then washed twice in stain/wash buffer. Cells were then fixed in 2% (w/v) formaldehyde overnight at 4°C. For intracellular staining, cells were then permeabilized by washing once and resuspending with 0.1% (w/v) saponin (Sigma), 1% FBS, and 0.1% NaN3 in PBS (permeabilization buffer). Rat anti-human IL-2 PE was then added. After 30 min at 4°C, cells were again washed in permeabilization buffer twice and once in stain/wash buffer, then read on an Epics Profile flow cytometer (Coulter, Miami, FL). Control staining was performed with directly conjugated isotype- and fluorochrome-matched Ig, and a minimum of 2 x 104 events was counted per test sample.

Scrape loading

Bacteriologic petri dishes (35 mm) were coated with poly-L-lysine (20 µg/ml) in PBS for at least 2 h at 37°C, then blocked with 2% (w/v) BSA for 1 h at room temperature. Cells were washed four times in RPMI 1640, and resuspended at 5 x 106 cells/ml in RPMI 1640 with the indicated treatments. These cells were then plated (5 x 106 cells) onto PBS-washed poly-L-lysine dishes and incubated for 30 min at indicated temperatures. Almost all of the cells became firmly attached within this time frame (data not shown). After the 30-min incubation, cells were removed physically from the surface with a cell scraper (Costar) in the presence of 500 µl of RPMI 1640 containing the test reagents or controls. These cells were then collected and placed in a 37°C 5% CO2 humidified incubator for at least 1 h. Cells were then washed once in complete medium and used for tests. All Abs for scrape loading were used at 50 µg/ml, unless otherwise noted. Control buffers for scrape loading were identical with the formulation in which commercial Abs were resuspended.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Scrape loading of resting T lymphocytes

The technique of scrape loading requires firmly adherent cells. The use of this method to introduce mAbs inside lymphocytes is problematic because, in general, PB-T cells exist in a low avidity adhesive state. Although activation treatments such as cross-linking the TCR or the addition of cytokines or chemokines can dramatically increase cell adhesion, these types of protocols had to be avoided because they would interfere with the later goal of studying processes involved in driving a lymphocyte from the resting state. Since we had shown previously that T lymphocyte adhesion to poly-L-lysine, even in the presence of mAb specific for the CD3 complex, does not promote T cell proliferation or induce cytokine production (18), poly-L-lysine was chosen as a nonspecific cell attachment factor for the scrape loading of resting T lymphocytes.

After washing in RPMI 1640, 1 ml of freshly isolated human PB-T cells (5 x 106/ml) was plated on poly-L-lysine- or BSA-precoated dishes and incubated on ice (4°C), at room temperature (20°C), or at 37°C for 30 min in the presence of fluoresceinated IgG (GAR-FITC, 100 µg/ml) to indicate scrape-loading efficiency. The cells were removed physically from the petri dish surface with a cell scraper. Once scraping was complete, cells were transferred to a 37°C humidified 5% CO2 incubator for 2 h. Cellular incorporation of GAR-FITC was measured by FACS analysis, and shown in Figure 1Go. T cells scrape loaded on poly-L-lysine (Fig. 1Go, black histograms) incorporated Ig at all temperatures, whereas cells plated in the BSA-coated dishes (Fig. 1Go, gray histograms) did not demonstrate any increase in fluorescent staining over controls. Control staining was performed by mixing GAR-FITC (100 µg/ml) with cells without scraping (data not shown). In this representative experiment (Fig. 1Go), scrape loading at 4°C demonstrated cells were 15.9% positive for GAR-FITC, and the mean fluorescence intensity (MFI) increased from 0.58 to 1.16 after scrape loading. However, maximal GAR-FITC was incorporated into PB-T cells when the loading was performed at 37°C. The MFI increased from 0.62 to 3.40, and the percentage of cells positive was 41.8. Although there was apparent heterogeneity in the incorporation of GAR-FITC, as demonstrated by the complex histograms of Figure 1Go, measurement of relative uptake of GAR-FITC by different PB-T cell subpopulations did not indicate substantial differences. For example, 52% of the CD4+ population incorporated GAR-FITC with a MFI of 8, the CD4- population was 43.4% with a MFI of 9.1, the CD45RO+ population was 51.5% with a MFI of 8.9, and the CD45RO- population was 54.6% with a MFI of 8.1. Digital fluorescent images of cells scrape loaded on poly-L-lysine indicated intracellular incorporation of GAR-FITC (Fig. 2GoB), as compared with cells that were scrape loaded on BSA (Fig. 2GoA). Exposure times for both images were identical. This staining pattern in Figure 2GoB is similar to that obtained with intracellular vital dyes (data not shown). Thus, scrape loading is a rapid method to intracellularly incorporate macromolecules such as Ig into freshly isolated human T lymphocytes.



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  		FIGURE 1. Scrape loading of GAR-FITC into freshly isolated peripheral blood T cells. PB-T cells were scrape loaded (see Materials and Methods) with GAR-FITC (100 µg/ml) on BSA (nonadherent substrate, gray histograms) or poly-L-lysine (adhesive substrate, black histograms) at indicated temperatures. Results are representative of three experiments performed.

 


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  		FIGURE 2. T cells scrape loaded with fluorescent Ab. PB-T cells were plated on BSA (A) or poly-L-lysine (B) and scraped in the presence of GAR-FITC, as described in Materials and Methods. Bright field images are shown on the left, and fluorescent images on the right. Digital images were obtained as described in Materials and Methods. Original magnification was x400, and equal exposure time was used for both images.

 
Physical parameters and recovery after scrape loading of PB-T cells

One hour after cells were scrape loaded with GAR-FITC, the cells were fixed in 2% formaldehyde, and physical parameters such as size and granularity were measured by FACS analysis. As shown for three different temperatures in Figure 3GoA, size as measured by forward light scatter (FLS), and granularity as measured by side light scatter (SLS) remained unchanged for cells that were scraped off poly-L-lysine-coated petri dishes, as compared with nonscraped cells removed from BSA-coated petri dishes. Fixation with formaldehyde did not change these physical parameters, as compared with unfixed samples (data not shown). Recovery of viable scraped cells was also measured (Fig. 3GoB). Approximately 70% of the initial input population of cells were recovered, and almost all cells were viable, as measured by trypan blue exclusion (not shown) when cells are scraped at both 20°C and 37°C, while approximately 40% of the input cells were recovered viable at 4°C. Thus, relatively high yields of scraped cells can be recovered at the optimal temperature for scrape loading (37°C), with no observed differences in the physical parameters of these cells.



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  		FIGURE 3. Physical parameters remain unchanged and cellular recovery is high after scrape loading. A, Forward light scatter (FLS) and side light scatter (SLS) were measured on an Epics profile flow cytometer for PB-T cells scraped or not scraped at three different temperatures. One representative experiment of three is shown. B, PB-T cells were plated on BSA or poly-L-lysine at three different temperatures and scraped as described in Materials and Methods. Cell recovery was determined by dividing the number of trypan blue excluding cells recovered after scraping cells off poly-L-lysine by the initial input number of cells. Equal numbers of cells were applied to both conditions. Error bars are the SE of the mean and represent the averages from two separate experiments.

 
Scrape-loaded cells retain functional activity, as measured by costimulation of proliferation and production of IL-2

The technique of scrape loading is a valuable tool only if the functional integrity of the scraped cells remains intact. To test whether the method of scrape loading would have deleterious effects on the ability of T cells to be activated, costimulation tests were performed on T cells that had been scrape loaded in the presence of a control, soluble protein (50 µg/ml BSA). Known costimulators of T cell proliferation such as CD28 and ß1 integrins were tested for their ability to costimulate T cells when coimmobilized with anti-CD3 mAb OKT3. As Figure 4GoA demonstrates, cells costimulated with immobilized anti-CD3 in conjunction with anti-CD28, FN, or anti-ß1 integrin were unaffected by the technique of scrape loading, as there was no real difference between proliferation of scrape-loaded and non-scrape-loaded cells. Proliferation assays, however, require approximately 3 days of incubation, which is well past events mediating early transcription factor induction, which could have been transiently affected by the scrape-loading procedure.



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  		FIGURE 4. Scrape-loaded PB-T cells maintain functionality, as determined by costimulation of proliferation and the induction of IL-2 production. A, Freshly isolated PB-T cells were scrape loaded on poly-L-lysine (gray bars) and compared with unscraped cells (black bars) in a proliferation assay. Proliferation was induced by coimmobilization of anti-CD3 mAb OKT3 in conjunction with anti-CD28, FN, or anti-ß1 integrin mAb. Data are expressed as the average ± SEM from triplicate determinations. B, Scraped PB-T cells maintain functional responses to pharmacologic activation stimuli. Intracellular IL-2 was measured as described in Materials and Methods. Unactivated PB-T cells (left panel) and PB-T cells activated with PMA (50 ng/ml) and ionomycin (2 µg/ml) (right panel) were intracellularly stained with rat anti-human IL-2 directly conjugated to PE. Both groups were incubated in the presence of 4 µM monensin during the activation period. The number of events counted was greater than 5 x 104. C, PB-T cell subsets maintain functionality with respect to costimulation of IL-2 production by CD3/CD28 ligation after scrape loading of nonspecific RAM Ig. RAM scrape-loaded PB-T cells (right panels, top and bottom) were compared with nonscraped cells in their ability to respond to CD3/CD28 costimulation. Multiparameter flow cytometry was used to analyze intracellular IL-2 accumulation within CD45RO+/- and CD4+/- subpopulations of PB-T lymphocytes. Gating for CD45RO and CD4 was set in reference to fluorochrome/isotype-matched control Ig (not shown). The left panel (control stain, top and bottom) represents background fluorescence of the CD45RO+/- and CD4+/- cell populations stained with control rat IgG2a PE, and therefore serves as a control for the rat anti-human IL-2 IgG2a PE. A minimum of 2 x 104 events was counted per test. The results in Figure 4GoC are summarized in Table IGo.

 
To test whether very early T cell activation events remain functional after the scrape-loading manipulation, the technique of intracellular cytokine staining was used (19). Freshly isolated T cells were scrape loaded on poly-L-lysine with rabbit anti-mouse (RAM) Ig, let rest for 1 h at 37°C, then activated in the presence of PMA (50 ng/ml) and ionomycin (2 µg/ml) in complete media containing 4 µM monensin. After 6 h of incubation in a humidified 37°C 5% CO2 incubator, cells were fixed overnight in 2% formaldehyde at 4°C. Staining for intracellular IL-2 was performed, as described in Materials and Methods. Control gating was set with fluorochrome/isotype-matched controls (rat IgG2a PE, not shown). As demonstrated in Figure 4GoB (right panel), 20.3% of the scrape-loaded cells contained detectable intracellular IL-2 within 6 h of T cell activation. Unactivated cells scrape loaded with RAM demonstrated no intracellular staining for IL-2 (Fig. 4GoB, left panel). This demonstrates that the physical removal of T cells adherent to poly-L-lysine with the concomitant introduction of Ab retains T cell functionality, as measured through the costimulation of IL-2 production.

Because the previous experiment was performed using high concentrations of pharmacologic inducers of T cell activation, it was important to determine whether scrape loading influenced early events in T lymphocytes coactivated under more relevant circumstances, such as ligation of the TCR/CD3 complex in conjunction with the costimulatory molecule CD28. In addition, the previous experiment dealt with a purified PB-T cell population as a whole, rather than certain discrete lymphocyte subsets. In Figure 4GoC (summarized in Table IGo), RAM scrape-loaded PB-T cells (Fig. 4GoC, right panels, top and bottom) were compared with non-scrape-loaded cells (middle panels, top and bottom) in their ability to respond to CD3/CD28 costimulation. Three-color flow cytometry was used to analyze intracellular IL-2 accumulation within CD45RO+/- and CD4+/- subpopulations of PB-T lymphocytes after both scraped and nonscraped cells were activated for 6 h in the presence of 4 µM monensin. Gating for CD45RO and CD4 was set in reference to fluorochrome/isotype-matched control Ig (not shown). The left panel of Figure 4GoC ("control stain," top and bottom) represents background fluorescence of the CD45RO+/- and CD4+/- cell populations. These had been stained with rat IgG2a PE after RAM scrape loading, and CD3/CD28 costimulation in the presence of 4 µM monensin, therefore, serves as a control for the rat anti-human IL-2 IgG2a PE stains. Unactivated cells, or cells plated on anti-CD3 mAb alone did not demonstrate detectable intracellular IL-2 accumulation (data not shown). Since the population of T cells is highly purified, the CD45RO- and CD4- populations may be considered as CD45RA+ and CD8+, respectively. In this experiment, there was only a slight effect on CD28/CD3 T cell coactivation, as measured by intracellular IL-2 accumulation due to the scrape-loading procedure. For example, costimulation caused 57.5% of the CD45RO+ (top middle panel, calculated from the top and bottom right quadrants) subset of T lymphocytes to become positive for intracellular IL-2 (see Table IGo). In the CD45RO+ subset that had been scrape loaded with RAM (top right panel), 52.5% of the population was positive for intracellular IL-2 after costimulation (see Table IGo). This demonstrates that RAM scrape-loaded T cells retain their functional integrity and can immediately be used for studies on early events regulating T cell activation.


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  		Table I. PB-T lymphocytes retain their ability to be constimulated by CD3 and CD28 coligation even after scrape-loading of Ram

 
Also noteworthy from the multiparameter flow-cytometric analysis shown in Figure 4GoC and summarized in Table IGo is that costimulation of PB-T cells with anti-CD3 and anti-CD28 mAbs demonstrates that IL-2 production is from both the CD45RO+ (memory) and CD45RO- (naive) subpopulations of T cells. However, the amount of cells responding to CD28/CD3 costimulation is usually skewed toward the CD45RO+ and CD4+ subsets. To test whether this were a general phenomenon, PB-T lymphocytes from 12 different donors were purified and tested for subpopulation expression of IL-2 after CD28/CD3 costimulation. Consistent with the results obtained in the RAM scrape-loaded cells (Fig. 4GoC), unmanipulated PB-T lymphocytes also demonstrated this skewed production of IL-2 (Fig. 5GoA). Although the range of total IL-2 production between the donors was broad, averages obtained from the different cell subsets displayed a distinct pattern of IL-2 production. Upon costimulation by CD28 and CD3 mAbs, CD45RO+ T cells on average were 43.4% (range = 13.1–79.2) positive for intracellular IL-2, whereas the CD45RO- subsets were on average only 23.5% (range = 5.6–50) positive (p < 0.0001, paired Student’s t test). Likewise for CD4+ subsets, the average percentage of cells IL-2 positive was 42.6 (range = 8.3–73.6). Of the CD4- cells, only 12.4% (range = 1.7–27.2) of the cells on average were IL-2 positive (p < 0.0001, paired Student’s t test). In no donor was this trend not apparent. Even when the total amount of IL-2 produced was low, the amount always conformed to the rule that CD45RO+ > CD45RO-, and CD4+ > CD4-. Since these are purified populations of T cells, and less IL-2 was produced in the CD45RO- and CD4- subsets, by inference the CD45RA+ and CD8+ subsets should produce less IL-2 than CD45RA- and CD8- subsets, respectively. This is in fact the case (Fig. 5GoB). In donor A, IL-2 production was greatest in the CD45RO+ and CD4+ subpopulations. In donor B, in which CD45RA and CD8 were stained instead of CD45RO and CD4, IL-2 production was greatest in the CD45RA- and CD8- subpopulations. Therefore, naive cells (CD45RO- or RA+) and CD8+ cells generally produce less IL-2 when costimulated with CD28 and CD3.



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  		FIGURE 5. CD28 costimulation of IL-2 production occurs in CD4+ and CD45RO+ subsets preferentially. A, Purified peripheral blood T lymphocytes were collected from a total of 12 donors. CD45RO and CD4 cell surface molecules were stained, and intracellular IL-2 was calculated for each subpopulation, according to the following formula: percentage of IL-2 positive = ((IL-2%+ for given subset)/(total percentage of a given subset in the whole population)) x 100. B, In donor A, CD45RO and CD4 cell surface molecules were stained along with intracellular IL-2 after costimulation by Abs to CD28/CD3 in the presence of 4 µM monensin. In donor B, CD45RA and CD8 were stained in conjunction with intracellular IL-2 after costimulation by Abs to CD28/CD3. Left panels for donor A and donor B are isotype-matched control stains for intracellular IL-2 analyses, and a minimum of 2 x 104 events was measured for each test.

 
CD28-dependent costimulation of PB-T cells, as measured by the intracellular accumulation of IL-2, is inhibited by cyclosporin A

As demonstrated in previous experiments, the induction of IL-2 production can be analyzed readily by intracellular staining of activated cells. However, this procedure requires the activation of PB-T cells in the presence of monensin, which stagnates Golgi transport by neutralizing its pH. In so doing, proteins normally secreted can accumulate in the Golgi and endoplasmic reticulum, augmenting their staining signal and facilitating FACS analysis. It was not clear, however, that this technique would be adequate to measure decreases in cytokine production if the amount that could be held in these vesicles was limited. Therefore, cells were activated with coimmobilized anti-CD3 and anti-CD28, in the absence or presence of cyclosporin A (CsA, 0.5 µg/ml) to determine whether decreases in IL-2 levels could be measured after 6 h of activation (Fig. 6Go). Cells were stained for CD45RO and intracellular IL-2. As summarized in Table IIGo, the percentage of cells positive for IL-2 decreased from 13.6 to 3.2 in the CsA-treated group of the CD45RO- subset of T cells. This represents a 76.5% decrease in the percentage of IL-2-positive cells. In the CD45RO+ subpopulation, the percentage of cells positive for IL-2 dropped from 40 to 12.6% in the CsA-treated cells. This represented a 68.5% decrease in the percentage of IL-2-positive cells after CsA treatment. It appeared then that the procedures involved in activating cells for intracellular IL-2 staining (particularly the use of monensin) would be conducive for the study of inhibition of IL-2 production.



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  		FIGURE 6. Inhibition of CD3/CD28 costimulation of IL-2 production by the pharmacologic agent CsA. The decrease in intracellular accumulation of IL-2 was measured when PB-T cells were costimulated with CD3/CD28 in the absence (left panel) or presence (right panel) of CsA (0.5 µg/ml). CD45RO+ gates were set from the analysis of fluorochrome/isotype-matched control mAbs (not shown). A minimum of 2 x 104 events was analyzed per test.

 

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  		Table II. Inhibition of CD28-induced intracellular IL-2 accumulation by CsA

 
Inhibition of pharmacologic (PMA/ionomycin) and physiologic (CD28/CD3) induction of IL-2 production by incorporation of a mixture of Abs specific for c-Fos and c-Jun transcription factors

Besides its own consensus site, AP-1 proteins are thought to associate with other transcription factors in as many as five other DNA binding sites, possibly acting as a master regulator of IL-2 induction (reviewed in 12 . Thus, it is a crucial component of the multifactor IL-2 promoter, which has been shown to require an association between all of its components for full promoter activity (8). To test the requirements of AP-1 in IL-2 induction, a mixture of polyclonal Abs to c-Fos and c-Jun was scrape loaded into primary human PB-T cells, and the cells were activated for 6 h, with high concentrations of PMA (50 ng/ml) plus ionomycin (2 µg/ml). The cells were then harvested, fixed overnight, and stained for intracellular IL-2. As Figure 7Go demonstrates, samples that were either not scraped (left panel), scrape loaded with RAM control (middle panel), or scrape loaded with polyclonal anti-c-Fos and anti-c-Jun Abs (right panel) all demonstrated significant production of IL-2. However, the sample that had been scrape loaded with c-Fos and c-Jun Abs showed a marked reduction in IL-2 production (a 41.6% decrease in IL-2+ cells from 25% to 14.6%). This effect was dose dependent and correlated with a decrease in IL-2 message, as measured by Northern blotting (data not shown). The inhibition of IL-2 seen when the anti-c-Fos and anti-c-Jun Abs were used alone was comparable with the inhibition seen when a combination of the Abs was used (data not shown).



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  		FIGURE 7. Scrape loading of Abs specific for c-Fos and c-Jun inhibits intracellular IL-2 production induced by PMA and ionomycin. Intracellular staining was performed as described in Materials and Methods. At least 2 x 104 events were measured for each condition, and PMA was used at 50 ng/ml, and ionomycin at 2 µg/ml. Gray histograms are nonspecific isotype control rat IgG1 PE. Black histograms are rat anti-human IL-2 IgG1 PE. These results are representative of three experiments performed.

 
Under a more relevant T cell activation setting, IL-2 production was induced by coimmobilizing mAbs specific for the CD3 complex and CD28. In Figure 8Go (summarized in Table IIIGo), RAM scrape-loaded PB-T cells (middle panels, top and bottom) were compared with scrape loading of a mixture of polyclonal Abs to c-Fos and c-Jun (right panels, top and bottom) in their ability to respond to CD3/CD28 costimulation. Three-color flow cytometry was used to analyze intracellular IL-2 accumulation within CD45RO+/- and CD4+/- subpopulations of PB-T lymphocytes, as described for Figure 4GoC. The left panel of Figure 8Go ("control stain," top and bottom panels) represents background fluorescence (irrelevant rat IgG2a PE) of the RAM-scraped CD45RO+/-- and CD4+/--activated cell populations, serving as a control for the rat anti-human IL-2 IgG2a PE. As summarized in Table IIIGo, inhibition of CD28/CD3 costimulation of IL-2 production occurred in all of the subsets measured. CD45RO+ subsets showed a decrease of 17.3% in the amount of IL-2-positive cells when scrape loaded with anti-AP-1 Abs. However, the CD45RO- subset (by default the CD45RA+ subset) demonstrated a decrease of 42.1%. The decrease in IL-2-positive cells from CD4+ and CD4- subsets was 26.3 and 49.3%, respectively. These results suggest that the c-Fos and c-Jun transcription factors are involved in primary human T cell activation, and that their requirement for IL-2 production is more stringent in the naive (CD45RO-) subset.



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  		FIGURE 8. CD28-induced costimulation of IL-2 production inhibited by scrape loading a mixture of polyclonal Abs specific for c-Fos and c-Jun transcription complexes. PB-T cells were either scrape loaded with RAM (RAM Scrape Loaded), or a mixture of rabbit Abs specific for c-Fos and c-Jun (Anti-c-Fos/c-Jun Scrape Loaded) was activated by costimulation with CD3 and CD28 mAbs. Three-color FACS analysis was performed as described in Materials and Methods. Control staining (left panels, top and bottom) represents nonspecific rat IgG2a PE Ab background staining of CD45RO+/- and CD4+/- subpopulations of costimulated T cells scrape loaded with RAM. A minimum of 2 x 104 events was analyzed per test.

 

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  		Table III. Scrape loading of a combination of polyclonal antibodies specific for c-Fos and c-Jun inhibit production of IL-2 induced by costimulation with CD3/CD28 mAbs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In T lymphocytes, a combination of signals emanating from the TCR/CD3 complex and costimulatory molecules culminates in transcription of the IL-2 gene. At this point of signal convergence, a number of transcription factors coordinately regulates maximal IL-2 gene expression. Since the first demonstration of the immediate upstream DNA sequence as a key element in inducible IL-2 gene expression (20), a large body of work has identified many transcriptional complexes suggested to be involved in the control of promoter activity (reviewed in Refs. 12 and 21). A variety of methods has been utilized to demonstrate the involvement of these transcription factors in the control of IL-2 gene expression, including in vitro and in vivo DNA footprinting (7, 8), electrophoretic mobility shift (and supershift) assays of nuclear protein extracts (9, 10), and transcriptional reporter assays involving extrachromosomal promoters transfected into T cells (11). Pharmacologic agents such as CsA or cAMP provide indirect evidence of transcription factor involvement in IL-2 gene induction, by inhibiting signal-transduction pathways before nuclear effects on transcription factor activity (22, 23). Although these techniques have been indispensable in the enumeration of factors interacting with the IL-2 promoter and their ability to promote transcription, interpretation of their physiologic activity in the IL-2 promoter of primary T lymphocytes, in which the IL-2 gene is within its correct chromosomal context, is complex. Correct chromosomal context is an important aspect, as it is becoming increasingly apparent that there are positional DNA effects regulating promoter activity. For example, genetic sequences can serve as boundaries between transcriptionally active and inactive DNA. These sequences are hypothesized to delineate domains of gene expression, avoiding promiscuous interactions between promoters and enhancers of spatially distinct genes (24). In addition, upstream of the IL-2 promoter region are poorly studied sequences thought to be involved in providing cell type specificity with respect to IL-2 production (12). Transiently transfected promoter/reporter constructs containing the bare minimal upstream elements lack these insulator or boundary sequences, and far upstream regulatory sequences, thus not reflecting promoter activity within the original chromosomal context.

To date, the only methods available for determination of transcription factor activity in the promotion of gene expression under physiologic conditions in which promoters are maintained within their natural context have been the use of genetically altered cell lines or mouse models. These alterations have come in the form of gene ablation studies such as transcription factor knockout (KO) experiments or mutational analysis (from either chemically induced mutations or homologous recombination) of transcription factor DNA-binding sequences that prevent DNA/factor interactions. Although the transcription factors c-Fos (14), c-Jun (15), NF-AT1 (25), and NF-{kappa}B proteins (RelA, c-Rel, RelB, p50, and I{kappa}B{alpha}) (26, 27, 28, 29, 30) have been studied in mouse KO models, only those of the NF-{kappa}B family have demonstrated involvement in the production of IL-2 after direct costimulation of T cells. In particular, c-Rel-/- mice showed drastic decreases in the induction of IL-2 after costimulation with CD28 and CD3 Abs, even though mutating the NF-{kappa}B site in the IL-2 promoter has only slight effects (13). In contrast, c-Fos and c-Jun KO experiments demonstrate that these transcription factors are either not involved in IL-2 transcription, or more likely, that there is functional redundancy within the family members of these genes, such that the adult animal has compensated for their absence in the regulation of IL-2 production. T cells generated from mice deficient in NF-AT1 have demonstrated Ag hyperresponsiveness, so it has been postulated that NF-AT1 is in the promoter of certain molecules involved in the down-regulation of the immune response, such as the CTLA-4 molecule.

This communication describes a method independent of genetic manipulation for analyzing the involvement of transcription factors in the induction of IL-2. This method involves scrape loading anti-transcription factor Abs into freshly isolated human T lymphocytes, whereby cellular functionality as measured by the costimulation of proliferation and IL-2 production is maintained. This has allowed the intracellular incorporation of Abs specific for AP-1, and the measurement of the effect these Abs have on IL-2 gene expression induced under different activation conditions. Since its first introduction (31), scrape loading has been used in a number of experimental systems for the incorporation of a variety of reagents: lucifer yellow for measurements of gap junction activity (32); the small GTP-binding protein p21ras (33, 34); DNA for transfection (35); fluorinated inositols (36); mAbs (37); restriction endonucleases (38); phalloidin (39); and Clostridium botulinum toxin C3 (40). The technique of scrape loading is based on the principle that when firmly adherent cells are physically scraped off their substrate, their lipid membranes are transiently perforated or torn and quickly reseal. As the membranes reseal, intracellular incorporation of some of the extracellular milieu occurs. Scrape loading has some unique advantages to other published techniques for the introduction of reagents intracellularly, in that it allows for the incorporation of nonspecific reagents for appropriate controls. For example, in the incorporation of mAbs, other methods of transiently perforating the cell membrane, such as in the case of Streptolysin O and that of the reversible detergent saponin, nonspecific Abs would be washed out in subsequent steps as the resealing of the membrane does not immediately follow initial perforation. With the technique of scrape loading, large quantities of cells can be loaded with excess specific and nonspecific material. As the resealing of the membranes in this instance is very fast, nonspecific Abs that are not retained within the cells by interaction with specific epitopes are theoretically incorporated in much the same amounts as Abs that are retained through Ab/Ag interactions. In this manner, the correct nonspecific controls are present for cellular testing.

The studies presented in this work indicate that the c-Jun and c-Fos gene products are in fact involved in the induction of IL-2 production in freshly isolated human T lymphocytes. Scrape loading specific Abs to c-Fos and c-Jun into isolated T cells substantially inhibited IL-2 production in naive (CD45RO-), memory (CD45RO+), CD4+, and CD4- T cell subsets when costimulated with CD28 and CD3 Abs. The data presented in this work substantiate molecular studies, such as in vitro and in vivo DNA footprinting, gel shift and supershift experiments, and promoter activity assays originally suggesting the importance of these transcription factors in IL-2 production. Scrape loading of a mixture of polyclonal Ab raised against c-Fos and c-Jun demonstrates a more pronounced effect on CD45RO- T cells and suggests the AP-1 complex is more stringently required in CD28/CD3 costimulation of IL-2 production in naive T cell subsets. This may be a reflection of the robust IL-2 production from CD45RO+ T cell subsets, as compared with CD45RA+ subsets.

There are a number of mechanisms by which the Abs may be inhibiting IL-2 production. For example, they may be sequestering transcription factors in the cytoplasm by preventing their nuclear translocation. Or, if the Ab can shuttle into the nucleus via association with the factor, then inhibition of either DNA binding or the formation of quaternary structures with DNA and other transcription factors may be the mechanism of action. Based on in vivo footprinting data, one would reason that if maximal inhibition of any one transcription factor involved in the IL-2 promoter were to occur, then almost complete inhibition of IL-2 production would result as the quaternary structure of the IL-2 promoter could not be established (8). This seems to be the case in RelA-/- mice, in which T cell IL-2 production is decreased ~50-fold when compared with normal mice. In the scrape-loading model with anti-c-Fos and anti-c-Jun Abs presented in this work, incomplete inhibition of IL-2 production was found, which may indicate submaximal inhibition of transcription factor activity. This is most likely a reflection of a decrease in nuclear AP-1 factors available to interact with the complex structure of the IL-2 promoter. This has allowed the recognition that more stringent conditions exist for AP-1 transcription factor involvement in CD28/CD3 costimulation of naive T cell subpopulations, which would have been overlooked if complete inhibition of T cell IL-2 production were to have occurred.

To date, the most compelling evidence for implicating certain effector molecules in T cell activation has come from genetic experiments involving ablations or mutations of effector molecules involved in signaling cascades or IL-2 transcription. Gene KO experiments involving c-Fos and c-Jun have been inconclusive in the determination of their role in IL-2 transcription (14, 15). In this communication, we provide a method to incorporate macromolecules such as Ig into freshly purified human peripheral blood T cells. Combining this with an approach for measuring intracellular IL-2 production, rapid measurements of the effects of specific reagents on components of intracellular signaling cascades can be made. We have used Abs specific for transcription factors that have been identified as possible players in the IL-2 promoter under a variety of in vitro experimental conditions. To our knowledge, this is the first evidence that these factors are involved in the production of IL-2 within single cells of the primary human T lymphocyte naive and memory subpopulations, in which the IL-2 promoter is within its correct chromosomal context. These techniques should prove fruitful for the study of signal-transduction pathways and T cell functional responses to a variety of stimuli.


   Acknowledgments
 
We thank J. N. Wygant for technical assistance and K. Ramirez for FACS analysis.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA62596 and Kleberg Foundation. Back

2 Address correspondence and reprint requests to Dr. Brad McIntyre, Department of Immunology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 180, Houston, TX 77030. Back

3 Abbreviations used in this paper: AP-1, activator protein-1; CsA, cyclosporin A; FN, fibronectin; GAR, goat anti-rabbit; MFI, mean fluorescence intensity; NF-AT, nuclear factor of activated T cells; NF-{kappa}B, nuclear factor-{kappa}B; PB-T, peripheral blood human T; PE, phycoerythrin; RAM, rabbit anti-mouse. Back

Received for publication June 5, 1997. Accepted for publication March 17, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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