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The Journal of Immunology, 1998, 160: 2735-2741.
Copyright © 1998 by The American Association of Immunologists

CD4+, But Not CD8+, T Cells from Mammary Tumor-Bearing Mice Have a Down-Regulated Production of IFN-{gamma}: Role of Phosphatidyl Serine1

Xiaofang Cheng and Diana M. Lopez2

Department of Microbiology and Immunology, University of Miami School of Medicine, and the Sylvester Cancer Center, Miami, FL 33101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-{gamma} production is dramatically reduced in T cells from mice bearing large mammary tumors. This inhibition of IFN-{gamma} gene expression occurs mostly in CD4+ T cells, as determined by ELISA and reverse transcriptase-PCR. The effects of known mammary tumor factors in normal T cells and its subsets were evaluated. Pretreatment with granulocyte-macrophage CSF resulted in increased IFN-{gamma} levels by T cells, while PGE2 pretreatment equally decreased the levels of this cytokine in CD4+ and CD8+ T cells from normal mice. Interestingly, phosphatidyl serine (PS) down-regulated the IFN-{gamma} production of CD4+, but not that of CD8+, T cells. Methylation analysis indicated that the CpG dinucleotide in SnaBI site of the IFN-{gamma} 5' promoter flank region was hypermethylated in CD4+, but not in CD8+, T cells of large tumor bearers and of normal mice pretreated with PS. Electrophoresis mobility shift assay using an oligonucleotide probe corresponding to the IFN-{gamma} promoter core region sequence showed a greatly reduced binding of a 90-kDa nuclear protein in CD4+ T cells from tumor bearers and in those from PS-pretreated normal mice. Since IL-2 production is not affected in either CD4+ or CD8+ T cells from tumor bearers, these studies indicate that IFN-{gamma} production can be regulated independently from that of other type 1 cytokines in vivo. Our data further suggest that PS is involved in IFN-{gamma} gene down-regulation during mammary tumorigenesis and contributes to the generalized immunosuppression associated with tumor growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IFN-{gamma} is a multifunctional cytokine known to be both an inhibitor of viral replication and a regulator of numerous immunologic functions. Produced by T cells and NK cells (1, 2, 3), its expression in tumor-bearing animals is often critical during tumor development for the immunologic defenses of the host. Studies using mice with disrupted IFN-{gamma} gene (4) have documented the importance of this cytokine in the activation of macrophages, which are important mediators of nonspecific antitumor activity. The activity of splenic NK cells is also greatly reduced in IFN-{gamma}-deficient mice (4). Furthermore, this cytokine appears to be necessary for the T cell response to allogeneic cells.

Previous studies from our laboratory have demonstrated that the growth of the D1-DMBA-3 mammary tumor causes multiple alterations in the phenotype and functions of lymphoreticular organs and their effector cells. Thus, we have shown that with increasing tumor burden there is a progressive loss of NK activity (5), delayed-type hypersensitivity (6), proliferative responses of splenic lymphocytes to mitogens and tumor-associated Ags (7), macrophage-mediated cytotoxicity (8), nitric oxide production (9), and the generation of allogeneic CTL (10). We have reported that IFN-{gamma} production is down-regulated in peritoneal (8) and splenic T cells (11) from tumor-bearing mice. This alteration is not the result of a shift from a Th1 to a Th2 phenotype, but is related to a down-regulation of IL-12 production (11). In previous investigations, we have found that the D1-DMBA-3 mammary tumor produces several factors that are capable of modulating several immune responses, i.e., PGE2 (12), granulocyte-macrophage CSF (GM-CSF)3 (13), and phosphatidyl serine (PS) (9). The latter has a potent inhibitory action on the production of nitric oxide and IL-12 by macrophages (9, 11). In the present study, we demonstrate that the impaired IFN-{gamma} production in tumor bearers is mostly due to a down-regulation of this cytokine in CD4+, but not in CD8+, T cells. Furthermore, we demonstrate that PS can directly affect the production of IFN-{gamma} in normal CD4+ T cells with similar characteristics as those occurring in the CD4+ T cells of tumor bearers, i.e., differential regulation at the level of methylation and of binding of transcriptional factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and tumors

BALB/c mice are maintained by brother-sister matings in our laboratory. The D1-DMBA-3 is a transplantable mammary adenocarcinoma derived from a nonviral, noncarcinogen-induced preneoplastic alveolar nodule in a BALB/c mouse after treatment with 7,12-dimethylbenzanthracene (14). The tumor is routinely transplanted in BALB/c by s.c. injection of 1 x 106 tumor cells. Tumor is apparent 8 days after implantation, and mice begin to die after 4 wk. Normal female BALB/c mice and large tumor bearers (4 wk after tumor implantation) were used in all experiments.

Reagents

RMPI 1640 supplemented with 100 U of penicillin and 100 µg/ml of streptomycin, 5 x 10-5 M 2-ME, 1 mM L-glutamine, 1% sodium pyruvate, and 10% endotoxin-free FCS (all from Life Technologies, Grand Island, NY) was used as culture medium. Con A was purchased from Boehringer Mannheim (Mannheim, Germany) and dissolved in serum-free medium and stored at -20°C. Recombinant GM-CSF (Genzyme, Boston, MA), PGE2 (Sigma Chemical Co., St. Louis, MO), and PS (Avanti Polar Lipids, Alabaster, AL) were purchased. PS was obtained as a dry crystal powder and dissolved completely in chloroform. After evaporation of the chloroform by nitrogen, PS was resuspended in culture medium and sonicated for 30 min just before use.

Preparation of T cells and subsets

Spleens from normal and 4-wk tumor-bearing mice were minced through sterile nylon mesh screens. The cell suspensions were centrifuged, and cell pellets were resuspended in 0.16 M Tris-buffered ammonium chloride for lysis of RBC. After washing twice with PBS, cells were loaded on prewarmed Sephadex G-10 (Pharmacia, Piscataway, NJ) columns to remove phagocytic cells according to the procedure of Ly and Mishell (15). T cells were purified by passing them through nylon wool columns twice. Cell purity was ascertained by fluorocytometric analysis in a FACScan (Becton Dickinson) as previously described (5). Routinely, the T cells or subset preparations had a purity of 90 to 95%.

To obtain CD4+ and CD8+ subsets, T cells were washed twice and resuspended in 5 x 106 cells/0.5 ml PBS-5% FCS. Twenty-five microliters (200 µg/ml) of rat anti-mouse CD4 or CD8 Abs (Caltag Laboratories, Hornby, Canada), incubated at 37°C for 45 min. Cells were washed three times with PBS. Dead cells were eliminated by centrifugation.

Purified T cells and T cell subsets were placed in 24-well tissue culture plates (Costar, Cambridge, MA) at a concentration of 106/ml and cultured in RPMI-10% FCS and 1% MEM-vitamins, 1% MEM-nonessential amino acids, 1% sodium pyruvate, 2% HEPES (all from Life Technologies). In some experiments, normal cells were pretreated with GM-CSF, PGE2, or PS for 18 h, washed three times with PBS, and cultured with Con A (final concentration, 5 µg/ml). After 18 h at 37°C at a 5% CO2 atmosphere, the supernatants were collected for ELISA tests.

Cytokine ELISA

The amounts of IFN-{gamma} and IL-2 present in the supernatants from unstimulated and stimulated T cells and subsets was measured by ELISA (Endogen, Boston, MA). The amounts of cytokine present in each well were quantitated by measuring absorbance at 450 to 550 nm using a Tecan SLT Rainbow Reader (Labinstruments, Research Triangle Park, NC). OD values were converted to picograms per milliliter by including dilutions of known amounts of recombinant murine cytokine in the ELISA. A standard curve was generated by plotting the OD of the standards vs their known cytokine concentrations.

RNA extractions

T cells and subsets for RNA extraction were obtained by incubating 20 x 106 cells in 150-mm tissue culture dishes. The cells were cultured with or without Con A (5 µg/ml) for 6 h at 37°C in 5% CO2. At the end of the culture period, plates were washed twice with PBS. RNA of T cells was purified by collecting cells in 15-ml tubes and adding 1 ml of Tri-Reagent (Molecular Research Center, Cincinnati, OH) following the procedure suggested by the manufacturer.

Reverse transcriptase (RT)-PCR

First-strand cDNA synthesis was performed using the Stratagene (La Jolla, CA) RT-PCR kit. Oligo(dT) (300 ng) was added to 10 µg of total RNA in a volume of 40 µl. Samples were heated to 65°C for 5 min, cooled to room temperature, and reacted with 20 U of murine Moloney leukemia virus RT, 20 mM dNTP, RNase inhibitor, and first-strand buffer. PCR was performed following the Invitrogen (San Diego, CA) protocol. Hot wax Mg2+ beads (1.5 mM, final concentration) were added to minimize nonspecific bands. Thirty-five cycles of 1 min at 94°C, 1 min at 54°C, and 2 min at 72°C, with 10 min at 72°C on the last cycle, were run using a Perkin-Elmer (Norwalk, CT) thermocycler. Primers for the mouse IFN-{gamma}, IL-2, and ß-actin were purchased from Stratagene (La Jolla, CA). The primer sequences used were 5'-TAC TGC CAC GGC ACA GTC ATT GAA-3' and 5'-GCA GCG ACT CCT TTT CCG CTT CCT-3' for IFN-{gamma}, 5'-GTC AAC AGC GCA CCC ACT TCA AGC-3' and 5'-GCT TGT TGA GAT GAT GCT TTG ACA-3' for IL-2, and 5'-GTG GGC CGC TCT AGG CAC CA-3' and 5'-CGG TTG GCC TTA GGG TTC AGG GGG G-3' for ß-actin. PCR products were separated on a 0.8% agarose gel in 0.5x TBE buffer (0.045 M Tris-borate and 0.001 M EDTA, pH 8). The gel was stained with ethidium bromide, and bands were visualized by UV illumination. PCR of IFN-{gamma}, IL-2, and ß-actin message produced fragments of 405, 451, and 245 bp, respectively.

Densitometric analysis

Images were scanned on a Howtek Scanmaster 3+ scanner (Hudson, NH). Band intensity was assessed on a Sun Sparcstation 5 computer using BioImage software (Mountain View, CA). The IFN-{gamma} and IL-2 band intensities were normalized with control ß-actin band intensity. Percentages were calculated relative to the maximum for each experiment.

Methylation rate of IFN-{gamma} gene by PCR

Genomic DNA was purified from cultured T cells using the Easy-DNA kit (Invitrogen) protocol. Ten micrograms of DNA was digested by restriction endoenzymes (either SnaBI or HpaII) at 37°C for 5 h. Cytosine methylation in CpG dinucleotide at sensitive sites (HpaII at -2622, -1290, -1233; SnaBI at -52) in the 5'-flanking region of murine IFN-{gamma} gene was measured by PCR (16, 17). The primers used for HpaII site -2622 were 5'-CCA ACA GAA AGA GAA GAG CCC-3' and 5'-CCA TGT GAC TTT TCA CTA TGG-3', those used for site -1290 were 5'-GGC CTT CAA GTC TCC AGC AGC-3' and 5'-CTT CAG CCA AAG GCT CAA CCA-3', those used for site -1233 were 5'-GGC CTT CAA GTC TCC AGC AGC-3' and 5'-CTA GCC TCG GGG TCT TGA GTC-3', and those used for SnaBI site -52 were 5'-CCA TAC CCT TTC CTT GCT TTT-3' and 5'-CCT GAT CGA AGG CTC CTC GGG-3'. All the primers were synthesized by Life Technologies. The enzyme-digested DNA mixtures were used for PCR under the same conditions as those used for the RT-PCR experiments. The 20 µl of PCR products were loaded on 1% agarose gel, and the results were analyzed by densitometry as detailed above.

Nuclear extract preparation

Unfractionated T cells or CD4+ or CD8+ T cell subsets (5 x 106) were cultured in six-well flat-bottom tissue culture plates and stimulated for 1 h with or without Con A (5 µg/ml). In some experiments normal T cells and subsets were pretreated for 18 h with PS (50 µg/ml). Cells were washed twice with PBS, then treated with cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 0.1 mM antipain, 10 µg/ml aprotinin, 0.1 mM chymostatin, 0.1 mM leupeptin, and 1 mM pepstatin). The cells were left to swell on ice for 15 min, after which 25 µl of 10% Nonidet P-40 was added and vortexed for 10 s. Nuclei were removed from cytosol by centrifugation at 14,000 rpm for 30 s. The supernatants were removed, and the nuclear pellets were resuspended in 50 µl of ice-cold buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM antipain, 10 µg/ml aprotinin, 0.1 mM chymostatin, 0.1 mM leupeptin, and 1 mM pepstatin), and the tubes were rocked vigorously at 4°C for 15 min on a shaking platform. The extracts were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were stored at -70°C. The protein concentration was measured using the Sigma protein determination kit.

Electrophoretic mobility shift assay (EMSA)

The oligonucleotide used for DNA-protein binding assay was 5'-AAA ACT GTG AAA ATA CGT AAT CCT CAG GAG A-3' (18) (synthesized by Life Technologies), which correspond to the human IFN-{gamma} promoter core region (-70 to -40). This human IFN-{gamma} promoter sequence has been reported to be identical with the sequence in the mouse promoter (-69 to -40), except for the replacement by TC at positions -48 and -47 instead of an additional CpG dinucleotide (18). Complementary single-stranded oligonucleotides were annealed into double-stranded oligonucleotides by heating to 65°C for 15 min, then slowly cooling to room temperature. To prepare a probe for the EMSA, the double-stranded oligonucleotide (100 ng) was added to forward reaction buffer (60 mM Tris-HCl (pH 7.8), 15 mM 2-ME, 10 mM MgCl2, and 0.33 µM ATP) along with 200 µCi of [{gamma}-32P]ATP and 5 U of T4 polynucleotide kinase in a volume of 10 µl and incubated for 1 h at 37°C. Unincorporated precursors were removed using Sephadex G-25 columns (Boehringer Mannheim), and the sample was resuspended in 200 µl of ultrapure water and stored at -20°C. Nuclear extracts (5 µg/sample) were incubated in a 20-µl volume with 1x binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 0.5 mM DTT), 10% glycerol, 0.05% Nonidet P-40, and 1 µg of poly(dI-dC)-poly(dI-dC) (Pharmacia) on ice for 10 min before addition of 32P-labeled target DNA (1 ng). After the completion of the binding reaction, 2 µl of 10x loading dye (250 mM Tris-HCl (pH 7.5), 0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) was added, and samples were electrophoresed at 4°C through a 5% polyacrylamide gel in 0.5x TBE buffer (0.045 M Tris-borate and 0.001 M EDTA, pH 8) that had been prerun at 10 V/cm for 2 h before sample loading. The gels were dried and visualized by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} protein production is down-regulated in CD4+ Th cells from tumor bearers

The levels of IFN-{gamma} protein produced by unstimulated and Con A-stimulated T cells and subpopulations from normal or large mammary tumor-bearing mice were analyzed in the culture supernatants by ELISA at the previously determined optimal conditions. As shown in Figure 1GoA, unseparated T cells and CD4+ cells from tumor-bearing mice had greatly reduced levels of IFN-{gamma} compared with those in normal mice. Interestingly, the CD8+ T cells from tumor-bearing mice produced similar amounts of IFN-{gamma} as CD8+ T lymphocytes from normal mice. The levels of IL-2 expressed by the CD4+ and CD8+ T cell subpopulations from normal and tumor-bearing mice were also determined. As shown in Figure 1GoB, no significant differences could be detected between the production of IL-2 by unseparated T cells or CD4+ and CD8+ T lymphocytes from normal and tumor-bearing mice.



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  		FIGURE 1. Cytokine production by splenic T cells. Unfractionated T cells and CD4+ and CD8+ subsets from normal and tumor-bearing mice were cultured in vitro. The supernatants from unstimulated or Con A (5 µg/ml)-stimulated T cells were collected from culture analysis after 18 h. The levels of cytokine were determined by ELISA. A, Levels of IFN-{gamma}; B, levels of IL-2. Striped indicates T cells from normal mice; solid black indicates T cells from larger tumor-bearing mice. Only the levels of IFN-{gamma} from unfractionated T cells and CD4+ T cells were significantly different (p > 0.02) in normal and tumor-bearing mice. All other values were not statistically different.

 
IFN-{gamma} mRNA expression parallels protein production

To determine whether the cytokine alterations in T cells from tumor-bearing mice were due to impaired transcription, the IFN-{gamma} RNA levels of unstimulated or Con A-stimulated T cells and subsets were determined by RT-PCR. Figure 2GoA shows that as was the case at the protein level, the IFN-{gamma} RNA levels expressed by Con A-stimulated unseparated T cells and CD4+ cells from tumor-bearing mice were significantly reduced compared with those expressed by T cells from normal mice. In contrast, the levels of RNA expressed by CD8+ T cells from tumor bearers were similar to those observed in normal CD8+ T cells. The levels of IL-2 RNA were likewise analyzed. As was the case in the ELISA studies, no significant differences could be detected in the production of this cytokine by T cells and subsets from normal and tumor-bearing mice (Fig. 2GoB).



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  		FIGURE 2. Detection of cytokine RNA in splenic T cells and CD4+and CD8+ T cell subsets from normal and tumor-bearing mice by RT-PCR. Total RNA was purified from cells cultured in the presence or the absence of Con A and reverse transcribed into cDNA followed by 40 cycles of PCR. The cytokine images were then analyzed by a densitometer and normalized with ß-actin band intensity. Lanes 1 and 3, T cells or subsets from normal mice;lanes 2 and 4, T cells or subsets from large tumor-bearing mice. A, IFN-{gamma} RNA; B, IL-2 RNA.

 
Effects of tumor-derived factors on the IFN-{gamma} production of T cells and subsets

In previous investigations we have documented the production of several tumor-derived factors that have biologic activity on several immune effector cells (9, 10, 11, 12, 13). We evaluated the possibility that one or more of these factors could be differentially affecting the T cell subsets in tumor-bearing mice. Normal T cells as well as its subpopulations were pretreated with various concentrations of GM-CSF, PGE2, or PS for 24 h, followed by Con A stimulation for 18 h. Figure 3Go shows the IFN-{gamma} levels in supernatants of such cultures as detected by ELISA. Addition of GM-CSF increased IFN-{gamma} levels in unseparated normal T cells and very slightly increased these levels in CD4+ and CD8+ T cell subsets. Addition of PGE2 severely diminished the production of IFN-{gamma} by T lymphocytes and CD4+ and CD8+ T cell subsets. Importantly, when various concentrations of PS previously shown to be released by the tumor cells (9) were used, a profound decrease in IFN-{gamma} levels could be observed in the pretreated normal unseparated T lymphocytes and CD4+ T cells. However, the IFN-{gamma} production of PS-pretreated CD8+ T cells was not down-regulated and was expressed at levels equal to those from the PS-untreated CD8+ normal T lymphocytes. This suggests that PS pretreatment had a differential effect on the IFN-{gamma} production of CD4+ and CD8+ T cells.



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  		FIGURE 3. The effect of tumor-derived factors on IFN-{gamma} production by unseparated T cells and CD4+ and CD8+subsets. T cells and subsets from normal BALB/c mice were pretreated with GM-CSF, PGE2, or PS for 18 h and stimulated with Con A (5 µg/ml) for an additional 18 h. Supernatants were collected, and IFN-{gamma} production was measured by ELISA. Solid white indicates total T cells; striped indicates CD4+ T cells; solid black indicates CD8+ T cells.

 
PS effects on IFN-{gamma} RNA in T cells and subsets

Using RT-PCR we measured IFN-{gamma} RNA levels in PS-pretreated cultured T cells from normal mice with or without Con A stimulation. As shown in Figure 4Go, the expression of IFN-{gamma} RNA was dramatically reduced in normal unseparated T cells and the CD4+ subset pretreated with PS, while the levels of IFN-{gamma} present in PS-pretreated CD8+ T cells were only slightly affected compared with those in untreated cells. These results closely parallel those in tumor-bearing mice.



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  		FIGURE 4. IFN-{gamma} RNA levels in T cells and subpopulations from normal mice untreated or pretreated with PS. Splenic T cells as well as CD4+ and CD8+ cells from normal BALB/c mice were untreated or pretreated with PS for 18 h and further treated with Con A for 18 h. IFN-{gamma} RNA levels were determined by RT-PCR, and the images were normalized by the intensity of ß-actin.

 
IFN-{gamma} gene methylation levels in T cells from tumor-bearing mice and PS-pretreated normal T cells

The data presented suggest that the inhibition of IFN-{gamma} production in CD4+ T cells from large tumor-bearing mice is an event occurring before transcription. Thus, we explored the possibility that the down-regulation of IFN-{gamma} gene expression at the molecular level is due to differential methylation levels of the T cell subsets. DNA was extracted from unseparated splenic T cells and CD4+ and CD8+ subsets from normal and tumor-bearing mice and digested with methylation-sensitive restriction enzymes. The digested DNA was used to measure the degree of methylation according to the quantitative PCR assay described by Singer-Sam et al. (16). As shown in Figure 5Go, the SnaBI site in inactivated T cells from both normal and tumor-bearing mice was highly methylated. Upon stimulation with Con A, there was a marked decrease in methylation in normal unseparated T cells as well as its subsets, correlating with the levels of IFN-{gamma} production described above. In contrast, unseparated T cells and CD4+ T cells in tumor bearers showed a hypermethylated state compared to those in normal mice, while the CD8+ T subset from the tumor-bearing mice exhibited a similar state of methylation as its normal counterpart. In additional experiments (Fig. 6Go), it was found that pretreatment of normal T cells and subsets with PS resulted in a pattern strikingly similar to that observed in tumor-bearing mice, i.e., the IFN-{gamma} gene SnaBI site of pretreated unseparated T lymphocytes and the CD4+ subset is hypermethylated, while the CD8+ cell subset has highly reduced levels of methylation compared with the CD4+ T cells pretreated with PS.



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  		FIGURE 5. PCR analysis of the degree of CpG methylation in the IFN-{gamma} promoter region. Genomic DNA of T cells and CD4+ and CD8+ subsets from normal and large tumor-bearing mice were purified and digested with SnaBI (10 U/mg of DNA) for 6 h at 37°C. Five microliters of digestion mix was used for detection of CpG methylation status by PCR as explained in Materials and Methods. The levels of methylation were determined on 1% agarose and analyzed by densitometry. Lanes 1 through 6, T cells from normal BALB/c mice; lanes 7 through12, T cells from large tumor-bearing mice; lanes 1, 2, 7, and 8, total T cells;lanes 3, 4, 9, and 10, CD4+ T cells; lanes 5, 6, 11, and 12, CD8+ T cells.

 


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  		FIGURE 6. Effect of PS on cytosine methylation at the CpG site of the IFN-{gamma} promoter of T cells. Unfractionated T cells as well as CD4+ and CD8+ subsets from normal mice, untreated or pretreated with PS (50 µg/ml), were stimulated with Con A (5 µg/ml). Genomic DNA was purified, and 10 µg from each sample was digested with SnaBI (10 U/µg of DNA) for 6 h at 37°C. Five microliters of digestion mix was used for detection of methylation at the SnaBI site by PCR according to the conditions detailed in Materials and Methods. The degree of methylation was determined on 1% agarose and analyzed by densitometry.Lanes 1 through 6, T cells from normal BALB/c mice; lanes 7 through 12, T cells from large tumor-bearing mice; lanes 1 through 4, total T cells; lanes 5 through 8, CD4+ T cells; lanes 9 through 12, CD8+ T cells.

 
Differential nuclear binding patterns of CD4+ and CD8+ T cells from tumor-bearing mice and PS-pretreated normal cells

In addition to the altered patterns of methylation observed in T cells and subsets from tumor-bearing mice, it is possible that there is an alteration in the activation and/or binding of a relevant transcription factor. To explore this possibility, we performed EMSA with a labeled oligonucleotide corresponding to the IFN-{gamma} core promoter region. In Figure 7Go it can be seen that there are several complexes observed when Con A-activated nuclear extracts from T cells or CD4+ and CD8+ subsets from normal mice were used. A prominent 90-kDa band was observed in all these samples, and at least one other band could be clearly detected. When nuclear extracts from T lymphocytes from tumor bearers were used, there was a disappearance of the 90-kDa band. In CD4+ cells from tumor-bearing mice there was a light 70-kDa band that was more prominent in the CD8+ T cell subset from these animals.



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  		FIGURE 7. EMSA analysis of the pattern of nuclear binding to the IFN-{gamma} gene core region. Nuclear extracts were prepared from Con A-stimulated unfractionated T cells and CD4+ and CD8+subsets derived from normal and tumor-bearing mice. EMSAs were carried out by incubating nuclear extract with a 32P-labeled oligonucleotide corresponding to the IFN-{gamma} core region. The results were resolved on 4% polyacrylamide gels. Lanes 1through 3, Normal T cells; lanes 4 through6, T cells from tumor bearers.

 
Similar experiments were performed to compare the binding of nuclear extracts from Con A-stimulated untreated and PS-pretreated unseparated normal T cells and CD4+ and CD8+ subsets. As shown in Figure 8Go, pretreatment of PS followed by Con A stimulation resulted in a dramatic alteration of the binding pattern of the unseparated T lymphocytes and the CD4+ subset represented by the almost complete loss of the 90-kDa band observed in untreated unseparated T cells and CD4+ T cells. Regarding the nuclear extracts of the PS-pretreated CD8+ T cells, although there was a decrease in the intensity of the 90-kDa band compared with that on untreated cells, it was clearly detectable and much more pronounced than that observed in the PS-pretreated unseparated T lymphocytes and CD4+ T cells.



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  		FIGURE 8. Effect of PS on the nuclear protein binding patterns of T cells and subsets. Nuclear extracts were prepared from T cells as well as from CD4+ and CD8+ cells from normal BALB/c mice with or without PS pretreatment and Con A stimulation, followed by incubation with a 32P-labeled oligonucleotide corresponding to the IFN-{gamma} core region. EMSA results were resolved on 4% polyacrylamide gels. Lanes 1 through 4, Total T cells; lanes 5 through 8, CD4+ cells;lanes 9 through 12, CD8+ cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years there has been considerable interest in the patterns of cytokine secretion by T cell subsets. Although the initial studies concentrated on the Th cell subpopulations (19, 20), it is now apparent that CD8+ cells and other T cells, such as {gamma}{delta} T cells, also display similar patterns of cytokine production (21, 22, 23, 24). An imbalance of cytokine subsets has been postulated as an important element in some disease states (25, 26, 27), and some investigations have shown a shift from a predominant Th1 to a Th2 phenotype during tumor progression (18, 28). Much less attention has been devoted to the alterations of cytokine patterns between CD4+ and CD8+ T cells in disease.

We have previously shown that the impaired IFN-{gamma} production observed in mice bearing large mammary tumors is not due to a shift from a Th1 to a Th2 phenotype, since the levels of IL-2, IL-4, IL-6, and IL-10 are unchanged in T cells from tumor bearers (11). Surprisingly, analysis of IFN-{gamma} production in CD4+ and CD8+ T lymphocytes revealed a dichotomy between the levels of IFN-{gamma} RNA and protein in these T cell subsets in tumor-bearing mice. Carter and Dutton (24) have reviewed the existence of populations of T cells polarized toward the secretion of type 1 or type 2 cytokines. However, the nature of the secretion patterns in individual cells and their regulation are not well understood. Our results clearly show a dissociation between the synthesis of IL-2 and IFN-{gamma}, two type 1 cytokines that have been shown by several authors (19, 29) to be coordinately regulated. While this may occur in whole populations, the work of Assenmacher et al. (30) has clearly shown by flow cytometry that IL-2 and IFN-{gamma} are coexpressed in some, but not all, Th cells. Bucy and colleagues (31) showed heterogeneity of single cell cytokine gene expression in clonal T cell populations. Dieli et al. (32) have shown that during contact hypersensitivity to the hapten picryl chloride, the levels of proliferation and IFN-{gamma} and IL-2 production displayed by immune lymph node cells were Ag specific, but required different cell populations. Kelso (22) has presented a model to explain the diversity of cytokine coexpression patterns in individual cells and proposed that different cytokine regulators increase the probability of expression of some, but not all, cytokine genes.

DNA methylation in various CpG dinucleotide sites in the 5' upstream flanking region and first intron of IFN-{gamma} gene had been investigated by several laboratories (33, 34, 35). In this study, we analyzed the methylation status of the 5' promoter region of IFN-{gamma} gene in the CD4+ and CD8+ T cell subsets in normal mice and tumor bearers. A correlation was obtained between the impaired production of IFN-{gamma} and a high level of methylation in the T cell subsets. Our results are in agreement with those of other investigators. Thus, Young et al. (35) reported that hypermethylation of CpG dinucleotide within a TATA proximal regulatory element of the promoter correlates with the transcription of the IFN-{gamma} gene in murine Th1 and Th2 CD4+ T cell clones. The site in Th2 cells was >98% methylated, while that in Th1 cells was nearly completely demethylated. Melvin et al. (33) have shown that methylation conditions in IFN-{gamma} gene in CpG sites within or near transcriptional activator elements in the 5'-flanking and first intron were essentially different in primary T-lineage cell populations in vivo. In addition to the methylation state of the IFN-{gamma} gene promoter region, other mechanisms may be contributing to the differential expression of this cytokine in CD4+ and CD8+ T cells from tumor-bearing mice. It has been shown that in the 5' upstream flanking region of the IFN-{gamma} gene there is an important nuclear protein binding region that may function as an enhancer-like element or a suppressor element of the gene (36, 37, 38). Methylation of the transcriptional regulatory elements containing an internal CpG could influence gene expression by directly preventing the binding of transcription factors (33) or acting via an indirect mechanism (39). In our EMSA studies shown in Figure 6Go, an approximately 90-kDa protein was found to bind to the IFN-{gamma} promoter core region in nuclear extracts from Con A-activated T cells and subsets of normal mice. In contrast, diminished levels of this nuclear binding protein could be shown in T cells and CD4+ T cells from tumor bearers. Interestingly, the binding of this nuclear factor was not severely altered in the tumor bearers’ CD8+ T cells, suggesting that this protein is involved in the differential expression of the IFN-{gamma} gene observed in T cell subsets during tumorigenesis.

To further elucidate the mechanisms responsible for the dichotomy of IFN-{gamma} expression in CD4+ and CD8+ T cells from tumor bearers, we pretreated in vitro normal T cells and their subsets with factors that we have previously shown to be produced by the mammary tumor cells used in our studies (9, 10, 11, 12, 13). As has been previously described by others, PGE2 was capable of down-regulating IFN-{gamma} production by T cells (40, 41). However, we have found that PGE2 equally depressed the levels of IFN-{gamma} on both CD4+ and CD8+ T cells (Fig. 3Go). GM-CSF, a cytokine constitutively produced by our mammary tumor cells (12, 13) and by several other murine and human nonlymphopoietic tumors (42, 43, 44, 45), was found to exert up-regulatory effects on the production of IFN-{gamma} by unseparated normal T cells as well as on CD4+ and CD8+ T cells. Pretreatment of normal unseparated T cells with PS resulted in a profound decrease in their IFN-{gamma} levels at both the transcriptional and translational levels. Furthermore, PS showed remarkable differential effects on T cell subsets from normal mice, i.e., down-regulation of the IFN-{gamma} levels of CD4+ T cells, but not of the CD8+ T cells. These results, which mimic those observed in tumor-bearing mice, were further substantiated by the similar patterns of methylation (Figs. 5Go and 6Go) and binding of a nuclear factor to the IFN-{gamma} core promoter sequence (Figs. 7Go and 8Go) observed between PS-pretreated T cells and subsets and those from tumor-bearing mice. Our previous studies have shown that PS possess suppressive effects on macrophages. Thus, we have shown that PS greatly down-regulates the transcription of inducible nitric oxide synthase and the production of nitric oxide (9, 46, 47). Furthermore, we have also described the impairment of IL-12 production in macrophages pretreated with this phospholipid (11).

IL-12 is a cytokine that has been shown to be important for the induction of IFN-{gamma} in CD4+, CD8+, and NK cells (48, 49). As stated above, the production of this cytokine is greatly down-regulated in PS-pretreated macrophages as well as in macrophages from tumor-bearing mice. Although this finding may help explain the low levels of IFN-{gamma} production by CD4+ T cells from tumor bearers, the reasons for the intact production of IFN-{gamma} by CD8+ T cells are not clear. However, our new findings that PS can directly affect the production of IFN-{gamma} in CD4+ T cells, but not in CD8+ T cells, may provide an alternative explanation for the observed phenomenon.

The results presented herein and those reported in the literature indicate that IFN-{gamma} synthesis is regulated at multiple levels and in different manners in the various types of cells. The differential expression of IFN-{gamma} in CD4+ and CD8+ T cells during tumorigenesis represents a clear example of the independent cytokine regulation in T lymphocytes. Furthermore, the apparent selective effects of PS on CD4+ T cells attest to the specificity of the effects that tumor-derived factors can exert on the various types of lymphoreticular cells and their subsets.


   Acknowledgments
 
We are grateful to Mr. Mantley Dorsey, Jr., and Mrs. Lynn Herbert for excellent technical assistance, and to Michelle Perez for preparing this manuscript.


   Footnotes
 
1 This work was supported by Grant CA25583 from the U.S. Public Health Service. Back

2 Address correspondence and reprint requests to Dr. Diana M. Lopez, Department of Microbiology and Immunology, University of Miami School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101. Back

3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage colony-stimulating factor; PS, phosphatidyl serine; RT-PCR, reverse transcriptase-polymerase chain reaction; EMSA, electrophoretic mobility shift assay. Back

Received for publication August 1, 1997. Accepted for publication November 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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