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International Immunology, Vol. 13, No. 1, 43-51, January 2001
© 2001 Japanese Society for Immunology

Counter-regulation of cytolytic activity and cytokine production in HIV-1-specific murine CD8+ cytotoxic T lymphocytes by free antigenic peptide

Megumi Takahashi1, Yohko Nakagawa1, Jay A. Berzofsky2 and Hidemi Takahashi1

1 Department of Microbiology and Immunology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
2 Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, National Institute of Health, Bethesda, MD 20892-1578, USA

Correspondence to: H. Takahashi


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We have reported previously that the cytolytic activity of murine CD8+ cytotoxic T lymphocytes (CTL) specific for HIV-1 gp160 envelope glycoprotein was markedly inhibited by brief exposure to the free minimal antigenic peptide (I-10: 10mer peptide from gp160) by direct binding to class I MHC molecules of specific CTL in the absence of antigen-presenting cells (APC). Here, we show that treatment of such CTL with the peptide induced not only the inhibition of cytolytic activity but also IL-2Rß down-modulation, followed by the inhibition of IL-2-dependent growth. The peptide-mediated inhibition and restoration of expression of IL-2Rß were well correlated with changes in both cytolytic activity and IL-2-dependent growth of the CTL. Since enzymatic activity of granzyme B, and mRNA expression of granzyme B and perforin were significantly reduced in peptide-treated CTL, the inhibition of cytolytic activity was mainly caused by the exhaustion of cytolytic molecules. Moreover, treatment of the CTL with the epitopic peptide resulted in production of high levels of IL-2, IFN-{gamma}, tumor necrosis factor-{alpha} and MIP-1ß in the culture supernatant. Maximum amounts of cytokines were obtained in the culture supernatant when the level of cytolytic activity was the lowest. Thus, although the CTL temporarily lost their cytolytic activities, they simultaneously gained the abilities to produce cytokines for activation of various cell populations. These changes induced by free antigenic peptide in CD8+ CTL reveal an interesting counter-regulation between their cytolytic activities and cytokine production.

Keywords: cytokines, cytolytic molecules, cytotoxic T lymphocyte, free antigenic peptide, HIV-1 IL-2Rß


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
TCR-mediated induction of IL-2 and IL-2 receptor (IL-2R) expression are the most important events in T cell activation required for G1 progression and entry into S phase. However, in the absence of co-stimulatory signals, engagement of the TCR on T cells with antigenic peptide presented by MHC molecules prevents the subsequent production of IL-2 and results in anergy for autocrine proliferation (1,2). These anergic T cells are unable to produce IL-2, but they do proliferate in response to exogenous IL-2. On the other hand, when a subset of T cells that cannot produce but require IL-2 for their growth is re-stimulated through the TCR after primary stimulation with antigen, IL-2 unresponsiveness and cell growth arrest are induced (3). Thus, IL-2 production and IL-2 responsiveness in anergic states have been well characterized in antigen-specific CD4+ T cells.

In contrast, little is known about anergic states of antigen-specific CD8+ T cells. Stimulation of CD8+ cytotoxic T lymphocytes (CTL) incapable of producing IL-2 with immobilized anti-TCR mAb did not induce their proliferation (4–6) in response to subsequent antigen stimulation, whereas their specific cytolytic activities were retained. Otten et al. reported that CD8+ T cells capable of producing IL-2 were brought into anergy by exposure to antigen in the presence of fixed antigen-presenting cells (APC) (7). These anergic CD8+ T cells failed to produce IL-2, but retained their cytolytic activities. Thus, in the case of anergic CD8+ T cells, their cytolytic activities as well as their responses to antigens or IL-2 must be taken into consideration.

CD8+ CTL recognize peptide epitopes in association with MHC class I molecules on the target cells, and subsequently proliferate and kill the target cells. Several recent papers have described the inhibitory effects of free antigenic peptide on the CTL in the absence of other presenting cells. Various effects have been reported, e.g. self-lysis of the CTL (8,9), loss of ability to lyse the target cells without self-lysis (10,11) and CTL–CTL killing (12,13). However, the effects of peptide differed between studies and thus the mechanisms responsible for inhibition of CTL is still controversial.

We demonstrated previously that the cytolytic activity of murine CTL specific for HIV-1 gp160 envelope glycoprotein was markedly inhibited by brief exposure to minimal free antigenic peptide and this was not due to fratricide or suicide (apoptosis) (11). Since the inhibition of cytolytic activity by free antigenic peptide might affect the progress of viral diseases such as AIDS and chronic hepatitis, or of cancer, we performed the present study with a view to clarifying the mechanism responsible for the induction of CTL inhibition and to find a strategy for preventing CTL dysfunction or for their recovery from their inactive state.

In this study, we demonstrated that brief treatment of the CTL with free epitopic peptide induced not only the inhibition of cytolytic activity but also IL-2 unresponsiveness associated with the down-modulation of IL-2Rß on the CTL. These findings suggested the possibility of a close relationship between signaling through IL-2Rß and the expression of cytolytic molecules. Moreover, we observed that such treatment of the CTL resulted in the high-level production of various cytokines in the culture supernatants. Therefore, exposure of CTL to free epitopic peptide inhibits cytolytic activity but facilitates cytokine production. Based on these observations, we discuss here the unique feature of counter-regulation observed in CD8+ CTL generated by free antigenic peptide.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice and synthetic peptides
Female BALB/c mice, 6–8 weeks of age, were purchased from Charles River Japan (Tokyo, Japan). Peptides were synthesized on an Applied Biosystems (Foster City, CA) model 430A peptide synthesizer, using conventional t-Boc chemistry and cleaved from the resin by liquid HF. Synthetic peptides were purified by gel filtration on Bio-gel P-4 and analyzed by HPLC on a C18 reverse-phase column. Peptide fractions containing >90% of the desired product were used for the experiments. Peptide I-10 (RGPGRAFVTI) (14) and peptide MNT10 (IGPGRAFYAT) (15) represent the immunodominant CTL epitopes, both presented by the murine class I MHC molecule H-2d, in the V3 loop of HIV-1 gp160 glycoprotein found in strains IIIB and MN respectively.

Generation of CTL lines
BALB/c mouse spleen cells (5x106) from mice previously immunized with 1x107 p.f.u. of vSC25 (recombinant vaccinia virus expressing HIV envelope glycoprotein gp160 of the IIIB isolate) (16) were stimulated with mitomycin C (MMC)-treated HIV-1-IIIB gp160 gene-transfected BALB/c3T3 cells (1x105 cells, named 15-12) in vitro (17) in 24-well plates containing 1.5 ml of complete T cell culture medium which was composed of RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, mixture of vitamins, 1 mM HEPES, 100 U/µl of penicillin, 100 µg/ml of streptomycin, 50 µM 2-mercaptoethanol, heat-inactivated 10% FCS and 10% rat T-STIM (Collaborative Biomedical Products, Bedford, MA). The CTL lines were established and maintained by bi-weekly stimulation with MMC-treated 15-12 cells and named LINE-IIIB cells.

CTL assay
Cytotoxicity was assessed in a standard 4–6 h 51Cr-release assay as described previously (17), with 51Cr-labeled 15-12 cells as targets at various E:T ratios in 96-well U-bottomed culture plates. After incubation, the plates were centrifuged and 100 µl of cell-free supernatants were collected to measure the radioactivity using a {gamma}-counter. The percentage of specific 51Cr release was calculated as: 100x(experimental release – spontaneous release)/(maximum release – spontaneous release). Maximum release was determined from supernatants of cells lysed by addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells. SEM of triplicate cultures was always <3% of the mean.

Measurement of cell proliferation
Peptide I-10 represents the minimal active epitope in the V3 loop of HIV-1 IIIB gp160 restricted by the Dd class I MHC molecule (14,18). The CTL were treated or untreated with 1 µM of peptide I-10 for 1 h, followed by washing to remove free peptide. The I-10-treated cells were seeded at a density of 1 or 5x104 cells/well in 96-well flat-bottomed microtiter plates and cultured in the presence or absence of 50 U/ml of human rIL-2 for 1, 2 or 3 days. Proliferation was measured by addition of 0.5 µCi [3H]thymidine/well for the last 16 h, and the plates were harvested and counted using a ß-counter (1450 Microbeta Trilux; Wallac, Gaithersburg, MD).

Colorimetric granzyme B assay
Granzyme B activity was measured using the granzyme B-specific synthetic substrate Boc-Ala-Ala-Asp thiobenzyl ester (BLT; Calbiochem, San Diego, CA) in PBS. A 180 µl volume of the reaction mixture [0.2 mM Boc-Ala-Ala-Asp thiobenzyl ester and 0.1 mM 5,5-dithiobis(2-nitrobenzoic acid) in 0.2 mM Tris buffer, pH 8.0] was added to 20 µl of cell lysates. The absorbance at 405 nm was measured after incubation for 60–90 min at 37°C. An absorbance of 0.01 was arbitrarily defined as 1 U of esterolytic activity.

FACS analysis
Cells were pelleted and resuspended at a concentration of 5x105 cells in 100 µl of PBS with 0.1% NaN3 containing phycoerythrin (PE)-labeled rat anti-mouse IL-2Rß (clone PC61; PharMingen, San Diego, CA ) or FITC-labeled rat anti-mouse IL-2R{alpha} (clone TM-ß1; PharMingen) mAb. After 30 min incubation on ice, cells were washed and resuspended in PBS for analysis by FACScan (Becton Dickinson, Mountain View, CA). For negative controls, the cells were incubated with rat IgG labeled either with PE or FITC. Fluorescence data were analyzed by the Lysys II software program (Becton Dickinson).

Semiquantitative RT-PCR
Total RNA was prepared from the CTL using Isogen (Nippon Gene, Toyama, Japan). Then 1 µg of RNA was incubated for 1 h at 42°C after adding 20 U of RNase inhibitors (TaKaRa Biomedicals, Otsu, Japan), 0.2 mM deoxynucleoside triphosphates, 2.5 nM random primers, 11 U of Rous associated virus-2 reverse transcriptase (TaKaRa) and reverse transcriptase buffer to a final volume of 20 µl. Quantification of cDNA was performed by amplifying the ß-actin housekeeping gene. Quantified cDNA was then amplified in the presence of 0.2 mM deoxynucleoside triphosphates, 2.5 U of Taq polymerase (TaKaRa) and 0.5 mM of each primer. The thermal cycle consisted of 30 s at 94°C, 45 s at 55°C for ß-actin or 60°C for IL-2, IFN-{gamma}, TNF-{alpha}, MIP-1ß, granzyme B and perforin, and 45 s at 72°C. The numbers of PCR cycles (ß-actin, 23 cycles; IL-2, 35 cycles; IFN-{gamma}, 32 cycles; TNF-{alpha}, 26 cycles; MIP-1ß,28 cycles; perforin, 31 cycles; granzyme B, 31 cycles) were chosen to generate PCR product during the exponential phase of amplification. PCR products were resolved on 2% agarose gels containing ethidium bromide and visualized under UV light illumination. Primers used in the present study are listed as follows: ß-actin sense: 5'-ATGGATGACGATATCGCT-3', ß-actin antisense: 5'-ATGAGGTAGTCTGTCAGGT-3'; IL-2 sense: 5'-AACAGCGCACCCACTTCAA-3', IL-2 antisense: 5'-TTGAGATGATGCTTTGACA-3'; IFN-{gamma} sense: 5'-AACGCTACACACTGCATCT-3', IFN-{gamma} antisense: 5'-TGCTCATTGTAATGCTTGG-3'; TNF-{alpha} sense: 5'-GAAAGCATGATCCGCGACGTGG-3', TNF-{alpha} antisense: 5'-GTAGACCTGCCCGGACTCCGCAA-3'; MIP-1ß sense: 5'-CCACAATAGCAGAGAAAVAGCAAT-3', MIP-1ß antisense: 5'-AACCCCGAGCAACACCATGAAG-3'; perforin sense: 5'-AGCCAGCGTCTCCAGTGAAT-3', perforin antisense: 5'-CGCTTCGGGTTCTGTTCTTC-3'; granzyme B sense: 5'-GAAGATGCCACCAGTCCTG-3', granzyme B antisense: 5'-GAAGATGCCACCAGTCCTG-3'.

Northern blotting analysis
Aliquots of 10 µg of total RNA prepared from peptide I-10-treated or untreated CTL were fractionated by electrophoresis in 1.2% agarose gels containing 6% glyoxal and transferred on to nylon membranes in 20xSSC (175.3 g of NaCl and 88.2 g of sodium citrate in a total volume of 1000 ml, pH 7.0). The blots were then hybridized with 32P-labeled probe in Rapid-hybrid buffer (Amersham, Little Chalfont, UK) at 55°C for 2 h. Radioactivity levels were measured using a Fujix BAS 2000 bio-imaging analyzer (Fuji Photo Film, Tokyo, Japan).

Measurement of cytokine activity and contents in culture supernatants
CTLL-2 cells (1x104 cells/well), an IL-2-dependent T cell line, were cultured with the samples for 24 h at 37°C. Proliferation of CTLL-2 cells was assessed by the MTT method (19). Concentration of IFN-{gamma} and TNF-{alpha} protein contents were measured by mouse IFN-{gamma} and TNF-{alpha} ELISA kits (BioSource International, Camarillo, CA) respectively. For quantitation of MIP-1ß, a combination of goat anti-mouse MIP-1ß antibody (R & D Systems, Minneapolis, MN) for capturing and biotinylated goat anti-mouse MIP-1ß antibody (Genzyme, Cambridge, MA) for detection was used. Neutralization tests were performed using anti-murine IFN-{gamma} mAb (clone XMG1.2; PharMingen, San Diego, CA), anti-murine TNF-{alpha} mAb (clone MP6-XT22; PharMingen) and anti-murine IL-2 mAb (clone S4B6; PharMingen), and anti-murine MIP-1ß polyclonal antibody (R & D Systems).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Time course analysis of the cytolytic activity after peptide I-10 treatment
The CD8+ CTL (LINE- IIIB cells, 1x106 cells/ml) specific for HIV-1 gp160 envelope protein were incubated with 1 µM of peptide I-10 for 1 h and then washed to remove free peptide. The treated cells were further incubated for an additional 1, 2 or 3 days in the absence of antigenic peptide and the cytolytic activity was assessed at the indicated time points. As previously reported (11), treatment of the CTL with peptide I-10 resulted in marked inhibition of target cell lysis. Time course studies demonstrated that maximum inhibition of the cytolytic activity occurred on day 1 and the cytolytic activity was restored to a level almost equal to that in untreated controls over time (Fig. 1AGo). The inhibition of cytolytic activity was dependent on the peptide I-10 concentration, and half-maximum inhibition was achieved between 0.01 and 0.1 µM (Fig. 1BGo).



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  		Fig. 1. Time course analysis of the cytolytic activity after peptide I-10 treatment. (A) The CTL (LINE-IIIB cells) were incubated with 1 µM epitopic peptide I-10 for 1 h and washed 3 times to remove free peptide. After incubation of treated cells for an additional 1 (•), 2 ({blacksquare}) or 3 ({blacktriangleup}) days, the cells were incubated with 51Cr-labeled 15-12 cells in 96-well round-bottomed microtiter plates for the 4 h CTL assay. {circ}, Untreated cells. (B) The CTL were incubated with 1, 1x10–1, 1x10–2, 1x10–3, 1x10–4, 1x10–5 or 1x10–6 µM peptide I-10 for 1 h and washed 3 times to remove free peptide. After treatment, cells were incubated with 51Cr-labeled 15-12 cells in 96-well round-bottomed microtiter plates for 4 h followed by 51Cr-release assay.

 
Effects of peptide I-10 treatment on IL-2-dependent growth of the CTL
Although the CTL used in this study were unable to secrete detectable levels of IL-2, they required IL-2 for their proliferation. When the CTL were treated with epitopic peptide I-10, IL-2-dependent growth was strongly inhibited (Fig. 2AGo). Since the cell viability determined by the MTT method was not affected by treatment with the peptide (data not shown), the unresponsiveness was not ascribed to cell death. The inhibition of IL-2-dependent CTL growth was corresponding to the peptide concentration. The minimum effective concentration of the peptide was 0.01 µM (Fig. 2BGo).



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  		Fig. 2. Effects of peptide I-10 treatment on the IL-2-dependent growth of the CTL. (A) The CTL were incubated with or without 1 µM peptide I-10 for 1 h and washed 3 times to remove free peptide. After incubation of 5x104 cells/well with or without human rIL-2 (50 U/ml) for an additional 1 day, cells were pulse labeled with [3H]thymidine for the last 16 h to measure proliferative responses. (B) The CTL were incubated with 1, 1x10–1, 1x10–2, 1x10–3, 1x10–4, 1x10–5 or 1x10–6 µM peptide I-10 for 1 h and washed to remove free peptide. After incubation of 5x104 cells/well with (shaded columns) or without (filled columns) human rIL-2 (50 U/ml) for an additional 1 day, cells were pulse labeled with [3H]thymidine for the last 16 h.

 
Effects of peptide I-10 treatment on the expression of IL-2R on the CTL
Because treatment of the CTL with peptide I-10 induced IL-2 unresponsiveness, we examined the levels of IL-2R{alpha} and ß chain expression on the CTL. The CTL treated with peptide I-10 showed marked down-modulation of IL-2Rß but not IL-2R{alpha} expression. The level of IL-2Rß down-modulation was highest 1 day after treatment and was gradually restored to the control level (Fig. 3AGo). IL-2Rß down-modulation was dependent on peptide concentration, which was effective at >=0.01 µM (Fig. 3BGo). Although both peptide I-10 and peptide MNT10 should bind the H-2Dd molecules expressed on the LINE-IIIB cells, only peptide I-10 could be recognized by the TCR of the CTL. Treatment of the CTL with peptide MNT10 induced no effect on either IL-2R{alpha} or ß chain expression (data not shown). Therefore, the IL-2Rß down-modulation could not be explained by simple binding of free epitopic peptide onto MHC class I molecules of the CTL.



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  		Fig. 3. Effects of peptide I-10 treatment on the expression of IL-2Rß on the CTL. The CTL were incubated with (filled curves) or without (open curves) 1 µM peptide I-10 for 1 h and washed 3 times to remove free peptide. After incubation of treated cells for an additional 1, 2 or 3 days (A) or just after treatment (B), cells were stained with PE-conjugated anti-mouse IL-2Rß chain mAb. After 30 min incubation on ice, cells were washed 3 times and resuspended in PBS for analysis by FACScan.

 
Effects of peptide I-10 treatment on granzyme B activity of the CTL
To better understand the mechanism by which peptide I-10 inhibits CTL activity, we examined the effects of peptide I-10 on granzyme B activity. We used a colorimetric assay to detect granzyme B enzymatic activity in the CTL lysates. Cell lysates were obtained at various time points after treatment of CTL with 1 µM peptide I-10 for 1 h. Significant reduction of granzyme B activity was observed on day 1 and the activity was gradually restored thereafter (Fig. 4Go).



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  		Fig. 4. Effects of peptide I-10 treatment on granzyme B activity of the CTL. The CTL were incubated with (shaded columns) or without (filled columns) 1 µM of peptide I-10 for 1 h and washed to remove free peptide. After the incubation of treated cells for an additional 1, 2 or 3 days, cells were incubated with 0.5% NP-40 in PBS for 30 min at 4°C to prepare cell lysates. The lysates were used for BLT assay to measure granzyme B activity.

 
Effects of peptide I-10 treatment on granzyme B, perforin and Fas ligand (FasL) mRNA expression by the CTL
We next examined the effects of I-10 treatment on the expression of genes associated with T cell-mediated cytotoxicity. Total RNA was isolated from the CTL treated with peptide I-10 for 1 h and cultured for 24 h. Granzyme B, perforin and FasL gene transcription were assessed by Northern blotting analysis. Treatment of the CTL with peptide I-10 resulted in marked reduction in both Granzyme B and perforin mRNA expression. In contrast, the levels of FasL mRNA was not changed (Fig. 5Go).



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  		Fig. 5. Effects of peptide I-10 treatment on granzyme B, perforin and FasL mRNA expression. The CTL were incubated with (left lane) or without (right lane) 1 µM peptide I-10 for 1 h and washed to remove free peptide. After incubation for an additional 1 day, total RNA was isolated and mRNA levels were determined by Northern blotting analysis.

 
Effect of rapamycin (RAP) on the cytolytic activity and the expression of cytolytic molecules
RAP has potent immunosuppressive properties reflecting the inhibition of T cell activation subsequent to the binding of IL-2 to its high-affinity receptor IL-2Rß (20). To confirm the relationship between signaling through IL-2Rß and expression of cytolytic molecules, we examined the effect of RAP on the cytolytic activity and the gene expression of cytolytic molecules in LINE-IIIB cells. Treatment of the CTL with 10 ng/ml RAP (Research Biochemicals International, Natick, MA) for 3 days led to a marked reduction of the cytolytic activity and the gene expression of granzyme B and perforin (Fig. 6Go)



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  		Fig. 6. Effect of RAP on the cytolytic activity and the expression of cytolytic molecules. The CTL of LINE-IIIB were incubated with (filled curves) or without (open curves) 10 ng/ml RAP for 3 days. (A) After incubation, the cells were incubated with 51Cr-labeled 15-12 cells in 96-well round-bottomed microtiter plates for 4 h CTL assay. (B) Total RNA was isolated from RAP-treated or untreated cells, and perforin and granzyme B mRNA levels were determined by semiquantitative RT-PCR.

 
Cytokine activity and contents in the culture supernatants
The culture supernatants of the CTL (I-10 sup) were harvested 1, 2 or 3 days after peptide treatment. I-10 sup harvested on day 1 contained large amounts of IFN-{gamma}, TNF-{alpha}, MIP-1ß and IL-2, and the levels of these cytokines were reduced over time (Table 1Go). RT-PCR analyses of cDNA from the CTL harvested at various time points after treatment with peptide I-10 showed that the levels of IL-2, IFN-{gamma}, MIP-1ß and TNF-{alpha} transcripts reached a maximum at 5 h (Fig. 7Go). Treatment of the CTL with Dd-binding control peptide MNT10 had no effect on the production of these cytokines (data not shown). Therefore, the I-10 sup contained various cytokines produced and secreted by I-10-treated CD8+ CTL.


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  		Table 1. Cytokine production by LINE-IIIB cells
 


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  		Fig. 7. Effects of peptide I-10 treatment on IL-2, IFN-{gamma}, TNF-{alpha} and MIP-1ß mRNA expression. The CTL were incubated with or without 1 µM peptide I-10 for 1 h and washed 3 times to remove free peptide. After the incubation of treated cells for an additional 5 h, or 1, 2 or 3 days, total RNA was isolated and mRNA levels were determined by semiquantitative RT-PCR.

 
Effects of I-10 sup on the CTL
We asked whether the CTL exposed to free peptide secreted factors that affected the activity of other CTL. The CTL were treated with 1 µM peptide I-10 for 1 h and washed to remove free peptide. After incubation for 24 h, the culture supernatants (I-10 sup) were harvested and used for the following experiments. At first, to examine the effect of the I-10 sup on the cytolytic activity, unstimulated CTL were cultured with I-10 sup for 24 h and the cytolytic activity was determined. We observed marked enhancement of the cytolytic activity by treatment of unstimulated CTL with I-10 sup (Fig. 8A and BGo). However, the degree of inhibition of cytolytic activity caused by free peptide I-10 remained the same in I-10 sup-treated and untreated CTL (Fig. 8AGo). To clarify the actual elements in I-10 sup responsible for the enhancement of cytolytic activity, we added anti-IFN-{gamma}, TNF-{alpha}, MIP-1ß or IL-2 neutralizing antibody to I-10 sup. Addition of anti-IFN-{gamma}, TNF-{alpha} or MIP-1ß only marginally affected the enhancement of cytolytic activity by I-10 sup. However, the enhancement was significantly but partially inhibited by the addition of anti-IL-2 antibody. As has shown in Table 1Go, I-10 sup contained ~100 U/ml of IL-2. Nevertheless, the enhancement of the cytolytic activity mediated by a high concentration of recombinant IL-2 was not comparable to that obtained by I-10 sup (Fig. 8BGo). This finding strongly indicates the existence of other factors in I-10 sup which cooperate with IL-2 to enhance the cytolytic activity.



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  		Fig. 8. Enhancement of cytolytic activity by culture supernatants from peptide I-10-treated CTL. The CTL were incubated with 1 µM peptide I-10 for 1 h and washed to remove free peptide. After 24 h incubation, the culture supernatants (I-10 sup) were harvested and used for the following experiments. (A) Untreated CTL were incubated with ({square}) or without ({circ}) I-10 sup for 24 h, and the CTL activity were assessed by 51Cr-release assay. Next, untreated CTL was incubated with ({blacksquare}) or without (•) I-10 sup for 24 h, then the CTL were washed and incubated with 1 µM peptide I-10 for 1 h. After washing to remove free peptide, the CTL activity was assessed by 51Cr-release assay. (B) I-10 sup was treated with 10 µg/ml (final concentration) of anti-IFN-{gamma}, TNF-{alpha}, MIP-1ß or IL-2 antibody at 37°C for 1 h, then the CTL were added to each treated I-10 sup and cultured for 24 h. After washing with PBS, the CTL activity was assessed by 51Cr-release assay. Data represent the results at an E:T ratio of 2.5:1 and similar results were obtained in three independent experiments. A, no treatment of the CTL (NTC); B, addition I-10 sup to NTC; C, addition anti-IFN-{gamma} antibody-treated I-10 sup to NTC; D, addition anti-MIP-1ß antibody-treated I-10 sup to NTC; E, addition anti-TNF-{alpha} antibody-treated I-10 sup to NTC; F, addition anti-IL-2 antibody-treated I-10 sup to NTC; G, addition human rIL-2 (1000 U/ml) to NTC

 
Effect of antigenic peptide on the cytolytic activity and cytokine production of ovalbumin (OVA)-specific CTL
To confirm whether the findings in this report, in which treatment of the CTL with their antigenic peptide induce a marked inhibition of cytolytic activity but enhancement of cytokine production, are generalizable, we performed similar experiments using another set of CTL and their specific peptide: H-2b-restricted OVA-specific CTL and peptide (amino acids 257–264, SIINFEKL) (21,22) (Y. Nakagawa and H. Takahashi, unpublished observation). As expected, treatment of OVA-specific CTL with their antigenic peptide resulted in marked inhibition of cytolytic activity, whereas both IFN-{gamma} production and gene expression in peptide-treated CTL were significantly enhanced (Fig. 9Go). These observations indicate that counter-regulation between cytolytic activity and cytokine production in the CTL by their free antigenic peptides seems to apply to CTL of multiple specificities.



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  		Fig. 9. Counter-regulation of cytolytic activity and cytokine production in OVA-specific CTL. OVA-specific CTL were incubated with (filled curves) or without (open curves) 4 µM OVA peptide for 1 h and washed 3 times to remove free peptide. (A) After incubation of treated cells for an additional 1 day, the cells were incubated with 51Cr-labeled E.G7-OVA transfectant (32) in 96-well round-bottomed microtiter plates for 4 h CTL assay. (B) Total RNA was isolated from OVA-treated or untreated cells and IFN-{gamma} mRNA levels were determined by semiquantitative RT-PCR. IFN-{gamma} protein contents in the 1 day culture supernatants were: untreated cells, below detection sensitivity; treated cells, 6050 ± 250 pg/ml.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Treatment of the LINE-IIIB CTL with the epitope peptide I-10 in the absence of APC inhibited both their ability to lyse target cells and their IL-2-dependent growth. At the same time, down-modulation of IL-2Rß expression was observed, indicating that unresponsiveness to IL-2 might be due to IL-2Rß down-modulation. Moreover, granzyme B mRNA expression and its enzymatic activity in peptide I-10-treated cells were markedly reduced as compared with those in untreated control cells. In contrast, since treatment with peptide I-10 did not decrease FasL mRNA levels in the CTL and cytolytic activity was not affected by anti-FasL antibody (data not shown), the inhibition of cytolytic activity may not be due to CTL damage or elimination by apoptosis, but may mainly be due to the exhaustion and reduction of granzyme B and perforin content.

IL-2 has been shown to be required for generation and proliferation of the CTL in vitro, and for up-regulation of the expression of perforin, granzyme B and FasL (20,22,23). However, the relevance of signaling through the IL-2R to the expression of the cytolytic molecules remains to be elucidated. Recently, Makrigiannis and Hoskin (20) reported that CTL generated in the presence of RAP exhibited reduced cellular proliferation and cytolytic activity against the target cells. They demonstrated that this suppressive effect of RAP on CTL induction was partially due to the inhibition of granzyme B and perforin gene expression. As RAP inhibited the activity of a cAMP-responsive element (CRE) binding protein induced by IL-2, and both granzyme B and perforin genes contain CRE and CRE-like binding motifs, they speculated on a mechanism by which RAP could reduce granzyme B and perforin gene transcription. Their findings suggested that signaling through the IL-2R was involved in the regulation of cytolytic molecule expression. Since the blockage of the IL-2R signaling by RAP also reduced the expression of cytolytic activity, granzyme B and perforin in our CTL, down-modulation of IL-2Rß by peptide I-10 might be implicated in the inhibition of the cytolytic response by down-regulating the expression of granzyme B and perforin.

If signaling through IL-2R is linked to the expression of cytolytic molecules, the IL-2R pathway should regulate not only T cell proliferation but also the cytolytic activity. In this study, we demonstrated that the reduction and restoration of peptide-induced changes in the expression of IL-2Rß were well correlated with those in both the cytolytic activity and IL-2-dependent cell growth. Although we have not yet determined the pathway responsible for IL-2Rß down-modulation, our findings suggest that there might be intracellular signal pathways through the TCR that regulate IL-2Rß expression, followed by induction of both cytolytic activity and IL-2-dependent growth.

Recently, Slifka et al. showed that activated CD8+ T cells can cycle cytokine production on/off by association/dissociation with viral peptides bound to APC, while retaining constitutive cytolytic activity (24). In contrast, as shown in this study, treatment of the CTL with free epitopic peptide in the absence of APC induced the inhibition of cytolytic activity. However, we also observed large amounts of cytokines—IL-2, IFN-{gamma}, MIP-1ß and TNF-{alpha} were produced in the culture supernatants of the peptide-treated CTL. It is speculated that these cytokines are involved in prevention of viral infection and disease progression mediated by CD8+ T cells. Baeker et al. have demonstrated that exposure of CD8+ T cells from both long-term HIV-1-infected survivors and some progressors to IL-2 enhanced their ability to suppress HIV replication (25). IFN-{gamma} and TNF-{alpha} may contribute to host defense by local and direct antiviral effects in place of the inactive CTL. In addition, IFN-{gamma} induces MHC class I and II molecule expression on a variety of cells, stimulating the recognition of infected cells by the CTL (26).

In this study, we showed that when unstimulated CTL were cultured with I-10 sup, their cytolytic activity was markedly enhanced. Although this enhancing effect of I-10 sup on the CTL activity was significantly inhibited by anti-IL-2 antibody, such enhancement could not be fully substituted with recombinant IL-2. Therefore, other factors in I-10 sup must also up-regulate the cytolytic activity in the presence of IL-2. For example, IL-15, which also uses the IL-2Rß receptor, may be involved (27,28). However, since there is no anti-IL-15 antibody for the mouse, we cannot block with that as we did with the other cytokines. The epitopic peptide-treated CTL showed loss of their cytolytic activity, but at the same time they gained the ability to produce cytokines enhancing unstimulated T cell functions. These findings suggest that treatment with peptide I-10 transiently converted the CD8+ CTL into cells of Th cell function.

The highest levels of cytokines were produced in the culture supernatant when the cytolytic activity level was the lowest. Sad et al. showed that CD8+ T cells from perforin-deficient mice were not cytolytic but produced Th1-type cytokines in contrast to those from wild-type mice (29). This inverse relationship between cytolytic activity and type 1 cytokine production in the same cells strongly indicates that expression of the CTL functions is regulated in a manner reciprocal to their helper functions.

In the case of HIV-1 infection, HIV-1-specific CD8+ CTL are thought to be key cells involved in prevention of both viral proliferation and disease progression (30). The antiviral effects of HIV-1-specific CTL are achieved by target cell lysis and a soluble inhibitory factor-mediated pathway (31,32). Such CTL-derived inhibitory factors include type 1 cytokines, MIP-1{alpha}, MIP-1ß, RANTES and other as yet undefined factors (31–33). In the present study, the cytolytic activity of the CTL was inhibited temporarily by direct interaction of the CTL with free antigenic peptides, but at the same time large amounts of soluble factors were secreted by the inhibited CTL. A similar phenomenon of counter-regulation by free antigenic peptide was observed in OVA-specific CTL. Our findings in this study may be helpful in clarifying the mechanisms responsible for the induction of CTL inhibition by epitopic peptide and may provide new therapeutic approaches for the treatment of AIDS through enhancing CD8+ CTL activities with factors produced by the CTL.


   Acknowledgments
 
This work was supported in part by grants from the Ministry of Education, Culture and Science, from the Ministry of Health, and CREST, JST, Japan.


   Abbreviations
 
APC antigen-presenting cell
CRE cAMP-responsive element
CTL cytotoxic T lymphocyte
FasL Fas ligand
IL-2R IL-2 receptor
MMC mitomycin C
OVA ovalbumin
PE phycoerythrin
RAP rapamycin
TNF tumor necrosis factor

   Notes
 
Transmitting editor: K. Okumura

Received 24 May 2000, accepted 26 September 2000.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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M. Takahashi, E. Osono, Y. Nakagawa, J. Wang, J. A. Berzofsky, D. H. Margulies, and H. Takahashi
Rapid Induction of Apoptosis in CD8+ HIV-1 Envelope-Specific Murine CTLs by Short Exposure to Antigenic Peptide
J. Immunol., December 1, 2002; 169(11): 6588 - 6593.
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