IL-12 is produced by antigen-presenting cells stimulated with soluble {alpha}ß TCR and restores impaired Th1 responses

Keiko Kawamoto, Vipin Paliwal1, Rajani Ramabhadran1, Marian Szczepanik2, Ryohei F. Tsuji3, Hiroshi Matsuda4 and Philip W. Askenase1

Department of Veterinary Surgery, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan
1 Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8013, USA
2 Department of Immunology, Jagiellonian University College of Medicine, 31–121, Krakow, Poland
3 Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken 278, Japan
4 Faculty of Veterinary Clinic, Department of Agriculture, Tokyo University of Agriculture and Technology, 3-8-5 Saiwai-cho, Fuchu, Tokyo 183, Japan

Correspondence to: P. W. Askenase


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Contact sensitivity (CS) is a cutaneous Th1 response that is induced by skin painting with reactive hapten. In prior in vivo studies of CS, we showed that recombinant soluble {alpha}ßTCR (sTCR) acted non-specifically to protect CS-effector T cells from suppression, but no molecular mechanism was determined. In the current study, we employed an in vitro system to investigate the mechanism of how sTCR protect CS-effector T cells from suppression. Immune CS-effector cells and appropriate hapten-conjugated antigen-presenting cells (APC) were incubated together with down-regulatory culture supernatant produced by suppressive spleen cells from mice tolerized i.v. with specific hapten, which produced strong inhibition of IFN-{gamma} production by the CS-effector cells. Importantly, addition of two different sTCR, of unrelated specificity, reversed this down-regulation and thus restored IFN-{gamma} production. We found that the APC, and not the CS-effector T cells, were the locus of the sTCR-mediated protection and showed direct binding of sTCR to APC by flow cytometry. Further, addition of anti-IL-12 showed that sTCR protection was due to IL-12 induced by sTCR and released by the APC, and was confirmed by ELISA measurement of IL-12 induced in APC supernatants by sTCR incubation. These results indicated a possible new regulatory loop in which suppression was reversed by IL-12 derived from APC, following direct surface binding of sTCR, and enhanced by IFN-{gamma} production from the Th1 CS-effector cells.

Keywords: T cell suppression, delayed-type hypersensitivity, contact sensitivity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cutaneous contact hypersensitivity (CS) is a T cell-mediated immune reaction in the epidermis in response to topically applied reactive hapten. Such haptens covalently couple to cell surface molecules; probably most importantly to self peptides in the groove of MHC class II molecules of antigen-presenting cells (APC); mainly epidermal Langerhans cells (13). During the afferent or sensitization phase of CS, these local Langerhans APC migrate, carrying the hapten from the sensitization area in the epidermis to the draining lymph nodes, and subsequently present the hapten–self peptide–MHC class II complexes to particular antigen–MHC-specific TCR of an appropriate subset of the peripheral recirculating T cell repertoire, that continually passes through the draining lymph nodes (4,5). Following this sensitization, subsequent CS responses are elicited by topical painting challenge with the hapten antigen, on a different skin site in the ear. This probably again results in hapten conjugation of self peptides in MHC class II molecules on local APC, leading this time to elicitation of the delayed time course CS responses; due to local infiltration of a few circulating antigen-specific immunized T cells, derived from the previously sensitized T cell repertoire and to their subsequent local activation (2,6). These few antigen-activated, antigen-specific, CS-effector T cells then produce certain Th1 cytokines, such as IFN-{gamma}, which is the crucial cytokine in CS reactions, to recruit and then activate large numbers of non-specific bone marrow-derived leukocytes, to constitute the inflammatory response of CS (7,8).

We recently demonstrated that an antigen-specific up-regulatory factor that was produced by an established T cell hybridoma contained determinants of {alpha}ß TCR and had a protective effect in vivo on CS T cell responses, specifically by preventing down-regulation due to suppressive cells or factor (911). More recently we employed an analogous up-regulatory recombinant soluble {alpha}ßTCR (sTCR) in an in vivo adoptive cell transfer system and found that pretreatment of CS-effector cells with recombinant sTCR prior to adoptive cell transfer also protected the cells from the suppressive factor TsF (12,13). However, and importantly, in this system the recombinant sTCR did not act antigen specifically to protect CS-effector cells (12,13), suggesting that both antigen-specific and antigen-non-specific potential effects of sTCR could be involved in regulating the function of CS-effector T cells.

Although it is generally recognized that tolerized cells from recipients treated i.v. with high doses of antigen and their soluble suppressive factors can down-regulate immune responses, the nature of these regulatory T cells and their factors have not been characterized fully (14,15). Several other laboratories, working in CS systems, have shown that {alpha}ß TCR genes encode portions of other analogous down-regulatory soluble mechanisms and these analogs of sTCR can in part suppress CS responses (1621). However, the mechanisms of how up-regulatory and protective sTCR restore suppressed CS responses are not understood.

In the current study, we investigated the non-antigen-specific up-regulatory effect of recombinant {alpha}ß sTCR on CS-effector T cells, by employing an in vitro culture system. We used recombinant sTCR that were derived from either D10 T cells (a CD4+, Th2, MHC class II-restricted clone) or from 2C T cells (a CD8+, cytotoxic T lymphocyte, MHC class I-restricted clone). We purified sTCR from BW5147 thymoma cells that were transfected with {alpha}ß TCR cDNA derived from these two T cell clones. Thus, to obtain a soluble form of recombinant sTCR, the BW5147 thymoma cells were co-transfected with {alpha} and ß chain cDNA of D10 or 2C TCR, spliced to murine Thy-1 cDNA, encoding phosphatiylinositol (PI) membrane anchor sequences, to provide each {alpha}ß TCR heterodimer with a PI cell surface link (19). Then, we obtained sTCR molecules by enzymatically cleaving the PI-linked TCR from the cell surface, following treatment with PI-specific phospholipase C (PI-PLC) and purified the released sTCR on an anti-TCRß mAb affinity column (1113). This procedure allowed us to obtain a sufficient amount of relatively pure (>95%) sTCR to examine the immunoregulatory activity of TCR in vitro. We determined whether recombinant sTCR could protect CS-effector cells from suppression in vitro, by measuring antigen-specific IFN-{gamma} production by CS-effector cells that were stimulated in vitro with specific hapten-conjugated APC. Here we report that recombinant sTCR bound directly to APC and overcame suppression in vitro. The mechanism of up-regulation by sTCR involved binding and stimulation of APC to release IL-12 that strengthened CS-effector Th1 T cells for production of IFN-{gamma}.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Male CBA/J mice, 6–8 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME), and were kept in filter topped microisolators and rested for at least 1 week before use.

Reagents
Picryl chloride [trinitrophenyl (TNP)] chloride (PCl) (Chemica Alta, Edmonton, Alberta, Canada), recrystallized from ethanol/H2O and then stored in a light-protected desiccator at room temperature, trinitro-benzene sulfonic acid (TNBSA) (Wako Pure Chemicals, Osaka, Japan), methotrexate, (Calbiochem, La Jolla, CA), PI-PLC, mitomycin C, biotinylated-BSA, (Sigma, St Louis, MO), and Protein A–Sepharose and CNBr-activated-Sepharose 4B (Pharmacia, Piscataway, NJ) were obtained from the manufacturers. RPMI, MEM, FBS, DMEM (Gibco/BRL, Grand Island NY), biotin-NHS, BCA protein assay reagent and horse IgG protein standard (Pierce, Rockford, IL) also were obtained from the manufacturer.

Cytokines and antibodies
Murine recombinant IL-12 and sheep polyclonal antibody to murine IL-12 were gifts of Dr Stanley F. Wolf (Genetics Institute, Cambridge, MA). FITC-conjugated Mac-3 mAb, rat anti-mouse IFN-{gamma} (IgG1, clone 18181D) and biotinylated-rat anti-mouse IFN-{gamma} (IgG1, clone XMG1.2) were obtained from PharMingen (San Diego, CA). Rat anti-mouse IL-12 mAb (clones C15.6 and C17.8) were obtained from Genzyme (Cambridge, MA). Using procedures recommended by the manufacturer, we conjugated anti-mouse IL-12 mAb (C17.8) with biotin to employ in an IL-12-specific sandwich ELISA. Anti-TCR Cß chain mAb was purified from supernatant of cultured hybridoma (H57-597), that was a kind gift from Dr R. T. Kubo (22) (Cytel, La Jolla, CA).

Cell lines
BW5147 thymoma cells that were co-transfected with {alpha} and ß chain cDNAs from D10 (CD4+, a class II-restricted Th2 cell clone) and 2C (CD8+, a class I-restricted cytotoxic T cell clone) were used. BW5147 thymoma cells transfected with D10 and 2C TCR {alpha} and ß chain cDNAs were engineered to contain a PI linkage derived from Thy-1 cDNA, and were provided by Dr Alfred Bothwell, Yale University School of Medicine (23). Cell lines were routinely negative on testing for mycoplasma.

Preparation of recombinant sTCR
D10 and 2C TCR transfected BW5147 cells were grown in DMEM supplemented with 10% dialyzed FBS, penicillin/streptomycin and 110 µM methotrexate for maximum dihydrofolate reductase gene-mediated amplification of {alpha}ß TCR heterodimer production and for maximal surface expression of TCR. Cells were harvested when their density reached 106/ml. About 3x109 cells were pooled, washed once with 50 ml of plain MEME, and then the pellet was resuspended in 9 ml of RPMI 1640 containing 1 mM HEPES and 1 µM sodium pyruvate. Then the cells were incubated with 2.5 U/ml PI-PLC enzyme for 1.5 h at 37°C. After 45 min, 0.1 ml of 1 M NaHCO3 (pH 11.0) was added to adjust the pH to 7.0. Then the cells were pelleted at 5000 r.p.m. for 10 min at 4°C in a Sorvall SS34 rotor. Supernatants were dialyzed against PBS overnight and then purified by anti-TCR ß chain mAb (H57), linked to a protein A–Sepharose affinity column. The concentration of base (pH 11)-eluted-affinity purified sTCR was determined by quantitative sandwich ELISA (24). Just before use, sTCR was heat-aggregated at 62°C for 30 min, followed immediately by chilling on ice (13).

Final sTCR preparations were screened for lipopolysaccharide (endotoxin) content employing the Limulus Amebocyte Lysate Pyrochrome Chromogenic Test Kit (Woods Hole, MA). Four different preparations of sTCR (each >150 µg/ml stock concentration) had on the average 0.2 ng/ml of LPS, which is a level far below what is known to activate murine cells (25).

Hapten conjugation of splenic APC
Naive mouse spleen cells were treated with 100 µg/ml of mitomycin C in complete RPMI 1640 medium at 37°C for 30 min at a cell density of 2x107/ml. After intensive washing with PBS, cells were incubated with 10 mM TNBSA, pH 7.2 in PBS at 37°C for 10 min and resulting TNP hapten-conjugated spleen cells were washed with PBS.

Contact sensitization of mice
Mice were contact sensitized by topical application of 100 µl of 5% PCl in an ethanol:acetone mixture (3:1) to the shaved abdomen and four footpads (2,6).

Cell culture for IFN-{gamma} production by CS-effector cells
A single-cell suspension of lymph node cells (LNC), obtained from 4 day contact sensitized mice, was prepared under aseptic conditions. For stimulation with hapten-conjugated TNP-APC, 4x105 immune LNC from PCl-sensitized mice were co-cultured with 4x105 TNP-APC, unless otherwise stated, in flat-bottomed 96-well tissue culture plates (Falcon, Franklin Lakes, NJ), in 0.2 ml RPMI 1640 containing penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 5x10–5 M 2-mercaptoethanol and 10% FBS. Culture supernatants were harvested to assess IFN-{gamma} production 48 h later and stored at –20°C until assayed.

TNP-specific T cell suppressive factor (TsF)
TsF was prepared as described previously (913). Briefly, mice were tolerized by i.v. injection twice, on days 0 and 3, with 0.35 ml of 1% TNBSA in PBS neutralized to pH 7.2 with 1 N NaOH. On day 7, mice were boosted by skin painting with 5% PCl in acetone:ethanol (1:3). The next day, spleen cell suspensions were harvested, and these tolerized spleen cells were cultured in RPMI 1640 with 5% FBS and penicillin/streptomycin for 48 h in stationary vertical flasks at a cell density of 3x107 cells/ml. Supernatants from these cells are hereafter referred to as TsF. The harvested supernatant of the cultured suppressive cells, that contained TNP-hapten-specific TsF, was used for suppression of CS-effector cell responses in the in vitro IFN-{gamma} production assay.

Quantitative ELISA for IFN-{gamma} and IL-12
Cytokine-specific sandwich ELISAs, for quantitative determination of IFN-{gamma} and IL-12, were performed by using separate capture and detection mAb, generally following the instructions of the manufacturer. Optimal concentrations of capture and detection mAb were determined in initial experiments using recombinant IFN-{gamma} and IL-12 as standards. Briefly, 96-well flat-bottom microtiter plates (Easy wash; Corning, Corning, NY) were coated overnight with capture mAb at 1 µg/ml in 0.1 M NaHCO3, pH 8.3, at 4°C. The plates then were washed and blocked with PBS containing 1% BSA and 0.05% Tween 20 for 1 h at 37°C. Then experimental samples, or standard recombinant IFN-{gamma} or IL-12 as positive controls, were added. After overnight incubation at 4°C, the plates were washed extensively and then incubated with biotin-conjugated detection mAb at 37°C for 45 min. Then the plates were washed and diluted (1:3000) horseradish peroxidase-conjugated streptavidin (Vector, Burlingame, CA) was added, and incubation continued for 30 min. For colorimetric development of wells, a TMB system (Kirkegaard & Perry, Gaithersburg, MD) was employed and absorbance was measured at 450 nm by an ELISA plate reader (TiterTek, Multiskan, EFLABov, Finland).

Biotinylation of sTCR
sTCR that were purified on an anti-{alpha}ß TCR mAb affinity column were dialyzed overnight against 0.1 M sodium bicarbonate buffer, pH 8.5, at 4°C and then mixed thoroughly with NHS-biotin reagent (Pierce, Rockford, IL). After incubation for 2 h on ice, the biotinylated sTCR were dialyzed overnight against PBS at 4°C.

Isolation of peritoneal exudate macrophages
Peritoneal exudate macrophages were induced by i.p. injection of 2 ml thioglycolate culture medium (Difco, Detroit, MI). Four days later peritoneal exudate cells (PEC) were harvested with 4 ml of ice-cold PBS containing 10 U/ml of heparin. PEC were washed 3 times with ice-cold PBS and resuspended at 107 cells/ml in complete RPMI 1640 medium, and then these cells were surface adhered by incubation for 1 h at 37°C in 100 mm glass Petri dishes in a 5% CO2 humidified incubator. Non-adherent cells were removed by aspiration and adherent cells were harvested by adding 0.05% trypsin. The enriched adherent macrophage population was assessed by flow cytometry for Mac-3 expression. Purity was always >95% and viability was >98%.

Flow cytometric analysis of cell surface binding of sTCR
Harvested macrophage-rich PEC were pelleted and resuspended in 0.1 ml of biotin-conjugated recombinant sTCR (10 µg/106 cells) in PBS containing 2% FBS (PBS/FBS). After 30 min incubation on ice, cells were washed twice with ice-cold PBS/FBS. As a control, cells were preincubated with unlabeled sTCR or with biotinylated BSA, to determine background fluorescence and then were incubated with PE-conjugated streptavidin (1:400 dilution) in PBS/FBS for 30 min on ice in the dark. Then the PEC were washed twice and fixed in 0.2 ml of 1% paraformaldehyde in PBS. An aliquot of 104 cells of each specimen was analyzed in a FACStar Plus cytometer (Becton Dickinson, Mountain View, CA).

Statistics
Student's t-test was used for evaluation of the significance of experimental differences and P < 0.05 was taken as level of statistical significance.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Suppressive factor inhibits IFN-{gamma} production in vitro by CS-effector cells
TsF was obtained from culture supernatants of spleen cells of mice tolerized with i.v. injection of a high dose of the water-soluble hapten antigen TNBSA. This TsF down-regulates CS responses in vivo, with genetic restriction and antigen specificity (911). However, the effect of the TsF has not been demonstrated clearly in vitro. To establish a system to study inhibition of CS mechanisms by TsF in vitro, we first measured IFN-{gamma} production by CS-effector cells as a positive response, since this cytokine is known to be one of the most important in elicitation of CS (7,8). Concentrations of IFN-{gamma} in 48 h culture supernatants of cells from contact sensitized mice were determined by specific sandwich ELISA. As shown in Fig. 1Go, TNP (PCl)-immune LNC produced IFN-{gamma} in response to specific TNP hapten-conjugated APC (Group B versus A). The production of IFN-{gamma} was both immunization and antigen specific, since no IFN-{gamma} production was detected (i.e. was below the limits of the assay) from co-incubation of non-immune LNC together with TNP-APC (Group A) or by incubation of TNP-immune LNC together with heterologous mismatched hapten (Oxazolone)-conjugated APC (Group C). Importantly, culture of TNP-immune CS-effector cells with TNP-conjugated APC, in the presence of various concentrations of TsF, resulted in inhibition of IFN-{gamma} production in a dose-dependent manner (Fig. 1Go, Groups D–G). In contrast, mock TsF culture supernatant (undiluted, 100%) from naive mouse spleen cells did not affect IFN-{gamma} production by CS-effector cells (data not shown). We concluded that antigen-specific IFN-{gamma} production served as an in vitro assay of CS-effector cell function. This assay appeared suitable for measuring inhibitory TsF activity in vitro that was consistent with previous in vivo studies, demonstrating TsF suppression of ear swelling responses produced by adoptive transfer of CS effector cells (913).



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Fig. 1. Down-regulation of IFN-{gamma} production by CS-effector T cells stimulated with TNP-APC. Four day immune CS-effector cells in LNC of PCl contact sensitized mice and TNP-conjugated APC were incubated together, with or without various concentrations of TsF supernatant (0–100%, i.e. nil to undiluted). The TsF was prepared from supernatants of i.v. TNBSA-tolerized mouse spleen cells. After 48 h co-incubation at 37°C, concentrations of IFN-{gamma} in the supernatants were determined by sandwich ELISA. Each value represents the mean ± SE of triplicates. *P < 0.001, compared to positive cultures without TsF (Group B).

 
Recombinant sTCR protect CS-effector cells from the suppressive effect of TsF
Our previous in vivo studies demonstrated that preincubation of CS-effector cells in vitro with recombinant sTCR, prior to incubation with TsF, protected CS-effector cells from suppression in an adoptive cell transfer of CS (1113). To assess the effect of sTCR on TsF suppression in vitro, we again incubated CS-effector LNC that were being stimulated with specific hapten-conjugated APC, but now with various concentrations of recombinant D10 sTCR without or together with a fixed dose of TsF (1:1 or a 50% concentration) (Fig. 2Go). The positive control level of IFN-{gamma} produced by CS-effector LNC (Group A) was reduced significantly (~60%) by TsF (Group B versus A, P < 0.001). Importantly, addition of D10 sTCR produced an apparent dose-dependent reversal of down-regulation (Groups C–E). Employing log10 increasing doses of sTCR, i.e. 1, 10 and 100 ng/ml sTCR respectively, progressively reversed TsF suppression of IFN-{gamma} production to 90, 99.4 and 128% of positive controls (Groups C–E versus Group A). Note that as little as 1 ng/ml recombinant sTCR was active (Group C, P < 0.001). Further, a high dose of 100 ng/ml D10 sTCR completely prevented the inhibitory effect of TsF on IFN-{gamma} production by CS-effector T cells (Groups D and E). In fact, sTCR enhanced IFN-{gamma} production by CS-effector cells dose-dependently, in the absence of TsF (Groups F–H).



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Fig. 2. sTCR overcomes down-regulation of IFN-{gamma} production. CS-effector LNC removed from mice contact-sensitized with 5% PCl were cultured without (Groups A and B) or with (Groups C–H) various concentrations of recombinant sTCR (1–100 ng/ml) in the presence (Groups C–E) or absence (Groups A and F–H) of down-regulatory 50% TsF (a 1:1 dilution of TsF supernatant). IFN-{gamma} produced in 48 h culture supernatants was analyzed employing a quantitative sandwich ELISA. Each value represents the mean ± SE of triplicate samples. *P < 0.001, compared between groups as indicated.

 
We concluded that recombinant sTCR acted to prevent TsF-mediated down-regulation of antigen-specific IFN-{gamma} production by CS-effector cells and that sTCR even in the absence of suppression was able to augment IFN-{gamma} production, suggesting that sTCR might act positively on the CS-effector cell mixture independently of added TsF. In additional studies, we also confirmed that another sTCR, that instead was derived from a cytotoxic T cell clone (2C), showed similar ability to enhance IFN production by CS-effector cells in vitro, as was mediated by D10 sTCR (data not shown).

Regarding antigen specificity and MHC restriction, the D10 TCR is known to bind a specific conalbumin peptide complexed with MHC class IIk, while 2C TCR recognizes its peptide bound to MHC class I Ld. Thus, these two TCR are quite different in antigen–MHC specificity. However, in soluble form these two {alpha}ß TCR molecules restored Th1 function of H-2k (CBA) effector cells. These results suggested that sTCR acted to augment IFN-{gamma} production in a non-antigen-specific and non-MHC-restricted manner, and thus that the antigen–MHC combining region of sTCR probably was not involved. However, as shown in Fig. 3Go, protective activity by D10 sTCR was observed in vitro only when CS-effector cells were cultured with specific hapten-conjugated APC. Thus, sTCR induced IFN-{gamma} production by PCl-immunized LNC that were cultured with TNP-APC, but did not augment IFN-{gamma} production when these TNP-immune LNC were cultured with unmatched APC that instead were conjugated with OX, the wrong hapten (Groups C and D versus G and H). Therefore, it appeared that the mechanism by which sTCR increased IFN-{gamma} production by CS-effector cells was independent of the variable region of the sTCR molecules encoding antigen–MHC specificity, but did depend on the quite different antigen–MHC specificity of the interaction between the CS-effector T cells and the APC.



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Fig. 3. sTCR overcomes down-regulation of antigen-specific IFN-{gamma} production. CS-effector cells were co-cultured with hapten-matched (TNP-APC: Groups A–D) or -unmatched (OX-APC: Groups E–H) APC with (Groups C, D, G and H) (10 ng/ml) or without (Groups A, B, E and F) recombinant sTCR in the presence (Groups B, D, F and H) or absence (Groups A, C, E and G) of 50% TsF. IFN-{gamma} produced in 48 h culture supernatants was assessed by a quantitative sandwich ELISA. Each value represents the mean ± SE of triplicate samples. *P < 0.001, compared between groups as indicated.

 
sTCR acts via an effect on the APC to overcome TsF down-regulation of CS-effector T cells
We next sought to determine the target cell(s) of recombinant sTCR in this culture system. The responses were largely due to two different interacting cell populations in the assay, i.e. CS-effector T cells and TNP-hapten-conjugated splenic APC. Thus, we separately preincubated each population with D10 sTCR for 60 min at 37°C and then washed to remove unbound sTCR. These separately sTCR-pulsed subpopulations were then combined with the other unpulsed subpopulation and then together were incubated with TsF. After 48 h, the concentrations of IFN-{gamma} in the supernatants were measured.

Preincubation of TNP-APC alone with sTCR produced a comparable protective effect (Fig. 4Go, Group F versus C), as when CS-effector cells and TNP-APC were cultured together with sTCR (Group D versus C). In contrast, CS-effector cells pulsed alone with sTCR before mixing did not reverse suppression (Group E versus C). We also conducted similar experiments in the absence of TsF. Preincubation of sTCR with TNP-APC prior to incubating with TNP-immune T cells enhanced IFN-{gamma} production without TsF (by only 10%) (Group B), while CS-effector cells incubated alone without TsF did not produce augmented IFN-{gamma} after combining with TNP-APC. Thus, we concluded that APC were the locus of action of recombinant sTCR, to restore the production of IFN.



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Fig. 4. APC probably are the locus of action of sTCR. CS-effector T cells isolated from 4 day PCl immune LNC or naive mouse spleen cells employed as APC, by conjugation with TNP, were preincubated separately with or without 10 ng/ml of recombinant sTCR for 1 h at 37°C. After extensive washing, separate populations of the sTCR-prepulsed immune LNC and TNP-APC were co-cultured in the presence (Groups C–G) or absence (Groups A and B) of down-regulatory 50% TsF (a 1:1 dilution of supernatant) for 48 h at 37°C and then were combined with the opposite population that had or had not been prepulsed with sTCR, and then were cultured together and then IFN-{gamma} production was determined in supernatants by ELISA. The data represent the mean ± SE of triplicate samples. *P < 0.05, **P < 0.001.

 
Cell surface binding of sTCR onto peritoneal exudate macrophages
The effect of sTCR on APC suggested that sTCR might bind to APC directly and that the APC might thus be stimulated by bound sTCR to somehow facilitate IFN-{gamma} production by the CS-effector cells, resulting in protection from suppression. To demonstrate possible direct binding of sTCR molecules to APC, we employed flow cytometry. We incubated biotin-conjugated sTCR with thioglycolate-induced peritoneal exudate macrophages, as an enriched (>95%) population of APC. When macrophages were incubated with 50 µg/ml, biotinylated D10 sTCR (Fig. 5aGo) or 2C sTCR (Fig. 5bGo) and subsequent staining with phycoerythrin (PE) or FITC–avidin was performed, then direct binding of sTCR onto the cell surface was observed. Staining was not obtained in controls mixed with PE or FITC–avidin alone. In fact, when APC were incubated with as little as 1 ng/ml biotin–D10 sTCR, a dose that caused protection from suppression (Fig. 2Go, Group C), staining of APC with FITC–avidin, was clearly greater than staining with FITC–avidin alone (Fig. 5aGo). In a concurrent experiment, macrophages were incubated with biotin-conjugated both D10 and 2C sTCR showed a similar ability as unbiotinylated sTCR to induce IFN-{gamma} production and protection from suppression (data not shown). Thus binding to APC and induction of IFN-{gamma} by T cells were associated properties and biotin conjugation did not interfere with this biologic property of sTCR.



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Fig. 5. Direct binding of sTCR to peritoneal exudate macrophages. Thioglycolate-stimulated peritoneal macrophages (95%) were incubated with or without, biotin-conjugated sTCR from D10 T cells (Fig. 4aGo) or 2C T cells (Fig. 4bGo) for 30 min on ice. Then cells were washed and stained with PE–avidin and then analyzed by flow cytometry. The data shown are representative of three separate similar experiments.

 
Effect of recombinant sTCR is mediated by IL-12 produced by the APC
The data presented above suggest that sTCR act on APC through direct binding to the APC surface. However the mechanism of eventual induced IFN-{gamma} production by T cells, leading to apparent overcoming of the TsF suppressive action, was still unclear. IL-12 is a heterodimeric (p40/p35) cytokine produced by monocyte/macrophages and B cells that stimulates growth of Th1 cells and induces IFN-{gamma} synthesis, while simultaneously inhibiting Th2 cytokine production (2628). Recent studies indicate that IL-12 plays an important role in potentiating in vivo Th1 cell-mediated immune responses, notably CS (29,30). Therefore, we investigated the possible involvement of IL-12 in augmentation of IFN-{gamma} production associated with protection from suppression. We added neutralizing sheep polyclonal antibody specific for mouse IL-12 to the cultures and then determined IFN-{gamma} production after 48 h. Figure 6Go shows that adding anti-IL-12 completely abrogated the production of IFN-{gamma} that was enhanced by sTCR in the presence of TsF (Fig. 6bGo, Group H versus G) or in the absence of TsF (Fig. 6aGo, Group D versus C). Addition of anti-IL-12 showed a slight decrease of IFN-{gamma} production by CS-effector cells in the absence (Fig. 6AGo, Groups A versus B) or presence of TsF (Fig. 6BGo, Group E versus F), suggesting that endogenous IL-12 may be involved in IFN production by CS-effector cells. Further, incubation of CS-effector cells with IL-12 itself (10 ng/ml) strongly overcame suppression by TsF and this effect was completely neutralized by addition of anti-IL-12 antibody (data not shown). Taken together, these results suggested that sTCR bound directly to the APC surface to induce IFN-{gamma} production. This IFN-{gamma} probably was produced by the Th1 CS effector T cells. Alternatively IFN-{gamma} could have come from the APC, as it has been shown recently that APC stimulated by IL-12 and IL-18 will produce IFN-{gamma} (28), but we found that APC required co-incubation with T cells (Fig. 4Go) and also APC did not produce IFN-{gamma} when stimulated by IL-12 alone in this system (data not shown).



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Fig. 6. Anti-IL-12 antibody neutralizes the sTCR effect. Anti-IL-12 polyclonal antibody (5 µg/ml) was added to mixed cell cultures of TNP-immune T cells and TNP-APC to determine the effect on IFN-{gamma} production in the absence (b) or presence (a) of 50% TsF and in various cases of sTCR addition to the cultures. After 48 h incubation, IFN-{gamma} concentrations were assessed by ELISA. *P < 0.005, **P < 0.001, compared to control (Groups A and E).

 
Production of IL-12 by peritoneal exudate macrophages stimulated by sTCR
The above results suggested that direct binding of sTCR to the cell surface may induce IL-12 production by APC. To confirm this possibility, we directly examined IL-12 production by peritoneal exudate macrophages incubated with recombinant sTCR. To determine potential elaboration of IL-12 protein, macrophages were incubated at a cell density of 106/ml with various concentrations of D10 or 2C sTCR for 48 h at 37°C. The concentration of IL-12 in harvested culture supernatants was determined by specific quantitative sandwich ELISA. We used LPS and IFN-{gamma} stimulation as positive controls, that are known to induce production of IL-12 in macrophages. Figure 7Go shows that similar to LPS and IFN-{gamma} (Groups B and C), both D10 sTCR (Fig. 7aGo, Groups D–F) and also 2C sTCR (Fig. 7bGo, Groups D–F) significantly induced IL-12 production from peritoneal exudate macrophages in a dose-dependent manner.



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Fig. 7. sTCR stimulate IL-12 production in peritoneal macrophages. Peritoneal exudate macrophages (106/ml) were incubated) for 48 h at 37°C with medium (Group A), IFN-{gamma} (10 ng/ml, Group B), LPS (1 µg/ml, Group C), or with various concentrations of D10 sTCR (a, Groups D–F) or 2C sTCR (b, Groups D–F). Then IL-12 concentrations in harvested supernatants were quantified by specific sandwich ELISA. The data shown are representative of three different experiments. *P < 0.001, compared to medium alone (Group A).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have demonstrated an in vitro functional effect of sTCR. Recombinant sTCR overcame down-regulatory effects of TsF on IFN-{gamma} production by immune CS-effector T cells that were responding to specific hapten-conjugated APC. Further, the apparent reversal of down-regulation was probably due to induction of IL-12 secretion that was derived from the APC, following binding and direct stimulation by the sTCR. Thus, sTCR stimulation of APC led to IL-12 that protected Th1 cells from suppression.

Classically, in vivo cell-mediated immune responses, such as the PCl CS system in CBA mice, are mediated by CD4+ {alpha}ßTCR+ Th1 effector T cells that characteristically produce IFN-{gamma}. In addition, recent studies have demonstrated that some CS and delayed-type hypersensitivity responses are mediated by CD8+ T cells in which haptens are presented via MHC-class I molecules on APC, but resulting responses still depend largely on final IFN-{gamma} secretion (29,30).

Adoptive cell transfer of CS with sensitized effector cells specific for chemically reactive haptens, such as PCl (TNP-Cl), is a well established in vivo model system for studying the role of the responsible CS-effector T cells and the putative modulatory role of soluble molecules derived from regulatory T cells (913). Using this CS model, we previously described an antigen-specific soluble regulatory molecule(s) released spontaneously from an established T cell hybridoma AF5 that blocked the down-regulatory effect of TsF on CS-effector cells and had antigenic determinants of {alpha}ß TCR, but not of {gamma}{delta} TCR (11). This suppression-preventing factor, that was derived from the AF5 T cell hybridoma cells, mediated its effect as released disulfide-linked heterodimeric molecules that were structurally, functionally and serologically analogous to {alpha}ß TCR (11). Partial characterization of this {alpha}ß TCR-related CS-protecting factor was carried out (11), but insufficient amounts of material were available for purification to yield enough material for more definitive characterization.

This led us to examine similar up-regulation of in vivo CS by more abundant recombinant sTCR that was derived from D10 and 2C T cell clones (23). Our recent findings showed that pretreatment with these recombinant sTCR, protected CS-effector cells from suppression by TsF, in a system employing adoptive cell transfer of CS in vivo (12,13). However, the mechanism of protection was unclear. In the current study we attempted to devise an in vitro system to analyze the mechanisms by which sTCR protected CS-effector T cells from suppression. We employed two different kinds of soluble recombinant {alpha}ß TCR that were derived from either D10, a CD4+ Th2 clone, or from 2C, a CD8+ cytotoxic T cell clone. We showed that both recombinant D10 sTCR [MHC class II (H-2k)-restricted and specific for conalbumin peptide] and recombinant 2C sTCR [restricted in specifity to MHC class I H-2d] had protective activity on TNP-immune, CBA/J, class II H-2k, CD4+, CS-effector Th1 cells. However, both sTCR we employed had in common release from T cells via PI-PLC, which could have contributed to the results. Thus, in the future we plan to explore this point by obtaining sTCR by other means. However, our prior study involved putative sTCR that had similar up regulatory function and were released from a T cell hybridoma spontaneously (11).

Our findings suggested that sTCR protection from suppression was neither antigen specific not MHC restricted and at the level of the sTCR, and thus confirmed prior in vivo findings that the same Th2 H-2k- and cytotoxic T lymphocyte H-2d-derived sTCR similarly protected H-2k CBA CS effector T cells in vivo, from similar suppression (13). Taken together, along with the fact that sTCR were found to stimulate IL-12 production from macrophages of different MHC and in the absence of any antigen, we concluded that the protective effect of these recombinant sTCR was neither antigen nor MHC specific or restricted. Since antigen–peptide–MHC specificity of TCR is due to recognition via the variable regions of conventional transmembrane TCR, these results suggest that protection of CS by recombinant sTCR may have been mediated via the sTCR constant region.

We demonstrated the presence of binding sites for sTCR on mouse peritoneal exudate macrophages by using immunofluorescence and this correlated with protective function. We postulated that sTCR may bind to specific putative binding sites on the surface of APC, although the nature of possible receptors on macrophages for sTCR molecules is at present unclear. Two different sTCR bound to APC, represented by peritoneal exudate macrophages, in an antigen- and MHC-independent manner, since no antigen was present, and H-2k, as well as H-2d-derived sTCR, bound to H-2k macrophages equally.

In addition to this apparent reversal of suppression, it was clear that sTCR led to IL-12 that stimulated CS-effector T cells in the absence of suppression. This finding suggested the possibility that in some instances IL-12 overcame suppression due to its direct and strong positive action, rather than via actual reversal. IL-12 is an important positive-acting cytokine that plays a crucial role in the regulation of cell-mediated immune responses, such as protective immunity in virus infections (31) or in parasite infestations like experimental Leishmaniasis (32,33). Accordingly, IL-12 mediates induction of protective Th1 immune responses and, importantly, IL-12 also promotes Th1 responses by suppressing development of counteracting Th2 cells and cytokines (28,29).

Accumulating evidence also has demonstrated the importance of IL-12 as a positive mediator in CS. For example, CS ear swelling responses were decreased significantly in animals injected in vivo with anti-IL-12 during sensitization (30), suggesting an important role of IL-12 in the induction of sensitized CS-effector T cells. Regarding tolerance to CS, the detailed mechanisms are not fully understood by which high-dose i.v. injection of hapten antigen induces an antigen-specific tolerance. However, we observed recently that IL-12 reversed this type of CS tolerance, even when established, suggesting that IL-12 can modulate elicited CS responses by acting positively on already developed CS-effector T cells and this effect was independent of IFN-{gamma} (34). Also, others have noted that IL-12 prevents induction of UV-induced hapten-specific tolerence (35) and can reverse established tolerance of CS effector cells induced by high-dose i.v. hapten (35). Taken together, these studies demonstrate that systemic treatment with IL-12 can critically up-regulate CS. Of course, it is possible that other secondary cytokines may also play a role in IL-12-mediated effects on CS, such as protection from suppression.

A new finding of this study was that sTCR could stimulate IL-12 production from peritoneal exudate macrophages. This suggested that sTCR stimulation of increased in vitro production of IFN-{gamma} by Th1 CS-effector cells was dependent on IL-12 derived from APC, that were stimulated by sTCR, following binding to the APC surface, implying that this may involve a receptor connected to positive signaling pathways in macrophages and other APC. Importantly, it was shown in another system that IL-12 can synergize with crucial B7/CD28/CTLA-4-mediated co-stimulation for Th1 cell proliferation and for production of cytokines such as IFN-{gamma} (36). This suggests a possible mechanism whereby sTCR binding to APC induces IL-12 perhaps by coordinating with or improving co-stimulatory signaling mechanisms, leading to enhanced IFN-{gamma} production. Besides exudate macrophages, perhaps other macrophage-related subpopulations, such as Langerhans cells (37) and dendritic cells (38), or even B cells (39) or keratinocytes (40), all of which are sources of IL-12, may similarly be activated by sTCR. Thus, if putative surface binding sites for sTCR exist on some of these other cell types, it may be possible that under certain circumstances that sTCR may also stimulate these cells to release IL-12 to augment Th1 responses or perhaps lead to release of other cytokines.

To date, there is only limited evidence for a naturally occurring soluble forms of TCR, as we have employed TCR in the recombinant form in the current in vitro studies. However, several studies favor the possible existence of sTCR in vivo. Firstly, a variety of several cell surface membrane molecules related to TCR, such as MHC class I heavy chain (41), CD4 (42) and CD8 (43), are known to be enzymatically cleaved from the cell surface after activation, and then are released to circulate in the serum in soluble form, as is true of many other cell surface receptors (44). Recently it was suggested that some of these soluble molecules in serum may be possible modulators of immune responses. For example, the serum level of HLA class I molecules increased during transplantation (45) and viral infections (46), and decreased with immunosuppressive therapy (45). Secondly, several laboratories have described the occurrence of soluble molecules expressing TCR{alpha} determinants, as well as soluble molecules possibly related to {alpha}ß TCR, which in contrast to our findings mediate immunosuppression (1621). Thirdly, in contrast, we have described spontaneously released putative sTCR (11), and in addition recombinant and unambiguous sTCR (12,13), that act positively in CS. Taken together, these results favor the existence of sTCR in vivo. Also, it was reported recently that a specific and very sensitive ELISPOT assay could detect sTCR released at the single-cell level. By using this assay, it was claimed that sTCR were released by T lymphoma cells, but not by B lymphoma, hepatoma or dead cells (47). Further studies using this new assay for T cells and also employing a sensitive ELISA that we reported to quantitate low levels of sTCR (24) may in the future provide useful information to clarify the possible role of sTCR in vivo.

In summary, we have shown that recombinant sTCR bind to APC and induce IL-12 production, which in turn protects Th1 CS-effector cells from suppression, allowing IFN-{gamma} production. It is possible that following some activation in vivo that {alpha}ß sTCR are released locally from T cells and then bind to local APC to subsequently stimulate IL-12 production. This positive loop might then act to strengthen Th1 immune responses in vivo, by maintaining or augmenting secretion of effector cytokines, such as IFN-{gamma}.


    Acknowledgments
 
We thank Dr Alfred Bothwell for providing BW5147 cells transfected with cDNA of {alpha}ß TCR from D10 and 2C T cell clones, resulting in expression of D10 and 2C {alpha}ß TCR on the surface in PI-linkage. We are also grateful to Dr Ralph T. Kubo for providing invaluable hamster B cell hybridoma cells producing mAb to the ß chain of the murine TCR (H57-597). We also thank Marilyn Avallone and Joanne Pacelli for their excellent secretarial skills. This work was supported in part by grants from the NIH to P. W. A. (AI-43371, AI-07174 and SCOR P50 HL-56389).


    Abbreviations
 
APC antigen-presenting cell
CS contact sensitivity
LNC lymph node cells
LPS lipopolysaccharide
PCl picryl chloride (trinitrophenyl chloride)
PE phycoerythrin
PEC peritoneal exudate cells
PI phosphatidylinositol
PI-PLC phosphatidylinositol-specific phospholipase C
sTCR soluble {alpha}ß TCR
TNBSA trinitro-benzene sulfonic acid
TNP trinitrophenyl
TsF T cell-derived suppressive factor

    Notes
 
Transmitting editor: K. Okumura

Received 9 July 1999, accepted 8 October 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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