J. Exp. Med.
© The Rockefeller University Press
0022-1007/96/12/2129/12 $2.00
Volume 184 December 1996 2129-2140

T Cells from Tolerized T Cell Receptor (TCR)-deficient Mice Inhibit Contact Sensitivity-Effector T Cells In Vivo, and Their Interferon- Production In Vitro

By Marian Szczepanik,^* Laurel R. Anderson,^§ Hiroko Ushio, Wlodzimierz Ptak,^* Michael J. Owen, Adrian C. Hayday,^§ and Philip W. Askenase

From the ^* Department of Immunology, College of Medicine, Jagiellonian University, Krakow, Poland;  Section of Allergy and Clinical Immunology, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8013; ^§ Department of Biology and Section of Immunobiology, Kline Biology Tower, Yale University, New Haven, Connecticut 06520; and  Imperial Cancer Research Foundation, London, England WZCA 3PX

Summary

Materials and Methods

Results

Discussion

Footnotes

References

Summary

Cutaneous contact sensitivity (CS)¹ responses to contact sensitizing hapten Ag such as picryl chloride (PCl) and oxazolone (OX) are classical manifestations of T cell-mediated immunity in vivo. In previously sensitized hosts, CS and the related delayed-type hypersensitivity (DTH) reaction, are manifest as macroscopically measurable inflammation (skin swelling) that peaks at 24-48 h after topical cutaneous Ag challenge (1).

It appears that early after challenge with hapten, local tissue mast cells (2, 3) and blood platelets (4) are induced to release the vasoactive amine serotonin (2) that facilitates local extravasation and subsequent migration and activation of Ag-specific CS-effector lymphocytes. Available data strongly suggest, but do not prove, that the responding lateacting lymphocytes are exclusively MHC class II-restricted, TCR⁺, CS-effector T cells (7, 8), which customarily make Th1-type effector cytokines, such as IFN- (9).

By contrast to the conventional CS-effector response, which is induced by immunization via skin painting with reactive hapten Ag, mice respond to high intravenous doses of soluble hapten Ag by developing tolerance. This high dose Ag tolerance is of particular significance with regard to the capacity of individuals that become tolerant toward contact hapten allergens, and may also underlie aspects of tolerance to self Ag. Numerous studies have demonstrated that high dose tolerance can be associated with the development of regulatory T cells that limit the response of CSeffector T cells (12). In this regard, it is unclear whether or not such CS-downregulatory T cells are exclusively T cells, or whether they may include T cells.

An assay commonly used to demonstrate high dose downregulation of CS, is the mixing together in vitro of spleen cells from mice tolerized intravenously with high doses of Ag, together with CS-effector cells from contact sensitized mice. Thereafter, the mixed regulatory and CS-effector cells are adoptively transferred to naive recipients, and a subsequent measurement is made of the transferred CS-effector response in the recipients. The studies reported in this manuscript use this mixing assay and other established assays, along with use of TCR-deficient ^/ mice, to assess the cells responsible for CS elicitation and for CS downregulation, respectively. The studies show that T cells cannot substitute for T cells as effectors of CS elicitation, but that T cells can medite downregulation of both CS effectors in vivo and IFN- production by these CS effectors in vitro. The studies are particularly pertinent given the reported capacity of T cells to negatively regulate T cell-driven responses in allergic (18, 19) and other immune responses.

Materials and Methods

Mice. 5-7-wk-old 129/J (H2^b), BDF₁ (H2^{b × d}), C57Bl/6 (H2^b), BALB/c (H2^d), and CBA/J (H2^k) mice were obtained from Jackson Labs (Bar Harbor, ME). TCR^/ and TCR^+/ mice (20) with different but defined MHC backgrounds that included H2^b, H2^d, and H2^b+d, were supplied locally. All mice were maintained in microisolator cages, and changed in a laminar flow hood. MHC haplotypes were determined by FACS^® with antiMHC class I^b (PE-conjugated), and class I^d (FITC-conjugated) mAb (PharMingen, San Diego, CA).

Reagents. Picryl chloride TNP chloride (PCl) obtained from Chemica Alta (Edmonton, Alberta, Canada) was recrystallized from methanol/H₂O, and protected from light and humidity. Trinitro-benzene sulfonic acid (TNBSA) (Eastman Fine Chemicals, Rochester, NY); oxazolone (4-ethoxymethylene-2-phenyloxazolone) (OX) (British Drug Houses via Gallard-Schlesinger Inds. Inc., Carle Place, NY); and anti-hamster IgG antibodycoated magnetic beads (1 µm iron magnetic particles; Advanced Magnetics, Inc., Cambridge, MA) were obtained from the manufacturers. Anti-TCR mAb (clone UC7-135D5, hamster IgG), was obtained from Dr. J. Bluestone (University of Chicago, Chicago, IL; 23). Low-tox rabbit complement C was obtained from Pel-Freeze Biologicals (Brown Deer, WI).

Active Immunization. Mice were actively sensitized by topical application of 0.15 ml of 5% PCl or 3% OX in 1:3 acetone-ethanol mixture, to the shaved abdomen, chest, and hind feet on day 0. 4 d later, zero time ear thickness was measured with an engineer's micrometer (Mitutoyo Mfg. Co., Tokyo, Japan) before local ear skin challenge via topical application to both sides of both ears of one drop of 0.8% PCl or OX solution in olive oil (for 129/J, BALB/c, TCR^+/ and ^/), or 1% PCl to less reactive BDF₁ or C57Bl/6. The subsequent optimal increase in ear thickness was measured at 24 h, and occasionally 48 h, after challenge as reported (1), and expressed in units of 0.01 mm ± SE. In all experiments, a group of nonimmune control animals was also challenged on the ears with 0.8% PCl or OX, and the resulting background increase in ear thickness (~2 U at 24 h) was subtracted from responses of experimental groups to yield net ear swelling responses, shown in the figures.

Induction of High Ag Dose Tolerance. For induction of TNP- specific high dose tolerance, mice received two intravenous injections of 3 mg TNBSA (1% TNBSA in distilled H₂0, readjusted to pH 7.2 with 1 M NaOH) in 0.3 ml on days 0 and 3. On day 7, spleens from these TNBSA-injected mice were harvested as a source of putative regulatory cells. Alternatively, mice were tolerized by intravenous injection of TNP-conjugated syngeneic spleen cells.

Adoptive Cell Transfer of CS Responses and Cell Mixing Assay. Donors of CS-immune effector cells were contact sensitized with 5% PCl or 3% OX on days 0 and 3. Immune lymph node and spleen cells were harvested on day 7 and 7 × 10⁷ were injected intravenously into normal syngeneic recipients. Immediately after transfer, recipients were challenged on the ears with 0.8% PCl or OX in olive oil. The increase in ear swelling was determined 24 h, and sometimes 48 h later. It was compared to actively sensitized positive controls and to negative controls that were simply challenged on the ears with 0.8% PCl or OX.

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Transfer into Tolerized Mice to Confirm Active Tolerance and Agspecificity. To test tolerance and Ag specificity of downregulatory cells in another system, ^/ mice were tolerized with a high dose of TNBSA or with OX-MRBC intravenously on days 0 and 3 before serving as cell transfer recipients. Then on day 7, nontolerized control or TNP- or OX- tolerized ^/ mice received 7 × 10⁷ CS-effector cells from normal TCR^+/+ mice (BALB/c) that were Ag homologous, or were of unrelated Ag specificity (TNP versus OX). ^/ recipient mice were then challenged immediately on the ears with homologous hapten (TNP or OX), and ear tested for 24 h ear swelling.

Immuno-magnetic Bead Cell Fractionation. Spleen cells (1.5 × 10⁸) from four TNBSA-tolerized ^+/+ 129/J, or ^/ donor mice were incubated in 20 ml UC7 hamster anti-TCR mAb (12 µg/ml) hybridoma supernatant for 60 min on ice. After mAb coating, cells were washed three times with PBS + 2% FCS, and resuspended in 40 ml PBS + 2% FCS containing goat anti-hamster Ig-coated paramagnetic beads at 5-10 beads per target cell. The cells were then incubated in a flat vertical 50-ml flask for 30 min on ice, after which a magnet (Advanced Magnetics Inc., Cambridge, MA) was applied to one side of the flask. 10 min later, magnetic bead nonadherent cells were recovered, and then the magnetic bead adherent cells were recovered. Nonseparated regulatory cells were used in experiments shown in Figs. 3, 5, and 6 b.

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Mixing of CS-effector Cells and Magnetic Bead-enriched Regulatory Cells. Immune CS-effector cells (7 × 10⁷) were incubated with 5 × 10⁷ unseparated regulatory T cells, with an equivalent number of separated magnetic bead positive cells, or with an equivalent number of magnetic bead nonadherent cells (i.e., TCR⁺-remaining cells) originating from either tolerized 129/J ^+/+ mice or TCR^/ mice for 30 min at 37°C. The washed effector and regulatory cell mixture was then injected intraperitoneally into naive 129/J recipients that were tested for CS responses by challenging ears with 0.8% PCl or OX in olive oil the next day. Recipients were then measured for ear swelling 24 h after Ag challenge to the ears.

In Vitro Culture of CS-effector Cells and Hapten-conjugated APC for Elaboration of IFN-, and Regulation by Tolerized Cells. A single cell suspension of just CS-effector lymph node cells was obtained aseptically from PCl contact sensitized mice. Suspensions of regulatory cells were from spleen cells of mice that were injected with TNBSA or OX-MRBC intravenously on days 0 and 3, or from control nonimmune mice. In some experiments, spleen cells from TNBSA-tolerized mice, or nonimmune controls, were incubated with 1 µg biotinylated anti- TCR (GL3; PharMingen) per 10⁶ cells at 2 × 10⁷ cells/ml for 20 min on ice. Then, 10 µl of streptavidin-conjugated magnetic microbeads (Miltenyi, Sunnyvale, CA and BioTek Instrs. Inc., Sunnyvale, CA) per 10⁷ cells were added and incubated for 15 min on ice. After washing, this cell and bead suspension was applied to a MiniMACS magnetic separation column (Miltenyi). After recovering of nonadherent -depleted cells and washing the column, -enriched adherent cells were eluted. Equivalent numbers of -enriched, or depleted cells were added in vitro as regulatory cells, to the CSeffector cells (1:1; 2 × 10⁵/well), and then were added together for 72 h to mitomycin C-treated TNP- or OX-conjugated normal spleen cells as APC (2 × 10⁵) in 0.2 ml of RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 25 mM Hepes, 5 × 10⁵ M/2-mercaptoethanol, and 10% fetal calf serum. At 48 h, culture supernatants were collected for IFN- quantitation.

ELISA Quantitation of IFN- in Culture Supernatants. Quantitative sandwich ELISA for IFN- was performed with two different mAb specific for mouse IFN- (clones R4-6A2 and XMG1.2; Pharmingen). 1 µg/ml of capture mAb to IFN- in 0.1 M Na₂CO₃, pH 8.3, was briefly coated onto 96-well microtrays (Corning Glass Incorporated, Corning, NY) overnight at 4°C. After blocking with 3% dry milk in PBS, samples and standard recombinant mouse IFN- (Genzyme Corp., Cambridge, MA) were applied to the wells and incubated overnight at 4°C. Then, 0.5 µg/ml of biotinylated separate mAb to IFN- was applied for 45 min at 25°C, followed by incubation with 1:3,000 dilution of horseradish peroxidase-conjugated streptavidin (Vector Labs., Inc., Burlingame, CA), for 30 min at 25°C. TMB (tetramethylbenzidine) microwell peroxidase substrate, and TMB one component stop solution (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) were used for reaction development and were read at OD 405.

Statistics. Double-tailed Student's t test was used to assess the significance of differences between groups, with P <0.05 taken as a minimum level of significance.

Results

TCR^/ Mice Have No 24 h CS Responses.

CS responses in contact sensitized TCR^/ mice and control 129/J mice were compared by sensitizing (via abdominal skin painting) with concentrated PCl (5%), followed 4 d later by topical challenge with dilute PCl (0.8%) applied to the ears, and subsequent measurement of ear swelling 24 h later. It was found that T cell-bearing 129/J mice developed a classical 24 h ear swelling response (Fig. 1, group A), whereas TCR^/ mice did not (Fig 1, group B).

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When ^/ mice were used as recipients of ⁺ cells from MHC-matched, immunized T cell-sufficient BDF₁ mice, they could mount CS upon ear challenge with hapten (Fig. 1, group D), similar to normal mice (Fig. 1, group C). This demonstrated that the vascular and cellular environment for eliciting CS was normal in TCR^/ knockout mice. The finding that the 24 h component of CS cannot be elicited in actively sensitized TCR^/ mice is consistent with other studies indicating that the active CSeffector cell type is TCR⁺ (7, 8, 24).

High Dose Ag Tolerance of TCR^/ mice Induces Regulatory Spleen Cells that Downregulate CS.

Because TCR^/ mice do not mount CS responses, it is impossible to determine directly whether high dose Ag tolerization of such mice induces downregulatory cells. Instead, CS-effector cells (7 × 10⁷) from contact-sensitized 129/J mice were mixed and incubated for 30 min at 37°C with 5 × 10⁷ spleen cells from ^/ mice, that were harvested on d 7 after two intravenous injections of tolerogenic TNBSA. Analogous TNBSA-tolerized cells were derived from TNBSA-treated MHC haplotype-matched TCR^+/ mice, or TCR^+/+ (129/J) mice. The cells were then transferred back to haplotype-matched recipient mice that were challenged with hapten.

Fig. 2 shows that upon transfer back to recipient mice, CS-effector cells elicit a 24 h response that can be significantly reduced by preincubation of the effector cells with cells from tolerized TCR^+/+ mice (Fig. 2, group B), TCR^+/ mice (Fig. 2, group C), or TCR^/ mice (Fig. 2, group D). Essentially the same results were obtained when effector cells from sensitized BDF₁ mice were mixed with cells from either TNBSA-tolerized BDF₁ mice or TNBSA-tolerized TCR^/ mice (Fig. 2, groups E-G).

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A prediction that follows from the data shown in Fig. 2 (groups A-G) is that TCR^/ mice will not support the actions of adoptively transferred TCR^+/+ CS-effector cells if the recipient ^/ mice are subject to high dose Ag tolerance beforehand. The data in Fig. 2, groups H and I confirm this prediction. This result is important in that it rules out that the detection of TCR^/ downregulatory cells in adoptive transfer experiments (Fig. 2, groups D and G) was artefactual, due to manipulation of the cells in vitro and their subsequent transfer. Furthermore, induction of high dose tolerance by TNBSA in TCR^/ mice was reproducibly demonstrated using transfers from individual TCR mice (data not shown), indicating the generality of the findings.

Expression of TCR on CS-regulatory Cells from Tolerized TCR^/ Mice.

To determine the phenotype of CS-regulatory cells induced by high Ag dose tolerance, splenocytes from TNBSA-tolerized mice were fractionated by use of anti-TCR mAb-coated magnetic beads. ⁺, , or unfractionated tolerized cells were then mixed for 30 min at 37°C with lymph node and spleen CS-effector cells (7 × 10⁷) from contact immunized, haplotype-matched mice, and the mixture transferred to recipients that were subsequently assayed for CS responses by ear challenge. In these experiments, anti- bead adherent cells ranged from 55 to 95% TCR⁺ when reanalyzed by FACS^® (see Materials and Methods). Thus, we estimate that 10⁶ cells were adoptively transferred into each recipient when unseparated TCR^/ cells were used, and about 7 × 10⁵ cells were transferred when magnetic bead -enriched cells were used.

The data in experiments 1 and 2 of Fig. 3 show that while the cells from TNBSA-treated ^+/+ 129/J mice that are responsible for downregulating CS effectors are exclusively TCR (group D), the most potent regulatory cell fraction from tolerized TCR^/ mice scored as TCR ⁺ (Fig. 3, group F). Similar results were obtained in experiment three, in which cells from H2^d TCR^/ mice were compared with cells from H2^d BALB/c mice for their response to high dose Ag tolerance. As before, when measuring the capacity of tolerized cells to downregulate a CSeffector response to PCl, the TCR ⁺ fraction from TCR^/ mice, but not from ^+/+ BALB/c mice, was again active (experiment 3, group C). Conversely, the active fraction from normal ^/ mice was TCR ⁺ (group D), and the TCR fraction from TCR^/ mice (group E) contained negligible residual activity.

T Cell-mediated Regulatory Activity Shows Antigen Specificity.

The discovery that T cells in TCR^/ mice can mediate downregulation of CS effector cells prompted an investigation of Ag specificity, since T cells and cells appear to have a different set of Ag specificities. To test this, cells from TCR^/ mice tolerized with high doses of OX or TNP, were mixed with CS-effector cells from TCR^+/+ mice sensitized with PCl or OX, and compared in their capacities to downregulate the 24 h CS response after transfer back to recipient mice. Data presented in Fig. 4 a show no capacity of cells from OX-tolerized TCR^/ mice to influence the CS response to TNP (group B), whereas TNP responses were inhibited significantly after mixing effector cells with cells from TNP-tolerized TCR^/ mice (Fig. 4 a, group C). Data in Fig. 4 a, groups D-F demonstrate that reciprocal Ag specificity was likewise apparent (i.e., cells from OX-tolerized TCR^/ mice downregulated effector cells from OX-sensitized mice, but not ^+/+ TNP-immune cells) (group E versus F).

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To confirm the above findings we tested Ag-specificity in tolerized ^/ mice by attempting to transfer ^+/+ CS-effector cells into these recipients. ^/ mice were tolerized with a high dose of TNBSA or OX-MRBC intravenously before cell transfer. Then, on day 7, control (nontolerized) or TNP or OX-tolerized ^/ mice received 7 × 10⁷ TNP or OX immune cells from normal ^+/+ (BALB/c) mice. The data presented in Fig. 4 b show there was no ability to adoptively transfer immunity into TNP tolerized ^/ mice with TNP-specific CS-effector cells (group B), while TNP-immune CS-effector cells transferred normal CS responses into OX-tolerized ^/ recipients (group C). Similar data were obtained when OX-specific immune cells were transferred (group D versus F). Thus, total inhibition of adoptive cell transfer was obtained when OXspecific immune CS-effector cells were transferred to OXtolerized ^/ mice (Fig. 4 b, group E), whereas OX CS was successfully transferred to TNP-tolerized ^/ mice (Fig. 4 b, group F). Thus, this system that did not involve cell harvest, cell mixing, nor cell transfer, confirmed the presence of active Ag-specific downregulation in high dose-tolerized ^/ mice.

T Cell-mediated Regulatory Activity is MHC Unrestricted.

To determine possible MHC restriction of downregulatory T cells, magnetic bead-enriched TCR⁺ cells from TNBSA-tolerized ^/ mice were incubated with 7 × 10⁷ PCl-immune CS-effector cells from MHC-compatible (129/J, H2^b), or -incompatible (CBA/J, H2^k) mice, for 30 min at 37°C, followed by subsequent transfer to recipients syngeneic with the CS-effector cells. The recipient mice were challenged immediately (before any significant host response to the allogeneic graft could develop), and ear swelling responses determined. The results in Fig. 5 show that ⁺ cells from TNBSA-tolerized TCR^/ mice downregulated CS-effector responses of allogeneic CBA/J H2^k CS-effector cells (group D), as effectively as the regulation of H2-matched CS-effector cells (group B).

Phenotype and dose-response Activity of CS-regulatory T Cells.

To establish the phenotype of T cells capable of inhibiting CS-effector responses, spleen cells from TNBSA-tolerized ^/ mice were treated with anti-CD4 or anti-CD8 mAb or PBS, and then rabbit complement 1:75. The three resultant populations of TCR^/ cells were then individually incubated with 129/J immune CS-effector cells for 30 min at 37°C, and each mixture of cells was subsequently assayed for CS-effector function after transfer to naive recipients. The results in Fig. 6 a demonstrate once more that TNBSA-treated TCR^/ mice are a reliable source of downregulation of CS-effectors (group B), and that this activity is unaffected by treatment with either antiCD4 (group C), or anti-CD8 (group D). Results are consistent with the active regulators being double negative (DN) T cells.

DN T cells have been reported in the lymphoid system of essentially all vertebrates examined, but they occur in mice in very low numbers relative to T cells. Therefore, to assess the potency of DN T cells to downregulate CS, a dose-response experiment was performed. 10fold dilutions (2.5 × 10⁵, 2.5 × 10⁴, or 2.5 × 10³) of 60% TCR⁺ cells (purified from TNBSA-tolerized ^/ mice by use of magnetic beads) were incubated, each with 7 × 10⁷ PCl-immune CS-effector 129/J cells for 30 min at 37°C, and then were harvested, washed, and transferred. Recipient ear swelling results (Fig. 6 b) showed a strong dose response of the assay to the inocula (groups B-D). Even the dose of 2.5 × 10³ T cells had activity in the assay (group D), albeit weaker than the other doses. The capacity of very small numbers of downregulatory T cells to score in this bio-assay is discussed below.

Cells From TNBSA-tolerized TCR^/ Mice Inhibit In Vitro Production of IFN- by TNP-specific CS-effector T Cells.

Since IFN- is a central cytokine in Th-1-mediated responses, such as DTH and CS, we determined whether IFN- production by CS-effector TNP-immune cells was affected by regulatory cells induced by high dose intravenous TNBSA or OX tolerogenesis. Using two different anti-IFN- antibodies in an ELISA assay, it was first established that 2 × 10⁵ lymph node cells from contact immunized mice, stimulated in vitro by TNP-conjugated APC, secreted >3 ng/ml IFN- over a 48 h period. Compared to production of IFN- by CS-effector cells mixed with nonimmune spleen cells (Fig. 7 a, groups A and D), IFN- production by CS-effector cells and TNP-APC was substantially inhibited by mixing with spleen cells from intravenous TNBSA tolerized normal ^+/+ mice (Fig. 7 a, group B) and also with spleen cells from intravenous TNBSA tolerized ^/ mice (Fig. 7 a, group E). Also, as controls, spleen cells from nonimmune and nontolerized animals incubated alone or in the presence of TNP or OX-APC did not secrete detectable levels (0.1 ng) of IFN- (data not shown).

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To determine the phenotype of regulatory cells responsible for the in vitro suppression of IFN- production, TCR⁺ or TCR cells in the spleens were separated, using anti- antibody coated magnetic beads that were used to separate spleen cells from nontolerized, TNBSA-tolerized, or OX-tolerized TCR^+/+ or TCR^/ mice, and then equivalent numbers of each cell subpopulation were added to TNP-immune CS-effector cells in vitro, and cultured together with TNP-hapten-conjugated APC. There was a striking suppression of IFN- production by ⁺ cells from TNBSA-tolerized ^/ mice (Fig. 7 b, group E, left) that did not occur with ⁺ cells from TNBSA-tolerized ^+/+ mice (Fig. 7 b, group B, left). In contrast, the inhibitory activity of TNBSA-tolerized ^+/+ mice was shown to be due to cells (Fig. 7 b, group B, right), and thus was probably due to T cells. In contrast to the downregulatory activity of ⁺ cells from TNBSA-tolerized ^/ mice, the cells of these mice were totally without inhibitory activity (Fig. 7 b, group E, right), as were OX-tolerized ⁺ and cells (Fig. 7 b, group C and F, left), again varifying Ag-specificity of ⁺ cells mediating tolerance.

These in vitro findings were consistent with the in vivo findings. Thus, we concluded that suppressive cells in Agtolerized ^/ mice were potent Ag-specific downregulatory T cells, both in vivo with contact sensitivity, and in vitro with IFN- production, while T cells that inhibited CS in vivo and IFN- in vitro that were obtained from normal ^+/+ mice, were not T cells, and probably were T cells. The failure of most cell mixtures tested in Fig. 7 b to reduce IFN- production demonstrated that selected reductions in IFN- production were not readily attributable to nonspecific effects of extra cells in the assay, such as binding of IFN- to IFN- receptors, or to degradation of secreted IFN-.

Discussion

T cell-deficient mice do not elicit the classical 24 h component of CS after active contact sensitization and subsequent skin challenge. This result extends previous findings implicating T cells as principal effectors in CS responses (7, 8, 25, 26): i.e., T cells and T cells are not redundant in their functional capacity as effector cells in CS. By contrast, TCR^/ mice can respond to high dose Ag tolerance by the induction of cells that can downregulate CS-effector activity from ^+/+ immunized donors. This was shown in two ways: splenocytes from tolerized TCR^/ mice downregulate CS-effector cells with which they are mixed in vitro, before cotransfer and elicitation of CS, and high dose Ag-treated tolerized TCR^/ mice inhibit the transfer of CS-effectors that function normally in untreated TCR^/ mice.

The experiments presented here demonstrate that cell- enriched fractions from TCR^/ mice are better mediators of high dose Ag tolerance than are cell-depleted fractions, suggesting that T cells are the active regulatory cells, as opposed to TCR⁺ cells that are a potential feature of TCR^/ mice (27). The lack of conventional MHC restriction shown by the TCR^/ regulatory cells, and their CD4CD8 phenotype are both consistent with most assays of function (29). The apparent hapten specificity of the regulatory cells demonstrated herein for the first time may be considered consistent with the hypothesis that the potential diversity (30) of the TCR is greater than that of any other Ag receptor (31). Thus, overall, downregulation can be mediated by Ag-specific T cells, and need not be solely mediated by specific T cells. However, in this study we have provided no evidence that the cells directly recognized either PCl/TNP or OX, nor whether the cells recognized either conventional APCs, or the effector T cells with which they were mixed, or some other cells. Conceivably all of these cells may have been haptenated.

Commonly, it has been argued that TCR^/ mice are deficient in immune effector responses because cells are uniquely capable of specifically responding to the challenging antigen. However, the apparent capacity of cells in TCR^/ mice to distinguish between CS-effector cells that are responsive to different hapten Ag raises questions about the completeness and/or validity of this explanation. Possibly, potential effector cells responsive to challenge by specific Ag may be unable to mount strong immune effector responses because they cannot interact efficiently with the professional antigen presenting cell system. This may be for physiologic reasons; e.g., cells may occur in an inappropriate anatomical location, in insufficient numbers, or produce inappropriate effector molecules. However, equally attractive is the idea that cells, like B cells, only rarely recognize molecular complexes of antigenic peptides and MHC. In such a case, cells would not "learn" that the body was infected from APCs and could not be expected to mount a conventional immune response of the kind that we associate with APC- T cell interactions (31).

Provocatively, the data presented here bear striking similarities with the role of cells reported in another system, namely the IgE allergic responsiveness of mice to protein antigen aerosols (18, 19). Thus T cells reduced ovalbumin-specific IgE responses in rats and mice that were tolerized with ovalbumin administered via repetitive respiratory aerosol administration (18, 19). Not only were T cells shown to be active in both cases, but strikingly low numbers of T cells per animal (~2 × 10³) were effective, as also was the case here (Fig. 6 b). Likewise, systemic oral tolerance to ovalbumin was suggested to be due to T cells (32). Recently it was also shown that mice deficient in T cells, and responding to infection with a natural intestinal pathogen, Eimeria, had exaggerated gut immunopathology attributable to unrestrained T cell function, or the effects thereof, suggesting a downregulatory function of T cells (33). In other studies, T cells from T. cruzi infected mice were shown to be suppressive in vivo and in vitro, correlating with autoimmunity (34). Finally, hepatic T cells could adoptively transfer tolerance to allogeneic skin grafts, and also reduced lymphokine production (35). T cells in tumor bearing mice were reported to decrease anti-tumor cytotoxic T cell activity (36). Thus, the data presented here, in the classical CS system, provide a clear demonstration of a fundamental functional phenotype of cells that is operative in numerous systems, including allergy to aerosolized antigen, oral tolerance, autoimmunity, transplantation, tumor immunity, and pathogenesis of infectious diseases. As such, the downregulation of T cells by the effects of T cells is emerging as a major and probably general functional capacity.

The downregulatory effect of T cells that we describe may in part be effected by a direct or indirect inhibition of IFN production by the CS-effector cells, as our results suggest. The mechanism for this inhibition has yet to be resolved. An obvious possibility is the production by T cells of cytokines that inhibit T cell production of IFN-, such as TGF, IL-4, or IL-10. The production by T cells of IL-4 would be consistent with the capacity of T cells from TCR^/ mice to support copious production of antibodies (22). Thus, regulation of CS by T cells may be another instance of Th2 regulation or diversion of Th1 cells (37). Alternatively, T cells may regulate CS-effector T cells by their being eliminated via injury, apoptosis, or cytotoxic lysis. There are many reports that T cells possess cytotoxic activity (38, 39). However, it is noteworthy that we have recent evidence that in vitro tolerance for IFN- production by splenocytes of TNBSA-tolerized mice can be reversed by IL-12 (Ushio, H., and P.W. Askenase, unpublished results), suggesting that cytotoxicity may not be the mechanism for T cell-mediated TNBSA tolerance.

In summary, DTH and CS are examples of important in vivo Th1 responses that can play a crucial role in defense against diverse microbial pathogens and tumor cells, and as effectors of some allergic and autoimmune diseases. This may be the reason for such tight regulation of these responses. It is possible that changes in immunoregulation could be a cause for decreased immune resistance in serious infections, or in cancers, or perhaps for the abnormally increased immune responses that occur in allergies and autoimmunity. The findings of the current study clearly establish that T cells can function as a newly recognized component of immune downregulation, and may be able to do so in an Ag-specific fashion, the underlying mechanism of which needs now to be defined.

Footnotes

Address correspondence to Philip W. Askenase, Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedal Street, New Haven, CT 06520-8013.

Received for publication 3 May 1996

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