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

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

From the * Department of Immunology, College of Medicine, Jagiellonian University, Krakow, Poland; Dagger  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 par  Imperial Cancer Research Foundation, London, England WZCA 3PX

Summary
Materials and Methods
Results
Discussion
Footnotes
References


Summary

Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using alpha beta T cell-deficient (TCRalpha -/-) mice, are presented here. The results clearly show that TCRalpha -/- mice cannot mount CS, implicating alpha beta T cells as the critical CS-effector cells. However, TCRalpha -/- mice can, after high dose tolerance, downregulate alpha +/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized alpha -/- mice are shown to include gamma delta TCR+ cells that also can downregulate interferon-gamma production by the targeted CS-effector cells in vitro. Downregulation by gamma delta cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that gamma delta T cells cannot fulfill CS-effector functions performed by alpha beta T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by gamma delta T cells. Comparisons are likewise considered with downregulation by gamma delta T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.


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, alpha beta TCR+, CS-effector T cells (7, 8), which customarily make Th1-type effector cytokines, such as IFN-gamma (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 alpha beta T cells, or whether they may include gamma delta 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 alpha -/- mice, to assess the cells responsible for CS elicitation and for CS downregulation, respectively. The studies show that gamma delta T cells cannot substitute for alpha beta T cells as effectors of CS elicitation, but that gamma delta T cells can medite downregulation of both alpha beta CS effectors in vivo and IFN-gamma production by these CS effectors in vitro. The studies are particularly pertinent given the reported capacity of gamma delta T cells to negatively regulate alpha beta T cell-driven responses in allergic (18, 19) and other immune responses.


Materials and Methods

Mice. 5-7-wk-old 129/J (H2b), BDF1 (H2b × d), C57Bl/6 (H2b), BALB/c (H2d), and CBA/J (H2k) mice were obtained from Jackson Labs (Bar Harbor, ME). TCRalpha -/- and TCRalpha +/- mice (20) with different but defined MHC backgrounds that included H2b, H2d, and H2b+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 Ib (PE-conjugated), and class Id (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 gamma delta 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, TCRalpha +/- and alpha -/-), 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.

To induce OX-specific tolerance, mice were injected intravenously once with 0.3 ml of 10% OX-conjugated mouse red blood cells (MRBC), or with OX-conjugated spleen cells (5 × 107) on days 0 and 3. The MRBC or spleen cells were labeled with OX as described previously (12). Briefly, 1 ml of packed MRBC was resuspended in 10 ml of DPBS (calcium- and magnesium-free PBS) and mixed for 30 min with freshly prepared 20 ml aqueous OX solution (containing 20 mg OX). The OXMRBC were then washed twice with PBS containing 5% FCS, and before use, the peletted OX-MRBC were resuspended to 10% in PBS. Spleens from mice injected intravenously with OXMRBC, or with OX spleen cells, were harvested on d 7 as a source of OX-specific regulatory 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.

For the cell mixing assay, 5-7 × 107 CS-effector immune lymph node and spleen cells from PCl or OX contact sensitized alpha +/+ donors were incubated for 30 min at 37°C with 5 × 107 regulatory spleen cells from mice intravenously tolerized with TNBSA or with OX-MRBC. Regulatory spleen cells were from tolerized alpha -/- mice, or control alpha +/+ 129/J, C57Bl/6, BALB/c, or BDF1 mice. Positive controls were immune CS-effector cells incubated without regulatory cells. After incubation, washed mixed cells from each group were transferred intravenously, and recipients were tested for CS responses by ear challenge with topical application of 0.8% PCl or OX, within 30 min immediately after transfer.

Transfer into Tolerized Mice to Confirm Active Tolerance and Agspecificity. To test tolerance and Ag specificity of downregulatory cells in another system, alpha -/- 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 alpha -/- mice received 7 × 107 CS-effector cells from normal TCRalpha +/+ mice (BALB/c) that were Ag homologous, or were of unrelated Ag specificity (TNP versus OX). alpha -/- 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 alpha +/+ 129/J, or alpha -/- donor mice were incubated in 20 ml UC7 hamster anti-TCR gamma delta 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.


Fig. 3. gamma delta T cells are responsible for CS downregulatory activity of high dose Ag tolerized TCRalpha -/- mice. In experiments 1 and 2, spleen cells (1.5 × 108) from TNBSA intravenous tolerized 129/J (group B), or TCRalpha -/- animals (group E), were separated into gamma delta TCR+ (groups C and F), and gamma delta TCR- (groups D and G) subpopulations by anti-gamma delta TCR mAb treatment in solution, and then incubation with anti-hamster Ig-coated para magnetic beads, and subsequently separated in vitro by exposure to a magnet. In experiment 3, similar cell fractionation used alpha +/+ BALB/c and their alpha -/- H2d counterparts. Immune CS-effector cells (7 × 107) were incubated for 30 min at 37°C with downregulatory, separated, magnetic bead positive cells (gamma delta TCR+) (groups C and F), or magnetic bead negative (gamma delta TCR-) cells (groups D and G), that were from either TNBSA-tolerized 129/J or BALB/c mice (groups C, D), or TNBSAtolerized TCRalpha -/- animals (groups F, G). Cell mixtures were then washed and injected intraperitoneally into naive recipients. As a positive control, CS-effector cells incubated without regulatory cells were transferred intraperitoneally (group A). The next day, animals were challenged on the ears with 0.8% PCl in olive oil and the increment in ear swelling was determined 24 h later. Statistical significance: group A vs B and D, P <0.01; group A vs group E, P <0.05; and group A vs group F, P <0.001.
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Fig. 5. Lack of MHC restriction of gamma delta downregulatory cells from TNBSA-tolerized TCRalpha -/- mice. Magnetic bead positive (gamma delta TCR+) cells from TNBSA-tolerized alpha -/- mice were incubated with 7 x 107 cells from MHC compatible 129/J H2b,d, or MHC incompatible (CBA/J, H2k) mice for 30 min at 37°C (groups B and D). As a positive control, we used immune cells from 129/J or CBA/J mice that were incubated without regulatory cells in medium alone (groups A and C). Then cells were washed and injected intraperitoneally into recipients that were syngeneic with the immune cells, and were skin tested for CS responsiveness. Statistical significance: group A vs B, and group C vs D, P <0.001.
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Fig. 6. Phenotype (a) and dose response of gamma delta downregulatory cells derived from TNBSA-tolerized alpha -/- mice (b). (a) Phenotype: To determine phenotype of downregulatory cells, spleen cells from TNBSA-tolerized alpha -/- mice were incubated with medium alone, or with mAb (antiCD4 or anti-CD8) for 40 min on ice. Cells were washed and incubated with rabbit complement for 45 min at 37°C, and were washed and resuspended in PBS. 7 × 107 CS-effector cells were incubated with medium alone (group A, positive control), with 5 × 107 untreated tolerized cells (group B), or with 5 × 107 anti-CD4 + C treated tolerized cells, (group C), or with 5 × 107 anti-CD8 + C treated tolerized cells, (group D). Statistical significance: group A vs B, and group A vs C P <0.001, and group A vs D P <0.01. (b)-Dose response: 7 × 107 immune CS effector cells were incubated with medium alone (group A, positive controls), or with 10-fold decreasing dilutions (2.5 × 105, 2.5 × 104, or 2.5 × 103 cells) of magnetic bead positive (gamma delta TCR+) downregulatory cells from alpha -/- TNBSA-tolerized mice, for 30 min at 37°C (groups B-D). After incubation, the cell mixtures were washed and injected into recipients that were challenged on the ears, and measured for CS responses at 24 h. Statistical significance: group A vs B and C, P <0.001, group A vs D, P <0.01.
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Recoveries of gamma delta cells were as follows. For 129/J TNBSA-tolerized donors, we harvested 15 × 107 separated spleen cells from three donors, obtaining ~106 anti-gamma delta magnetic bead positive cells that were 60% gamma delta + by FACS®. Thus, there were ~1.6 × 105 gamma delta cells transferred to each of four recipients, while there were 10 × 105 gamma delta cells in the starting spleen of one donor mouse. For alpha -/- TNBSA-tolerized donors, we harvested 20 × 107 spleen cells from three donors, obtaining 3.4 × 106 anti-gamma delta bead positive total cells that were on average 80% gamma delta + by FACS®, thus allowing for 7 × 105 gamma delta -enriched cells transferred to each of four recipients. FACS® analysis of the anti-gamma delta immunobead separated subpopulations was presented elsewhere (24), in which we reported that the percentage of gamma delta cells adhering to the anti-gamma delta beads can vary between 55-95% of the total adherent cells; the rest are not specifically bound, and on assay did not have biologic activity. Also, we noted that unavoidable splenic macrophage phagocytosis of some mAb-coated gamma delta cells that were attached to beads, probably contributed to variability in gamma delta T cell recoveries.

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 gamma delta cells, or with an equivalent number of magnetic bead nonadherent gamma delta - cells (i.e., alpha beta TCR+-remaining cells) originating from either tolerized 129/J alpha +/+ mice or TCRalpha -/- 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-gamma , 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-gamma delta 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 gamma delta -depleted cells and washing the column, gamma delta -enriched adherent cells were eluted. Equivalent numbers of gamma delta -enriched, or gamma delta 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 × 10-5 M/2-mercaptoethanol, and 10% fetal calf serum. At 48 h, culture supernatants were collected for IFN-gamma quantitation.

ELISA Quantitation of IFN-gamma in Culture Supernatants. Quantitative sandwich ELISA for IFN-gamma was performed with two different mAb specific for mouse IFN-gamma (clones R4-6A2 and XMG1.2; Pharmingen). 1 µg/ml of capture mAb to IFN-gamma 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-gamma (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-gamma 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

TCRalpha -/- Mice Have No 24 h CS Responses.

CS responses in contact sensitized TCRalpha -/- 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 alpha beta T cell-bearing 129/J mice developed a classical 24 h ear swelling response (Fig. 1, group A), whereas TCRalpha -/- mice did not (Fig 1, group B).


Fig. 1. Actively sensitized TCRalpha knock out mice (alpha -/-) have no classical late 24 h cutaneous CS responses, but can serve as adoptive cell transfer recipients. TCRalpha +/+ 129/J (group A) or TCRalpha -/- (group B) mice, were contact sensitized with 0.15 ml of 5% PCl skin painting. 4 d later, animals were challenged on the ears with 0.8% PCl, and CS responses were determined 24 h later by measuring increases in ear thickness with an engineer's micrometer. Results were expressed as U × 0.01 mm ± SE. Background ear swelling in PCl challenged controls that were not sensitized, were subtracted from experimental groups to give net ear swelling, as is shown. BDF1 (group C) alpha +/+ immune cells from PCl contact sensitized donors were transferred intravenously to BDF1 recipients, or to TCRalpha -/- H2b recipients (group D) that were ear challenged similarly, and then, 24 h ear swelling responses were determined.
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When alpha -/- mice were used as recipients of alpha beta + cells from MHC-matched, immunized alpha beta 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 TCRalpha -/- knockout mice. The finding that the 24 h component of CS cannot be elicited in actively sensitized TCRalpha -/- mice is consistent with other studies indicating that the active CSeffector cell type is TCRalpha beta + (7, 8, 24).

High Dose Ag Tolerance of TCRalpha -/- mice Induces Regulatory Spleen Cells that Downregulate CS.

Because TCRalpha -/- 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 alpha -/- 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 TCRalpha +/- mice, or TCRalpha +/+ (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 TCRalpha +/+ mice (Fig. 2, group B), TCRalpha +/- mice (Fig. 2, group C), or TCRalpha -/- 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 TCRalpha -/- mice (Fig. 2, groups E-G).


Fig. 2. High dose Ag tolerance of TCRalpha -/- mice induces cells that downregulate CS and generate an inhibitory environment. As an assay for generation of downregulatory cells, CS-effector cells (7 × 107) from PCl contact sensitized 129/J mice (groups A-D) were incubated for 30 min at 37°C, alone (group A, positive controls), or with 5 × 107 putative regulatory cells induced by two intravenous injections of TNBSA in TCRalpha +/+ 129/J (group B), TCRalpha +/- (group C), or TCRalpha -/- (group D) mice. The cell mixture was then washed and transferred intravenously into naive syngeneic 129/J recipients that immediately were challenged on the ears with 0.8% PCl. Groups B-D were significantly different (P <0.001) from control group A. In groups E-I, from a pooled separate experiment, TCRalpha +/+ BDF1 mice or their TCRalpha -/- H2b,d counterparts were used. BDF1 are poorly CS-reactive and thus were skin sensitized with PCl twice (days 0 and 3). On d 7, immune CS-effector lymph node and spleen cells (7 × 107), from these mice were injected intravenously into normal BDF1 mice (groups E-G), or into TCRalpha -/- recipients (groups H and I). Tolerized downregulatory cells were induced in BDF1 or TCRalpha -/- mice by two intravenous injections of 5 × 107 TNP-conjugated TCRalpha -/- spleen cells on days 0 and 3. Spleens from these tolerized mice served as a source of downregulatory cells respectively in groups F and G, in which 3.5 × 107 tolerized cells were mixed and incubated with BDF1 PCl-immune cells (7 × 107), before being injected intravenously together into the recipients. In group H, recipient TCRalpha -/- animals were tolerized via prior intravenous injection of TNP-conjugated spleen cells, i.e., they were tolerized by a procedure similar to inducing regulatory cells used in the mixing and adoptive transfer experiments (groups E-G). Statistical significance: group E vs F, I and H, P <0.005. Each group consisted of three animals. In group I, BDF1 CS-immune cells were transferred successfully into untreated TCRalpha -/- mice.
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A prediction that follows from the data shown in Fig. 2 (groups A-G) is that TCRalpha -/- mice will not support the actions of adoptively transferred TCRalpha +/+ CS-effector cells if the recipient alpha -/- 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 TCRalpha -/- 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 TCRalpha -/- mice was reproducibly demonstrated using transfers from individual TCRalpha - mice (data not shown), indicating the generality of the findings.

Expression of gamma delta TCR on CS-regulatory Cells from Tolerized TCRalpha -/- 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 gamma delta mAb-coated magnetic beads. gamma delta +, gamma delta -, 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-gamma delta bead adherent cells ranged from 55 to 95% gamma delta TCR+ when reanalyzed by FACS® (see Materials and Methods). Thus, we estimate that >=106 gamma delta cells were adoptively transferred into each recipient when unseparated TCRalpha -/- cells were used, and about >=7 × 105 gamma delta cells were transferred when magnetic bead gamma delta -enriched cells were used.

The data in experiments 1 and 2 of Fig. 3 show that while the cells from TNBSA-treated alpha +/+ 129/J mice that are responsible for downregulating CS effectors are exclusively TCR gamma delta - (group D), the most potent regulatory cell fraction from tolerized TCRalpha -/- mice scored as TCR gamma delta + (Fig. 3, group F). Similar results were obtained in experiment three, in which cells from H2d TCRalpha -/- mice were compared with cells from H2d 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 gamma delta + fraction from TCRalpha -/- mice, but not from alpha +/+ BALB/c mice, was again active (experiment 3, group C). Conversely, the active fraction from normal alpha -/- mice was TCR alpha delta + (group D), and the TCR gamma delta - fraction from TCRalpha -/- mice (group E) contained negligible residual activity.

gamma delta T Cell-mediated Regulatory Activity Shows Antigen Specificity.

The discovery that gamma delta T cells in TCRalpha -/- mice can mediate downregulation of CS effector cells prompted an investigation of Ag specificity, since gamma delta T cells and alpha beta cells appear to have a different set of Ag specificities. To test this, cells from TCRalpha -/- mice tolerized with high doses of OX or TNP, were mixed with CS-effector cells from TCRalpha +/+ 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 TCRalpha -/- 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 TCRalpha -/- 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 TCRalpha -/- mice downregulated effector cells from OX-sensitized mice, but not alpha +/+ TNP-immune cells) (group E versus F).



Fig. 4. (a) Reciprocal Ag specificity of splenic gamma delta + regulatory cells from TNP or OX-tolerized alpha -/- mice that act on TNP versus OX-immune CS-effector T cells. BDF1 mice were used as donors and recipients of PCl or OX immune cells, while their H2b,d TCRalpha -/- counterparts were used as donors of tolerized regulatory cells. To tolerize spleens of TCRalpha -/- mice, normal alpha -/- spleen cells were conjugated in vitro with TNP or OX, and 5 × 107 hapten-conjugated self cells were injected intravenously into alpha -/- mice on days 0 and 3, and then spleen cell suspensions from these tolerized recipients were prepared on day 7 as putative regulatory cells. BDF1 CS-effector cell donors were contact skin-sensitized with TNP (via PCl painting) (groups A-C), or with OX (groups D-F), on days 0 and 3, and then spleen and lymph node cells were transferred into BDF1 recipients on day 7, either alone (7 × 107 cells) (groups A and D), or mixed with Ag homologous downregulatory alpha -/- cells (groups C and E), or Ag heterologous downregulatory cells (5 × 107) from TCRalpha -/- mice (groups B and F). Adoptive cell recipients that were MHC homologous to CS-effector cells that were cotransferred, were challenged on the ears with TNP or OX hapten, and ear swelling measurements were made 24 h later. Each group consisted of three mice. Statistical significance: group A vs C, P <0.001; and group D vs E, P < 0.002; group D vs F NS. (b) Antigen-specificity of tolerance in alpha -/- mice tested as recipients of alpha +/+ CS-effector cells. To confirm Ag-specificity of tolerized alpha -/- mice we used an assay that did not involve transfers and manipulation of the putative regulatory cells. alpha -/- mice were tolerized by intravenously injection of TNBSA or OX-MRBC before serving as recipients for transfer of CS-effector cells from normal BALB/c mice that were of homologous or unrelated Ag (TNP vs OX) specificity. The experiments included groups of positive and negative controls. Mice were then challenged immediately on the ears with homologous hapten (TNP or OX), and then tested for CS ear swelling 24 and 48 h later. Statistical analysis at 24 h using the two tailed Student's t test showed: group B vs A, P <0.01; group B vs C, P <0.02; group E vs D, P <0.01; group E vs F, P <0.01. For 48 h: group B vs A, P <0.02; group B vs C, P <0.01; group E vs D, P <0.001; and group E vs F, P <0.01.
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To confirm the above findings we tested Ag-specificity in tolerized alpha -/- mice by attempting to transfer alpha +/+ CS-effector cells into these recipients. alpha -/- 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 alpha -/- mice received 7 × 107 TNP or OX immune cells from normal alpha +/+ (BALB/c) mice. The data presented in Fig. 4 b show there was no ability to adoptively transfer immunity into TNP tolerized alpha -/- mice with TNP-specific CS-effector cells (group B), while TNP-immune CS-effector cells transferred normal CS responses into OX-tolerized alpha -/- 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 alpha -/- mice (Fig. 4 b, group E), whereas OX CS was successfully transferred to TNP-tolerized alpha -/- 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 alpha -/- mice.

gamma delta T Cell-mediated Regulatory Activity is MHC Unrestricted.

To determine possible MHC restriction of gamma delta downregulatory T cells, magnetic bead-enriched gamma delta TCR+ cells from TNBSA-tolerized alpha -/- mice were incubated with 7 × 107 PCl-immune CS-effector cells from MHC-compatible (129/J, H2b), or -incompatible (CBA/J, H2k) 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 gamma delta graft could develop), and ear swelling responses determined. The results in Fig. 5 show that gamma delta + cells from TNBSA-tolerized TCRalpha -/- mice downregulated CS-effector responses of allogeneic CBA/J H2k 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 gamma delta T Cells.

To establish the phenotype of gamma delta T cells capable of inhibiting CS-effector responses, spleen cells from TNBSA-tolerized alpha -/- mice were treated with anti-CD4 or anti-CD8 mAb or PBS, and then rabbit complement 1:75. The three resultant populations of TCRalpha -/- 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 TCRalpha -/- 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) gamma delta T cells.

DN gamma delta 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 alpha beta T cells. Therefore, to assess the potency of DN gamma delta 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% gamma delta TCR+ cells (purified from TNBSA-tolerized alpha -/- 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 gamma delta T cells had activity in the assay (group D), albeit weaker than the other doses. The capacity of very small numbers of downregulatory gamma delta T cells to score in this bio-assay is discussed below.

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

Since IFN-gamma is a central cytokine in Th-1-mediated responses, such as DTH and CS, we determined whether IFN-gamma 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-gamma 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-gamma over a 48 h period. Compared to production of IFN-gamma by CS-effector cells mixed with nonimmune spleen cells (Fig. 7 a, groups A and D), IFN-gamma production by CS-effector cells and TNP-APC was substantially inhibited by mixing with spleen cells from intravenous TNBSA tolerized normal alpha +/+ mice (Fig. 7 a, group B) and also with spleen cells from intravenous TNBSA tolerized alpha -/- 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-gamma (data not shown).



Fig. 7. (a) gamma delta T cells in spleens of TNBSA intravenously tolerized alpha -/- mice Ag-specifically downregulate IFN-gamma production by CS-effector T cells. 2 × 105 TNP-immune CS-effector cells from alpha +/+ mice were cultured in vitro in the presence of TNP-conjugated normal spleen cells as APC, either with or without added regulatory cells (group A), or with regulatory cells of two different specificities: either TNBSA-tolerized or OXtolerized. These regulatory cells were either from alpha +/+ normal mice (groups B and C), or were harvested from TNBSA-tolerized or OX-tolerized alpha -/- mice (groups E and F, respectively). At the end of 48 h, supernatants from the individual microwell cultures were harvested and assayed subsequently for IFN-gamma content with a double mAb capture ELISA immunoassay. Statistics: group A vs B, P <0.004; group C vs A, P <0.01; group E vs D, P <0.004; and group F vs D, P <0.02. (b) Phenotype of in vitro gamma delta down regulatory cells: 2 × 105 lymph node CS-effector cells from PCl-sensitized donors were cultured in individual wells of flat-bottomed wells of microtrays in quadruplicate with TNP- or OX-conjugated syngeneic spleen cells as APC, either alone (groups A and D), or with regulatory cell subpopulations from TNBSA-tolerized or OX-tolerized donors (group B and groups C, E, and F). These regulatory cells came from either normal alpha +/+ mice (groups B and C), or from matched TCRalpha knockout alpha -/- mice. Prior to addition to microcultures, these regulatory cells were each separated by immunomagnetic bead techniques, using anti-gamma delta TCR mAb, into gamma delta enriched cells (gamma delta +, left), or gamma delta -depleted cells (gamma delta -, right). After 48 h culture of the various cell mixtures, supernatants were harvested from individual wells and assayed subsequently using a two mAb capture ELISA immunoassay for IFN-gamma content. Statistics: group A vs B (left), NS; group A vs B (right), P <0.001; group A vs C (right), P <0.001; group D vs E (left), P <0.002; group D vs F (left), NS; group D vs E (right), NS.
[View Larger Versions of these Images (16 + 17K GIF file)]

To determine the phenotype of regulatory cells responsible for the in vitro suppression of IFN-gamma production, gamma delta TCR+ or gamma delta TCR- cells in the spleens were separated, using anti-gamma delta antibody coated magnetic beads that were used to separate spleen cells from nontolerized, TNBSA-tolerized, or OX-tolerized TCRalpha +/+ or TCRalpha -/- 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-gamma production by gamma delta + cells from TNBSA-tolerized alpha -/- mice (Fig. 7 b, group E, left) that did not occur with gamma delta + cells from TNBSA-tolerized alpha +/+ mice (Fig. 7 b, group B, left). In contrast, the inhibitory activity of TNBSA-tolerized alpha +/+ mice was shown to be due to gamma delta - cells (Fig. 7 b, group B, right), and thus was probably due to alpha beta T cells. In contrast to the downregulatory activity of gamma delta + cells from TNBSA-tolerized alpha -/- mice, the gamma delta - cells of these mice were totally without inhibitory activity (Fig. 7 b, group E, right), as were OX-tolerized gamma delta + and gamma delta - cells (Fig. 7 b, group C and F, left), again varifying Ag-specificity of gamma delta + cells mediating tolerance.

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


Discussion

alpha beta 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 alpha beta T cells as principal effectors in CS responses (7, 8, 25, 26): i.e., alpha beta T cells and gamma delta T cells are not redundant in their functional capacity as effector cells in CS. By contrast, TCRalpha -/- mice can respond to high dose Ag tolerance by the induction of cells that can downregulate CS-effector activity from alpha +/+ immunized donors. This was shown in two ways: splenocytes from tolerized TCRalpha -/- mice downregulate CS-effector cells with which they are mixed in vitro, before cotransfer and elicitation of CS, and high dose Ag-treated tolerized TCRalpha -/- mice inhibit the transfer of CS-effectors that function normally in untreated TCRalpha -/- mice.

The experiments presented here demonstrate that gamma delta cell- enriched fractions from TCRalpha -/- mice are better mediators of high dose Ag tolerance than are gamma delta cell-depleted fractions, suggesting that gamma delta T cells are the active regulatory cells, as opposed to TCRbeta +alpha - cells that are a potential feature of TCRalpha -/- mice (27). The lack of conventional MHC restriction shown by the TCRalpha -/- regulatory cells, and their CD4-CD8- phenotype are both consistent with most assays of gamma delta function (29). The apparent hapten specificity of the regulatory gamma delta cells demonstrated herein for the first time may be considered consistent with the hypothesis that the potential diversity (30) of the gamma delta TCR is greater than that of any other Ag receptor (31). Thus, overall, downregulation can be mediated by Ag-specific gamma delta T cells, and need not be solely mediated by specific alpha beta T cells. However, in this study we have provided no evidence that the gamma delta cells directly recognized either PCl/TNP or OX, nor whether the gamma delta 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 TCRalpha -/- mice are deficient in immune effector responses because alpha beta cells are uniquely capable of specifically responding to the challenging antigen. However, the apparent capacity of gamma delta cells in TCRalpha -/- 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 gamma delta 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., gamma delta cells may occur in an inappropriate anatomical location, in insufficient numbers, or produce inappropriate effector molecules. However, equally attractive is the idea that gamma delta cells, like B cells, only rarely recognize molecular complexes of antigenic peptides and MHC. In such a case, gamma delta 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-alpha beta T cell interactions (31).

Provocatively, the data presented here bear striking similarities with the role of gamma delta cells reported in another system, namely the IgE allergic responsiveness of mice to protein antigen aerosols (18, 19). Thus gamma delta 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 gamma delta T cells shown to be active in both cases, but strikingly low numbers of gamma delta 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 gamma delta T cells (32). Recently it was also shown that mice deficient in gamma delta T cells, and responding to infection with a natural intestinal pathogen, Eimeria, had exaggerated gut immunopathology attributable to unrestrained alpha beta T cell function, or the effects thereof, suggesting a downregulatory function of gamma delta T cells (33). In other studies, gamma delta T cells from T. cruzi infected mice were shown to be suppressive in vivo and in vitro, correlating with autoimmunity (34). Finally, hepatic gamma delta T cells could adoptively transfer tolerance to allogeneic skin grafts, and also reduced lymphokine production (35). gamma delta 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 gamma delta 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 alpha beta T cells by the effects of gamma delta T cells is emerging as a major and probably general functional capacity.

The downregulatory effect of gamma delta T cells that we describe may in part be effected by a direct or indirect inhibition of IFNgamma 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 gamma delta T cells of cytokines that inhibit T cell production of IFN-gamma , such as TGFbeta , IL-4, or IL-10. The production by gamma delta T cells of IL-4 would be consistent with the capacity of gamma delta T cells from TCRalpha -/- mice to support copious production of antibodies (22). Thus, regulation of CS by gamma delta T cells may be another instance of Th2 regulation or diversion of Th1 cells (37). Alternatively, gamma delta T cells may regulate CS-effector alpha beta T cells by their being eliminated via injury, apoptosis, or cytotoxic lysis. There are many reports that gamma delta T cells possess cytotoxic activity (38, 39). However, it is noteworthy that we have recent evidence that in vitro tolerance for IFN-gamma 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 gamma delta 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 gamma delta 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

   The authors are especially grateful to Scott Roberts (University of Connecticut, Storrs, CT), Topher Dudley (National Institutes of Health), and Adrian Smith (Yale University, New Haven, CT) for review of the manuscript, and to Marilyn Avallone for her excellent secretarial skills.
   This work was supported by National Institutes of Health grants AI-12211, AI-26639, and AI-02174 to P.W. Askenase, and AI-27855 to A.C. Hayday, the Polish Committee of Scientific Research, Maria Sklodowska-Curie Fund II (Polish American Agreement) to W. Ptak, and the Markey Foundation to L.R. Anderson.
   1Abbreviations used in this paper: DN, double negative T cells (CD4-, CD8-); CS, contact sensitivity; DTH, delayed-type hypersensitivity; MRBC, mouse red blood cells; OX, oxazolone; PCl, picryl chloride; TMB, tetramethylbenzidine; TNBSA, Trinitro-benzene sulfonic acid.

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Copyright © 1996 by The Rockefeller University Press.