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Rudimentary TCR Signaling Triggers Default IL-10 Secretion by Human Th1 Cells¹

Gregory G. Burrows^2,*,, Yuan K. Chou^*,, Chunhe Wang^*, Justin W. Chang^*, Thomas P. Finn^||, Nicole E. Culbertson^||, Joseph Kim^*, Dennis N. Bourdette^*,||,#, Deborah A. Lewinsohn^,, David M. Lewinsohn^,**, Masayuki Ikeda, Tohru Yoshioka, Charles N. Allen^¶, Halina Offner^*,|| and Arthur A. Vandenbark^*,,||

Departments of ^* Neurology and Biochemistry and Molecular Biology, Division of Pediatric Infectious Diseases, Department of Molecular Microbiology and Immunology, and ^¶ Center for Research in Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, OR 97201; ^|| Neuroimmunology Research, ^# Neurology Service, and ^** Division of Pulmonary and Critical Care Medicine, Veterans Affairs Medical Center, Portland, OR 97201; and Department of Molecular Neurobiology, School of Human Sciences, Waseda University, Tokorozawa, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the 1 and 1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85–99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR -chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TCR of CD4⁺ T lymphocytes recognizes immunogenic peptide sequences bound within the groove of MHC class II molecules (1). Such recognition results in a signal that is transmitted through the TCR, activates intracellular signal transduction pathways, and culminates in the proliferation and differentiation of CD4⁺ T cells into effector cells with multiple downstream functions (2). Naive CD4⁺ T cells may differentiate along distinct lineages that include Th1 or Th2 cells (3). Th1 cells mediate inflammatory responses, activate macrophages, and stimulate cell-mediated immunity. Th2 cells activate B cells, stimulate humoral immunity, are prominent mediators of allergic responses, and may inhibit Th1 cells (4). The choice of differentiation into either Th1 or Th2 cells is a crucial step that determines the direction of the subsequent adaptive immune response and whether humoral or cell-mediated immunity will predominate. This choice is influenced by many factors, including the route of entry and antigenic composition of the pathogen, the type and state of the APC that initially responds to the presence of the pathogen (5, 6), and the strength of interaction between the TCR and the MHC-peptide, the "trimolecular complex" (7).

Our recent studies exploring the differential susceptibility of human CD4⁺ Th1 and Th2 cells to induction of anergy and apoptosis using ethylcarbodiimide-Ag-coupled APC (8) revealed that the signaling requirements for inactivation of Th1 and Th2 cells (and perhaps their activation as well) are different. What are the molecular events that control CD4⁺ T lymphocyte effector function and phenotype? Previous work has demonstrated that a longer duration of contact between the TCR and its ligand results in a full signal through the TCR and subsequent Th1 differentiation, whereas shorter duration contact or altered interaction, as in the case of altered peptide ligands, results in a partial signal that favors Th2 differentiation (7, 9). Cell surface features independent of the actual trimolecular complex itself can also modify its stability and in turn modulate downstream cellular differentiation and proliferation. These features include the density of the MHC-peptide ligand on the APC cell surface, the stabilization contributed by the CD4 coreceptor, which binds primarily to the MHC class II 2 domain (10), costimulatory interactions such as B7:CD28, and interactions between adhesion molecules that may serve to stabilize the cell-cell interface. In addition, the plasma membranes of T cells contain microdomains biochemically distinct from bulk plasma membrane, commonly referred to as "lipid rafts," that have a profound effect on the trimolecular complex and its ability to coordinate downstream signaling (11). The duration and stability of the trimolecular complex can theoretically be described in terms of its multiple dissociation constants, and an understanding of the relationship of these multiple interactions lays the groundwork for a fundamental understanding of how the signaling complex functions.

With these larger goals in mind, we have developed a "minimal" TCR ligand that would allow study of TCR signaling in primary T cells and T cell clones in the absence of costimulatory interactions that complicate dissection of the information cascade initiated by MHC/peptide binding to the TCR and TCR chains. A minimum TCR ligand conceptually consists of the surface of an MHC molecule that interacts with the TCR and the three to five amino acid residues within a peptide bound in the groove of the MHC molecule that are exposed to solvent, facing outward for interaction with the TCR.

In previous studies, we described the design, biochemistry, and biophysical characterization of recombinant TCR ligands (RTLs)³ derived from MHC class II (12, 13, 14). MHC class II molecules are membrane-anchored heterodimers on the surface of APC that bind to the TCR, initiating a cascade of interactions that result in Ag-specific activation and differentiation of clonal populations of T cells. Each polypeptide chain consists of four extracellular domains (12, 12), a transmembrane region, and a short cytoplasmic tail. We have used rational drug design and protein molecular engineering to express the 1 and 1 domains of HLA-DR2 as a single exon of 200 aa residues with various N-terminal extensions containing antigenic peptides. These HLA-DR2-derived RTLs fold to form the peptide-binding/T cell recognition domain of the native MHC class II molecule (14).

Rat MHC class II RT1.B-derived RTLs have been used successfully to protect and treat actively induced and passively induced experimental autoimmune encephalomyelitis (12, 13, 14, 15, 16). Here we address a relatively straightforward hypothesis: can the phenotype of autoreactive CD4⁺ human T cell clones be modulated therapeutically by treatment with human RTLs? Inflammatory Th1, CD4⁺ T cells are activated in a multistep process that is initiated by coligation of the TCR and CD4 with MHC-peptide complex present on APC. This primary, Ag-specific signal must be presented in the proper context, which is provided by costimulation through interactions of additional T cell surface molecules such as CD28 with their respective conjugate on APC. Stimulation through the TCR in the absence of costimulation, rather than being a neutral event, can induce a range of cellular responses from full activation to anergy or cell death (17). In this report, we describe experiments that document the ability of Ag-specific RTLs to induce a variety of human T cell signal transduction processes as well as modulate effector functions, including cytokine profiles and proliferative potential.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
RTLs

General methods for cloning, expression, purification, in vitro folding, and biochemical analysis of these molecules have been described previously (12, 13, 14, 15, 16). DNA constructs encoding the RTLs on pET-21d⁺ plasmid were transformed into BL21(DE3)-competent cells for protein expression. Positive clones were chosen for expression of the HLA-DR2 11-derived RTL303 and RTL311 covalent peptide-coupled molecules. Bacteria were grown in 1-liter cultures to midlogarithmic phase (OD₆₀₀ 0.6–0.8) in Luria-Bertani broth (BD Biosciences, Sparks, MD) containing carbenicillin (50 µg/ml) at 37°C. Recombinant protein production was induced by addition of 0.5 mM isopropyl--D-thiogalactoside. After incubation for 3 h, the cells were centrifuged and stored at -80°C before processing. All subsequent manipulations of the cells were at 4°C. The cell pellets were resuspended in ice-cold PBS, pH 7.4, and sonicated for 4 x 20 s with the cell suspension cooled in a salt-ice-water bath. The cell suspension was then centrifuged, the supernatant fraction was poured off, and the cell pellet was resuspended and washed three times in PBS and then resuspended in 20 mM ethanolamine, 6 M urea, pH 10, for 4 h. After centrifugation, the supernatant containing the solubilized recombinant protein of interest was collected and stored at 4°C until purification. RTL constructs were purified and concentrated by FPLC ion exchange chromatography using Source 30Q anion exchange media (Amersham Pharmacia Biotech, Piscataway, NJ) in an XK26/20 column (Amersham Pharmacia Biotech), using a step gradient with 20 mM ethanolamine, 6 M urea, 1 M NaCl, pH 10. Homogeneous peaks of the appropriate size were collected, dialyzed extensively against PBS at 4°C, and concentrated by centrifugal ultrafiltration with Centricon-10 membranes (Amicon, Beverly, MA). For purification to homogeneity, a finish step used size exclusion chromatography on Superdex 75 medium (Pharmacia Biotech) in an HR16/50 column (Amersham Pharmacia Biotech). The final yield of purified protein varied between 5 and 10 mg/L of bacterial culture, and the molecules used in these studies form well-defined aggregates that are highly soluble in aqueous buffers.

Synthetic peptides

MBP_85–99 peptide (ENPVVHFFKNIVTPR) and BCR-ABL b3a2 peptide (ATGFKQSSKALQRPVAS; CABL), (18) were prepared on an Applied Biosystems 432A (Foster City, CA) peptide synthesizer using fmoc solid phase synthesis. The myelin basic protein (MBP) peptide was numbered according to the bovine MBP sequence (19). Peptides were prepared with C-terminal amide groups and cleaved using thioanisole-1,2-ethanedithiol-dH₂O in trifluoroacetic acid for 1.5 h at room temperature with gentle shaking. Cleaved peptides were precipitated with six washes in 100% cold tert-butylmethyl ether, lyophilized, and stored at -70°C under nitrogen. The purity of peptides was verified by reverse phase HPLC on an analytical Vydac C₁₈ column.

T cell clones

Peptide-specific T cell clones were selected from PBMC of a multiple sclerosis (MS) patient homozygous for HLA-DRB1*1501 and a healthy donor homozygous for HLA-DRB1*07, as determined by standard serological methods and further confirmed by PCR amplification with sequence-specific primers (20). Frequencies of T cells specific for human MBP_85–99 and CABL were determined by limiting dilution assay. PBMC were prepared by Ficoll (American Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation and cultured with 10 µg/ml of either MBP_85–99 or CABL peptide at 50,000 PBMC/well of a 96-well U-bottom plate plus 150,000 irradiated (2500 rad) PBMC/well as APC in 0.2 ml medium (RPMI 1640 with 1% human pooled AB serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µg/ml penicillin G, and 100 µg/ml streptomycin) for 5 days, followed by adding 5 ng/ml IL-2 (R&D Systems, Minneapolis, MN) twice per week. After 3 wk, the culture plates were examined for "clump formation" by visual microscopy and the cells from the "best" 20–30 clump-forming wells among a total of 200 wells per each peptide Ag were expanded in 5 ng/ml IL-2 for another 1–2 wk. These cells were evaluated for peptide specificity by the proliferation assay, in which 50,000 T cells/well (washed three times) were incubated in triplicate with 150,000 freshly isolated and irradiated APC/well plus either medium alone, 10 µg/ml MBP_85–99 or 10 µg/ml CABL peptide for 3 days, with [³H]thymidine added for the last 18 h. Stimulation index (SI) was calculated by dividing the mean cpm of peptide-added wells by the mean cpm of the medium alone control wells. T cell isolates with the highest SI for a particular peptide Ag were selected and expanded in medium containing 5 ng/ml IL-2, with survival of 1–6 mo, depending on the clone, without further stimulations.

Subcloning and expansion of T cell number

Selected peptide-specific T cell isolates were subcloned by limiting dilution at 0.5 T cells/well plus 100,000 APC/well in 0.2 ml medium containing 10 ng/ml anti-CD3 (BD PharMingen, San Diego, CA) for 3 days, followed by addition of 5 ng/ml IL-2 twice per wk for 1–3 wk. All wells with growing T cells were screened for peptide-specific response by the proliferation assay, and the well with the highest SI was selected and continuously cultured in medium plus IL-2. The clonality of cells was determined by RT-PCR, with a clone defined as a T cell population utilizing a single TCR V gene. T cell clones were expanded by stimulation with 10 ng/ml anti-CD3 in the presence of 5 x 10⁶ irradiated (4500 rad) EBV-transformed B cell lines and 25 x 10⁶ irradiated (2500 rad) autologous APC per 25-cm² flask in 10% AB pooled serum (BioWhittaker, Walkersville, MD) for 5 days, followed by washing and resuspending the cells in medium containing 5 ng/ml IL-2, with fresh IL-2 additions twice a week. Either autologous or heterologous B cell lines and PBMC were usable, but heterologous cell lines were determined empirically to be even better in supporting T cell expansion. As professional APC, the transformed B cells were enriched with costimulatory molecules and related B cell-derived cytokines which were essentially required for T cell expansion using anti-CD3 stimulation. Otherwise, T cells stimulated with anti-CD3 alone or with inadequate costimulation would be turned to an anergic status. In this article, we report a method using an existing EBV-transformed heterologous B cell line and freshly isolated autologous PBMC, similar to a protocol reported by us previously (21). Expanded T cells were evaluated for peptide-specific proliferation and the selected, expanded T cell clone with the highest proliferation SI was used for experimental procedures.

AV and BV gene expression by RT-PCR

The clonality of the Th1 cells used in this study was determined by RT-PCR (22), using the AV and BV gene-specific primers in Table I. Briefly, total RNA was isolated from fresh or frozen pelleted cells using the Stratagene RNA Isolation Kit (Stratagene, La Jolla, CA). cDNA was synthesized in a 20-µl volume using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) and an oligo(dT)_12–18 primer (Life Technologies), following the manufacturer’s recommendations. For amplification of TCRBV cDNA, a panel of 26 BV and a single BC primer was used. A portion of the BC primer was labeled (either 2–3% was radioactively labeled with [³²P]ATP, or 50% was end labeled at the 5' end with the fluorochrome, Cy3 (Amersham Pharmacia Biotech). As a positive control for the reaction, two BC primers (forward and reverse) were used, and the reverse primer was labeled as above. The cDNA from 1500–2000 T cells was used in each 15-µl reaction, along with 0.3 µl of each primer, 0.5 U Taq DNA polymerase (Promega, Madison, WI), 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 0.2 mM dNTPs, and 2 mM MgCl₂. Amplification was conducted for 24–26 cycles (94.5°C for 30 s, 60°C for 1 min, 72°C for 1 min), followed by a final 5-min extension at 72°C. All PCR reactions were performed in a PerkinElmer GeneAmp 9600 thermocycler (PerkinElmer/Cetus, Norfolk, CT). For the amplification of TCRAV cDNA, a panel of 30 AV primers and an AC primer was used (the AC primer was partially labeled as above). As a positive control for the reactions, two AC primers (forward and reverse) were used, one labeled as above. PCR conditions were as described above. After amplification, 10 µl of each reaction were loaded on a 6% polyacrylamide gel and run at 250 V for 22 min. If the DNA was radioactively labeled, the gel was dried for 1 h, exposed to a phosphor screen for 30 min–1 h, and analyzed by phosphor imaging (Bio-Rad Molecular Imager FX; Bio-Rad, Hercules, CA). If the DNA was fluorescently labeled, the gel was directly imaged on a fluorescent imager (Bio-Rad Molecular Imager FX). In either case, the PCR products of the correct size were quantitated by measuring phosphor or fluorescent signal intensity, and the background was subtracted using an adjacent region below the bands.

Cell culture supernatants were recovered at 72 h and frozen at -80°C until use. Cytokine measurement was performed by ELISA as previously described (23), using cytokine-specific capture and detection Abs for IL-2, IFN-, IL-4, and IL-10 (BD PharMingen). Standard curves for each assay were generated using recombinant cytokines (BD PharMingen), and the cytokine concentration in the cell supernatants was determined by interpolation.

Flow cytometry

Two-color immunofluorescent analysis was performed on a FACScan instrument (BD Biosciences, Mountain View, CA) using CellQuest software. Quadrants were defined using isotype-matched control Abs.

-Phosphorylation assay

T cells were harvested from culture by centrifuging at 400 x g for 10 min, washed, and resuspended in fresh RPMI. Cells were treated with RTLs at 20 µM final concentration for various amounts of time at 37°C. Treatment was stopped by addition of ice-cold RPMI, and cells were collected by centrifugation. The supernatant was decanted, and lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride-HCl, 0.8 µM aprotinin, 50 µM bestatin, 20 µM leupeptin, 10 µM pepstatin A, 1 mM activated sodium orthovanadate, 50 mM NaF, 0.25 mM potassium bisperoxo(1,10-phenanthroline) oxovanadate, 50 µM phenylarsine oxide) was added immediately. After a mixing at 4°C for 15 min to dissolve the cells, the samples were centrifuged for 15 min, and cell lysate was collected. To the lysate was added an equal volume of sample loading buffer, mixed and boiled for 5 min, and then aliquots were separated by 15% SDS-PAGE. Protein was transferred to a polyvinylidene difluoride membrane for Western blot analysis. Western blot block buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 20, 1% BSA). Primary Ab was anti-phosphotyrosine, clone 4G10 (Upstate Biotechnology, Lake Placid, NY). Secondary and tertiary Ab was determined from an ECF Western blotting kit (Amersham). The dried blot was scanned using a Storm 840 scanner (Molecular Dynamics, Sunnyvale, CA), and chemifluorescence was quantified using ImageQuant version 5.01 (Molecular Dynamics).

ERK activation assay

T cells were harvested and treated with RTLs as for -phosphorylation assay. Western blot analysis was performed using anti-phospho-extracellular signal-related kinase (ERK) (Promega) at 1/5000 dilution or anti-ERK kinase (New England Biolabs, Beverly, MA) at 1/1500 dilution and visualized using an ECF Western blotting kit. Bands of interest were quantified as described for -phosphorylation assay.

Ca²⁺ imaging

Human T cells were plated on polylysine-coated 35-mm glass bottom dishes and cultured for 12–24 h in medium containing IL-2. Fura-2-acetoxymethyl ester (5 mM; Molecular Probes, Eugene, OR) dissolved in the culture medium was loaded on the cells for 30 min in CO₂ incubator. After a rinse of fura-2 and an additional 15-min incubation in the culture medium, the cells were used for calcium measurement. Fluorescent images were observed by an upright microscope (Axioskop FS; Zeiss, Oberkochen, Germany) with a water immersion objective (UmplanFL 60x/0.8; Olympus, Melville, NY). Two wavelengths of the excitation UV light (340 or 380 nm) switched by a monochromator (Polychrome 2; T.I.I.L. Photonics, Martinareid, Germany) were exposed for 73 ms at 6-s interval. The intensity of 380 nm UV light was attenuated by a balancing filter (UG11; Omega Optical, Brattleboro, VT). The excitation UV light was reflected by a dichroic mirror (FT 395 nm; Zeiss), and the fluorescent image was band-passed (BP500–530; Zeiss), amplified by an image intensifier (C7039-02; Hamamatsu Photonics, Middlesex, NJ), and exposed to a multiple format-cooled CCD camera (C4880; Hamamatsu Photonics). The UV light exposure, CCD control, image sampling, and acquisition were done with a digital imaging system (ARGUS HiSCA; Hamamatsu Photonics). The background fluorescence was subtracted by the imaging system. During the recording, cells were kept in a culture medium maintained at 30°C by a stage heater (DTC-200; Dia Medical Systems, Tokyo, Japan). The volume and timing of drug application were regulated by a trigger-driven superfusion system (DAD-12; ALA Scientific Instruments, Westbury, NY).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study was initiated to analyze the mechanism by which human RTLs affect human T cell clones in vitro. Two different MHC class II DR2-derived RTLs (HLA-DR2b: DRA*0101, DRB1*1501) were used in this study (Fig. 1). RTL303 (11/MBP_85–99) and RTL311 (11/CABL) differ only in the Ag genetically encoded at the N terminus of the single exon RTL. The MBP_85–99 peptide represents the immunodominant MBP determinant in DR2 patients (24), and the CABL peptide (18) contains the appropriate motif for binding DR2. The human T cell clones used in this study were selected from a DR2-homozygous patient and a DR7-homozygous healthy control.

View larger version (68K): [in this window] [in a new window]   FIGURE 1. Structure-based design of the human HLA-DR2-derived RTLs. A, Schematic scale model of an MHC class II molecule on the surface of an APC. The polypeptide backbone extracellular domain is based on the crystallographic coordinates of HLA-DR1 (Protein Data Bank accession code 1AQD) (24 25 ). The transmembrane domains are shown schematically as 0.5-nm cylinders, roughly the diameter of a polyglycine helix. Color scheme: -chain, red; -chain, blue. Residues conserved between human HLA-DR, HLA-DP, HLA-DQ, murine I-A and I-E, and rat RT1.B class II molecules are magenta. Bound peptide is green. The C termini of the MHC class II heterodimers are labeled. B, The HLA-DR2 11-derived RTL303 molecule containing covalently coupled MBP85–99 peptide. C, The HLA-DR2 11-derived RTL311 molecule containing covalently coupled CABL peptide. The view of the RTLs is symmetry related to the MHC class II molecule in A by rotation around the long axis of bound peptide by 45 degrees (y-axis) and 45 degrees (z-axis). Left, The same color scheme as in A, with primary TCR contact residues colored yellow. Middle, colored according to electrostatic potential (EP). The color ramp for EP ranges from red (most positive) to purple (most negative) (30 ). Right, colored according to lipophilic potential (LP). The color ramp for LP ranges from brown (highest lipophilic area of the molecule) to blue (highest hydrophilic area) (31 ). The program Sybyl (Tripos Associates, St. Louis, MO) was used to generate graphic images using an O2 workstation (Silicon Graphics, Mountain View, CA) and coordinates deposited in the Brookhaven Protein Data Bank (Brookhaven National Laboratories, Upton, NY). Structure-based homology modeling of RTLs was based on the refined crystallographic coordinates of HLA-DR2 complexed with MBP peptide (DRA*0101, DRB1*1501) (25 ). Amino acid residues in the HLA-DR2 MBP-peptide complex (Protein Data Bank accession number 1BX2) were substituted with the CABL side chains, with the peptide backbone of HLA-DR2 modeled as a rigid body during structural refinement using local energy minimization.

Sequence alignment of MHC class II molecules from human, rat, and mouse species provided a starting point for our studies, as previously described (13). Structure-based homology modeling was performed using the refined crystallographic coordinates of human DR2 (25) as well as DR1 (26, 27), murine I-E^k molecules (28), and scorpion toxins (29). Because a number of amino acid residues in human DR2 (Protein Data Bank accession number 1BX2) were missing/not located in the crystallographic data (25), the correct side chains based on the sequence of DR2 were substituted in the sequence, and the peptide backbone was modeled as a rigid body during structural refinement using local energy minimization. These relatively small (200-aa) RTLs were produced in Escherichia coli in large quantities and refolded from inclusion bodies, with a final yield of purified protein between 15 and 30 mg/L of bacterial culture (14). Fig. 1 shows a schematic scale model of an MHC class II molecule on the surface of an APC (Fig. 1A). The HLA-DR2 11-derived RTL303 molecule containing covalently coupled MBP_85–99 peptide (Fig. 1B, left) and the HLA-DR2 11-derived RTL311 molecule containing covalently coupled CABL peptide (Fig. 1C, left), are shown with the color scheme used in Fig. 1A with the primary TCR contact residues colored yellow for clarity. The P2 histidyl, P3 phenylalanyl, and P5 lysyl residues derived from the MBP peptide are prominent, solvent-exposed residues. These residues are important for TCR recognition of the MBP peptide. The corresponding residues in the CABL peptide (P2 threonine, P3 glycine, P5 lysine) are also shown. Immediately striking is the percentage of surface area that is homologous across species. When colored according to electrostatic potential (EP) (Ref. 30 ; Fig. 1, B and C, middle), or according to lipophilic potential (LP) (Ref. 31 ; Fig. 1, B and C, right), subtleties between the molecules are resolved that likely play a specific role in allowing TCR recognition of Ag in the context of the DR2-derived RTL surface.

The design of the constructs allows for substitution of sequences encoding different antigenic peptides using restriction enzyme digestion and ligation of the constructs. Structural characterization using circular dichroism demonstrated that these molecules retained the anti-parallel sheet platform and antiparallel helices observed in the native class II heterodimer, and the molecules exhibited a cooperative two-state thermal unfolding transition (14). The RTLs with the covalently linked Ag-peptide showed increased stability to thermal unfolding relative to "empty" RTLs, similar to that observed for rat RT1.B RTLs (13).

Human T cell clones

DR2 and DR7 homozygous donor-derived Ag-specific T cell clones expressing a single TCR BV gene were used to evaluate the ability of Ag-specific RTLs to directly modify the behavior of T cells. Clonality was verified by TCR BV gene expression (data not shown), and each of the clones proliferated only when stimulated by specific peptide presented by autologous APC. DR2 homozygous T cell clone MR#3-1 was specific for the MBP_85–99 peptide, and DR2-homozygous clone MR#2-87 was specific for the CABL peptide. The DR7 homozygous T cell clone CP#1-15 was specific for the MBP_85–99 peptide (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. T cell clones used in this study. DR2-restricted T cell clones MR#3-1, specific for MBP85–99 peptide, and MR#2-87, specific for CABL-b3a2 peptide, and a DR7-restricted T cell clone CP#1-15 specific for MBP85–99 peptide were cultured at 50,000 cells/well with medium alone or irradiated (2,500 rad) frozen autologous PBMC (150,000/well) plus peptide-Ag (MBP85–99 or CABL, 10 µg/ml) in triplicate wells for 72 h, with [3H]thymidine incorporation for the last 18 h. Each experiment shown is representative of at least two independent experiments. Bars represent cpm ± SEM.

We examined -chain phosphorylation in the DR2-homozygous T cell clones MR#3-1 and MR#2-87. MR#3-1 is specific for the MBP_85–99 peptide carried by RTL303, and MR#2-87 is specific for the CABL peptide carried by RTL311. The antigenic peptide on the N-terminal end of the RTLs are the only difference between the two molecules. The TCR- chain is constitutively phosphorylated in resting T cells, and changes in levels of -chain phosphorylation are one of the earliest indicators of information processing through the TCR. In resting clones, was phosphorylated as a pair of phosphoprotein species of 21 and 23 kDa, termed p21 and p23, respectively. Treatment of clone MR#3-1 with 20 µM RTL303 showed a distinct change in the p23:p21 ratio that reached a minimum at 10 min (Fig. 3). This same distinct change in the p23:p21 ratio was observed for clone MR#2-87 when treated with 20 mM RTL311 (Fig. 3). Only RTLs containing the peptide for which the clones were specific induced this type of -phosphorylation, previously observed after T cell activation by antagonist ligands (32, 33).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. TCR chain phosphorylation induced by RTL treatment is Ag specific. DR2-restricted T cell clones MR#3-1 specific for MBP85–99 peptide or MR#2-87 specific for CABL-b3a2 peptide were incubated at 37°C with medium alone (control) or with 20 µM RTL303 or RTL311. A, Western blot analysis of phosphorylated shows a pair of phosphoprotein species of 21 and 23 kDa, termed p21 and p23, respectively. B, Quantification of these bands showed a distinct change in the p21:p23 ratio that peaked at 10 min. Each experiment shown is representative of at least three independent experiments. Points represent mean ± SEM.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. RTLs induce a sustained high calcium signal in T cells. Calcium levels in the DR2-restricted T cell clone MR#3-1 specific for the MBP85–99 peptide were monitored by single-cell analysis. RTL303 treatment induced a sustained high calcium signal, whereas RTL301 (identical with RTL303 except a single point mutation, F150L) showed no increase in calcium signal during the same time period. Data are representative of 2 separate experiments with at least 14 individual cells monitored in each experiment.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. ERK activity is decreased in RTL-treated T cells. DR2-restricted T cell clone MR#3-1 specific for the MBP85–99 peptide or MR#2-87 specific for CABL b3a2 peptide were incubated for 15 min at 37°C with no addition (control), and with 20 or 8 µM RTL303 or RTL311. At the end of the 15-min incubation period, cells were assayed (A) for activated, P-ERK and T–ERK. B, Quantification of activated P-ERK, presented as the fraction of the total in control (untreated) cells. Each experiment shown is representative of at least three independent experiments. Bars represent mean ± SEM.

On activation with APC plus Ag, clone MR#3-1 (MBP_85–99 specific) and MR#2-87 (CABL-specific) showed classic Th1 cytokine profiles that included IL-2 production, high IFN-, and little or no detectable IL-4 or IL-10. As is shown in Fig. 6A, activation through the CD3- chain with anti-CD3 Ab induced an initial burst of strong proliferation and production of IL-2, IFN-, and surprisingly IL-4, but no IL-10. In contrast, on treatment with RTL303, clone MR#3-1 continued production of IFN- but in addition substantially increased its production of IL-10 (Fig. 6A). IL-10 appeared within 24 h after addition of RTL303, and its production continued for >72 h, to 3 orders of magnitude above the untreated or RTL311-treated control. In contrast, IL-2 and IL-4 levels did not show RTL-induced changes (Fig. 6A). Similarly, after treatment with RTL311, clone MR#2-87 (CABL specific) also showed a dramatic increase in production of IL-10 within 24 h that continued for >72 h above the untreated or RTL303-treated control (Fig. 6B). Again, IL-2 and IL-4 levels did not show detectable RTL-induced changes, and IFN- production remained relatively constant (Fig. 6B). The switch to IL-10 production was exquisitely Ag specific, with the clones responding only to the cognate RTL carrying peptide Ag for which the clones were specific. The DR7 homozygous T cell clone CP#1-15 specific for MBP_85–99 showed no response to DR2-derived RTLs (data not shown), indicating that RTL induction of IL-10 was also MHC restricted.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Direct Ag-specific modulation of IL-10 cytokine production in T cell clones was induced by RTL treatment. DR2-restricted T cell clones MR#3-1 and MR#2-87 were cultured in medium alone (control), anti-CD3 (-CD3) mAb, or 20 µM RTL303 (303) or RTL311 (311) for 72 h. Proliferation was assessed by [3H]thymidine uptake. Cytokines (picograms per milliliter) profiles were monitored by immunoassay (ELISA) of supernatants. Each experiment shown was representative of at least three independent experiments. Bars represent mean ± SEM. Clone MR#3-1 showed initial proliferation to anti-CD3 but not to RTLs.

As anticipated, anti-CD3-pretreated T cells were strongly inhibited, exhibiting a 71% decrease in proliferation and a >95% inhibition of cytokine production, with continued IL-2R (CD25) expression (Table II; Fig. 7), a pattern consistent with classical anergy (39). Clone MR#3-1 showed a 42% inhibition of proliferation when pretreated with 20 µM RTL303, and clone MR#2-87 showed a 57% inhibition of proliferation when pretreated with 20 µM RTL311 (Table II; Fig. 7). Inhibition of proliferation was also MHC class II specific, as clone CP#1-15 (HLA-DR7-homozygous donor; MBP_85–99 specific) showed little change in proliferation after pretreatment with RTL303 or RTL311 (Table II). Clone MR#3-1 pretreated with RTL303 followed by restimulation with APC-Ag showed a 25% reduction in IL-2, a 23% reduction in IFN-, and no significant changes in IL-4 production (Fig. 7). Similarly, clone MR#2-87 showed a 33% reduction in IL-2, a 62% reduction in IFN- production, and no significant change in IL-4 production. It was of critical importance, however, that both RTL-pretreated T cell clones continued to produce IL-10 on restimulation with APC-peptide (Fig. 7).

View larger version (35K): [in this window] [in a new window]   FIGURE 7. IL-10 cytokine production induced by RTL pretreatment was maintained after stimulation with APC-peptide. T cells showed a reduced ability to proliferate and produce cytokines after anti-CD3 or RTL treatment, and the RTL effect was Ag and MHC specific. IL-10 was induced only by specific RTLs, and IL-10 production was maintained even after restimulation with APC-Ag. T cell clones were cultured at 50,000 cells/well with medium, anti-CD3, or 20 µM RTLs in triplicate for 48 h and washed once with RPMI. After the wash, irradiated (2500 rad) frozen autologous PBMC (150,000/well) plus peptide-Ag (MBP85–99 at 10 µg/ml) were added, and the cells were incubated for 72 h with [3H]thymidine added for the last 18 h. Each experiment shown is representative of at least two independent experiments. Bars represent mean ± SEM. For cytokine assays, clones were cultured with 10 µg/ml anti-CD3 or 20 µM RTL303 or RTL311 for 48 h, followed by washing with RPMI and restimulation with irradiated autologous PBMC (2500 rad; T:APC, 1:4) plus peptide-Ag (10 µg/ml) for 72 h. Cytokines (picograms per milliliter) profiles were monitored by ELISA of supernatants. Each experiment shown is representative of at least three independent experiments. Bars represent mean ± SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results presented above demonstrate clearly that the rudimentary TCR ligand embodied in the RTLs delivered signals to Th1 cells and support the hypothesis of specific engagement of RTLs with the -TCR signaling complex. Signals delivered by RTLs have different physiological consequences than those that occur after anti-CD3 Ab treatment.

In our system, anti-CD3 induced strong initial proliferation and secretion of IL-2, IFN-, and IL-4 (Fig. 6). Anti-CD3-pretreated T cells that were restimulated with APC-Ag had markedly reduced levels of proliferation and cytokine secretion, including IL-2, but retained expression of IL-2R, thus recapitulating the classical anergy pathway (Fig. 7). In contrast, direct treatment with RTLs did not induce proliferation, Th1 cytokine responses, or IL-2R expression but did strongly induce IL-10 secretion (Fig. 6). RTL pretreatment partially reduced proliferation responses and Th1 cytokine secretion but did not inhibit IL-2R expression on restimulation of the T cells with APC-Ag. Importantly, these T cells continued to secrete IL-10 (Fig. 7). Anti-CD3 treatment down-modulates TCR off the surface of the clones, whereas RTL pretreatment does not (data not shown), another clear difference between anti-CD3 and RTL treatment. Thus, it is apparent that the focused activation of T cells through Ab cross-linking of the CD3 chain had vastly different consequences than activation by RTLs, presumably through the exposed TCR surface. It is probable that interaction of the TCR with MHC-Ag involves more elements and a more complex set of signals than activation by cross-linking CD3 chains, and our results indicate that signal transduction induced by anti-CD3 Ab may not accurately portray ligand-induced activation through the TCR, suggesting that CD3 activation alone likely does not comprise a normal physiological pathway.

The signal transduction cascade downstream from the TCR is very complex. Unlike receptor tyrosine kinases, the cytoplasmic portion of the TCR lacks intrinsic catalytic activity. Instead, the induction of tyrosine phosphorylation following engagement of the TCR requires the expression of nonreceptor kinases. Both the Src (Lck and Fyn) family and the Syk/Zap-70 family of tyrosine kinases are required for normal TCR signal transduction (40). The transmembrane CD4 coreceptor interacts with the MHC class II 2 domain (10). This domain has been engineered out of the RTLs. The cytoplasmic domain of CD4 interacts strongly with the cytoplasmic tyrosine kinase Lck, which enables the CD4 molecule to participate in signal transduction. Lck contains an SH3 domain that is able to mediate protein-protein interactions (42) and that has been proposed to stabilize the formation of Lck homodimers, potentiating TCR signaling after coligation of the TCR and coreceptor CD4 (43). Previous work indicated that deletion of the Lck SH3 domain interfered with the ability of an oncogenic form of Lck to enhance IL-2 production, supporting a role for Lck in regulating cytokine gene transcription (44, 45). T cells lacking functional Lck fail to induce Zap-70 recruitment and activation, which has been implicated in downstream signaling events involving the mitogen-activated protein kinases (MAPK) ERK1 and ERK2 (46).

Although the complete molecular signal transduction circuitry remains undefined, RTLs induce rapid antagonistic effects on -chain phosphorylation and ERK activation. The intensity of the p21 and p23 forms of increased together in a nonpeptide-Ag-specific fashion (Fig. 3A), whereas the p23:p21 ratio varied in a peptide-Ag-specific manner (Fig. 3B), due to a biased decrease in the level of the p23 moiety. The antagonistic effect on ERK phosphorylation also varied in a peptide-Ag-specific manner (Fig. 5A). RTL treatment also induced marked calcium mobilization (Fig. 4). The fact that all three of these pathways were affected in an Ag-specific manner strongly implies that the RTLs are causing these effects through direct interaction with the TCR. Experiments are currently under way to quantify the association and dissociation constants of the RTLs for soluble single-chain TCR. These studies will allow us to tailor the affinity of DR2-derived RTLs for engagement with cognate -TCR.

How do we reconcile the fact that the RTLs induce calcium (Fig. 4) but reduced MAPK activation? A sustained calcium flux implies activated ras, which in turn should activate MAP kinases. This sequence is thought to occur during T cell activation. The fact that it is altered with RTL treatment and/or coupled to antagonistic -chain phosphorylation may identify a breakpoint or keypoint of regulation in this signal transduction pathway-feedback loop. Signal transduction studies are under way to investigate the role of MAPK phosphatases in our model system to determine whether they are being specifically up-regulated by RTL treatment. A plausible hypothesis would be that MAPK phosphatases are up-regulated in the absence of coreceptor and/or costimulatory input, shifting the balance toward a lower level of activated MAPK.

The most important new finding presented above is the Ag specific induction by RTLs of IL-10 secretion. This result was unexpected, given the lack of IL-10 production by the Th1 clones when stimulated by APC-Ag or by anti-CD3 Ab. Moreover, the continued secretion of IL-10 on restimulation of the RTL-pretreated clones with APC-Ag indicates that this pathway was not substantially attenuated during reactivation. This result suggests that TCR interaction with the RTL results in default IL-10 production that persists even on re-exposure to specific Ag. The elevated level of IL-10 induced in Th1 cells by RTLs has important regulatory implications for autoimmune diseases such as MS because of the known anti-inflammatory effects of this cytokine on Th1 cell and macrophage activation (47).

It is likely that the pathogenesis of MS involves autoreactive Th1 cells directed at one or more immunodominant myelin peptides, including MBP_85–99. Conceivably, RTLs such as RTL303 could induce IL-10 production by these T cells, thus neutralizing their pathogenic potential. Moreover, local production of IL-10 after Ag stimulation in the CNS could result in the inhibition of activation of bystander T cells that may be of the same or different Ag specificity, as well as macrophages that participate in demyelination. Thus, this important new finding implies a regulatory potential that extends beyond the RTL-ligated neuroantigen-specific T cell. RTL induction of IL-10 in specific T cell populations that recognize CNS Ags could potentially be used to regulate the immune system while preserving the T cell repertoire and may represent a novel strategy for therapeutic intervention of complex T cell-mediated autoimmune diseases such as MS.

	Acknowledgments

 
We thank Dr. Kim Neve for help in setting up our signal transduction studies, Kevin Hicks and David Barnes for ELISA, and Leslie Spencer for help in processing human blood.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI43960 and ES10554 (to G.G.B.), the National Multiple Sclerosis Society (to G.G.B.), the Nancy Davis Center without Walls (to G.G.B.), the Department of Veterans Affairs (to A.A.V.), and the Japan Society for the Promotion of Science (to T.Y.).

² Address correspondence and reprint requests to Dr. Gregory G. Burrows, Department of Neurology L-219, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201. E-mail address: ggb{at}ohsu.edu

³ Abbreviations used in this paper: RTL, recombinant TCR ligand; MS, multiple sclerosis; MBP, myelin basic protein; CABL, BCR-ABL b3a2 peptide (ATGFKQSSKALQRPVAS); SI, stimulation index; ERK, extracellular signal-related kinase; EP, electrostatic potential; LP, lipophilic potential; P-ERK, phosphorylated ERK; T-ERK, total cellular ERK; MAPK, mitogen-activated protein kinase.

Received for publication April 5, 2001. Accepted for publication August 13, 2001.

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