The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thomas, S.
Right arrow Articles by Brumeanu, T.-D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thomas, S.
Right arrow Articles by Brumeanu, T.-D.
The Journal of Immunology, 2003, 170: 5981-5992.
Copyright © 2003 by The American Association of Immunologists

A Model for Antigen-Specific T-Cell Anergy: Displacement of CD4-p56lck Signalosome from the Lipid Rafts by a Soluble, Dimeric Peptide-MHC Class II Chimera1

Sunil Thomas, Rajeev Kumar, Anca Preda-Pais, Sofia Casares and Teodor-D. Brumeanu2

Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Soluble, dimeric peptide-MHC chimeras were shown to induce Ag-specific T cell anergy in vitro and in vivo. In this study, we describe a mechanism by which a soluble, dimeric peptide MHC class II chimera (DEF) induces Ag-specific T cell anergy. The anergic cells showed a displacement of the CD4-p56lck signaling module from the GM1-rich plasma membrane microdomains (lipid rafts), and subsequently an increase in p59fyn kinase activity, a dominant expression of p21 inhibitory TCR {zeta}-chain, and a poor phosphorylation and recruitment of {zeta}-associated protein of 70 kDa kinase to the TCR’s immunoreceptor tyrosine-based activation motifs. The Th1 and Th2 transcription was suppressed and the cells were arrested in the Th0 stage of differentiation. Recovery from DEF anergy occurred late and spontaneously at the expense of low thresholds for activation-induced cell death. In contrast to DEF, a combination of TCR and CD4 mAbs did not induce such alterations or anergy, indicating that the ligand-mediated topology of TCR and CD4 coengagement can differentially affect the T cell function. Our results argue for a model of anergy in which the defective partitioning of signaling molecules in lipid rafts is an early, negative signaling event in T cells. Physiological ligands like DEF chimeras may provide new tools for silencing the autoimmune processes, and may also help in deciphering new mechanisms of negative regulation in T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell anergy is defined as the state of live T cells that fail to display effector functions when stimulated through the TCR and other receptors required for full activation (1). Anergy can be induced in vitro by TCR ligation in the absence of costimulation (2), partial TCR signaling by altered peptide ligands (3), or by suppressive cytokines such as IL-10 and TGF-{beta} (4). Anergy can be induced in vivo by oral administration of Ag (5), i.v. administration of a high dose of soluble peptide Ag (6) or soluble, dimeric peptide-MHC chimeras (7, 8). One early negative signaling event leading to T cell anergy is the lack of phosphorylation and recruitment of {zeta}-associated protein of 70 kDA (ZAP-70)3 kinase to the TCR’s immunoreceptor tyrosine-based activation motifs (ITAMs). ZAP-70 is critical for the recruitment of protein commuters required for downstream activation of protein kinase C and Ras-mitogen-activated protein kinase signaling pathways (9). Several negative regulatory mechanisms of ZAP-70 kinase activity have been described: 1) inhibition of ZAP-70 catalytic tyrosine residues through the introduction of negative regulatory immunoreceptor tyrosine-based inhibitory motif-like motifs by CD31 (10) and CD5 (11); 2) phosphorylation of negative regulatory tyrosines of p56lck kinase by p50csk (12); the p56lck is a major protein tyrosine kinase that mediates phosphorylation of ZAP-70 (13); 3) dephosphorylation of ZAP-70 catalytic residues by phosphatases (14); and 4) Cbl-mediated ubiquitination and degradation of ZAP-70 protein (15). In this study, we describe a mechanism by which poor phosphorylation and recruitment of ZAP-70 to the TCR occurs in the context of p56lck sequestration in the nonlipid rafts of plasma membrane in T cells.

It has been demonstrated that the GM1 glycosphingolipid-rich microdomains of plasma membrane (lipid rafts) are foci of signal transduction (16). Various receptors and signaling molecules are differently distributed in the lipid rafts of resting T cells. For example, the CD4 coreceptor and its associated p56lck kinase are mostly located in the lipid rafts (17, 18), whereas the CD3 {epsilon}-chain and TCR reside mostly in the nonlipid rafts (19). In contrast, the TCR {zeta}-chain is recruited in the lipid rafts of resting T cells (20). During the T cell activation subsequent to the formation of immunological synapse as mediated by TCR interaction with the MHC-peptide complexes on APCs (21), various protein receptors like CD2, CD3, CD28, and TCR (22), and protein tyrosine kinases like p56lck, p59fyn, and phosphatidylinositol 3-kinase (23, 24, 25), as well as protein commuters like linker for activation of T cells and Grb2 (25, 26), are concentrated in the lipid rafts. Meanwhile, protein phosphatases with a negative regulatory effect on TCR signaling like Src homology protein-1 are recruited into the lipid rafts, while others like CD45 extracellular phosphatase are excluded (16). In this study, we found that the sole interaction of a soluble, dimeric peptide-MHC-II ligand (DEF) with the TCR and CD4 coreceptor on resting CD4 T cells at high receptor occupancy drastically altered the topology of the CD4-p56lck signalosome in the plasma membrane microdomains leading to T cell anergy.

Soluble, dimeric peptide-MHC chimeras built on an Ig scaffold were shown to induce Ag-specific T cell anergy in vitro and in vivo (Refs. 7, 8, 27 and 28). These chimeric molecules are physiological ligands genetically engineered to recognize the peptide-specific TCR and CD4 coreceptor on T cells. We have engineered the first soluble, dimeric (HA110–120)-I-Ed chimera (DEF) on an IgG2a scaffold (29), and showed that at low TCR and CD4 occupancy, DEF induces hemagglutinin (HA)-specific Th2 cell differentiation by a negative regulation of STAT4 phosphorylation (30), while at high receptor occupancy induces HA-specific T cell unresponsiveness by a blockade of CD3/TCR signaling (28). In this study, we found that the signaling blockade occurs due to the displacement of the CD4-p56lck signalosome from the GM1-rich microdomains of the T cell plasma membrane, followed by poor activation of ZAP-70 kinase with subsequent suppression of Th1 and Th2 transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Naive CD4 T cells were negatively sorted on mouse CD4 column kits (R&D Systems, Minneapolis, MN) from the spleen of nonimmunized HA-specific TCR transgenic mice on a BALB/c background (H-2d). The HA-specific CD4 T cells express the 14.3d TCR that recognizes the HA110–120 immunodominant epitope of PR8/A/34 influenza virus in context of I-Ed class II molecules (31).

TCR and CD4 ligands

The soluble dimeric peptide-MHC-II chimera (DEF) is a physiological ligand for the TCR and CD4 coreceptor, which consists of the I-Ed{alpha} and I-Ed{beta} extracellular domains stabilized through a murine Fc{gamma}2a fragment at the C termini of I-Ed{beta} chain, and expresses the HA110–120 immunodominant CD4 epitope of HA of PR8 influenza virus covalently linked to the N terminus of I-Ed{beta} chains (29). Negatively sorted, CD4 TCR-HA T cells were incubated with DEF for 4 h at 37°C at high TCR and CD4 receptor occupancy (50 µg/ml/106 cells). The CD4 (GK1.5) mAb is a rat IgG2a/k and the TCR V{beta}8 (F23) mAb is a mouse IgG1/k obtained by affinity chromatography from hybridoma-secreting cells. The removal of DEF and Ab ligands bound to the cells before carrying out the assays was achieved by washing the cells for 10 min with 0.1 M sodium acetate buffer, pH 4.5, on ice followed by washing with PBS.

[3H]Thymidine incorporation ([3H]TdR) and cytokine assays

Spleen cells (106) from TCR-HA Tg mice were exposed to the ligands at high TCR and CD4 occupancy (50 µg ligand/ml/106 cells) for 4 h, the ligands were removed, and the HA110–120 peptide (10 µg/ml) was added to the cultures for 3 days, in the presence or absence of rIL-2 (20 IU/ml/106 cells). The tritiated thymidine (1 µCi/well) was added for the last 24 h, cells were harvested on Skatron filter paper (Sterling, VA), and the cpm was measured in a {beta}-scintillation chamber. The production of IL-2, IFN-{gamma}, and IL-4 in the cell culture supernatants was measured in the second and third day of proliferation by ELISA kits according to the manufacturer’s instructions (BioSource International, Camarillo, CA).

Cytofluorometric analyses (FACS)

To determine the number of cell divisions, negatively sorted CD4 TCR-HA T cells were first incubated with 50 µM CFSE (Sigma-Aldrich, St. Louis, MO) for 2 h in RPMI 1640 medium supplemented with 10% FCS, cells were washed, and then treated with ligands as described. Some 2 x 105 cells were rested for 8 and 24 h in ligand-free medium, and the CFSE fluorescence intensity among 20,000 cells was acquired among gated HA-specific T cells (stained with 6.5.2 mAb-PE conjugate) by an Epics Profile Analyzer II cytofluorometer (Corixa, Hialeah, FL). To determine the annexin V expression on cells treated or not with ligands, the cells (2 x 105) were made free of ligands as described, resuspended in 1% BSA/PBS/0.05% NaN3, and doubly stained for 30 min on ice with annexin V mAb-PE (BD PharMingen, San Diego, CA) and 6.5.2 clonotypic mAb-FITC conjugates. Cells were washed with 1% BSA/PBS/0.05% NaN3, fixed in 1% paraformaldehyde, and 20,000 events were acquired by a FACS analyzer. To determine the expression level of HA-specific TCR and CD4 on T cells treated or not with ligands, cells were rested for 2 or 18 h in ligand-free medium, washed, and incubated or not with 50 µM cycloheximide, and then doubly stained with 6.5.2 mAb-FITC and GK1.5 mAb-PE conjugates (BD PharMingen). The signal-to-noise background was subtracted from each sample stained with FITC- and PE-labeled isotope control Abs.

Western blot analyses

Negatively sorted, splenic CD4 TCR-HA Tg T cells (20 x 106) incubated with ligands and then rested in ligand-free medium for 2, 12, or 24 h, were washed and lysed on ice in native conditions in the presence of a mixture of protease inhibitors (Roche Diagnostic, Indianapolis, IN). The total cell lysates were cleared by centrifugation for 10 min at 300 x g and the cell membranes were isolated by ultracentrifugation for 120 min at 120,000 x g. The membrane pellets were solubilized in native lysis buffer and the protein extracts were cleared for 2 h at room temperature with protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) bound to isotype control Abs. Some 50 µg of the membrane protein extracts were precipitated for 24 h at 4°C with 10 µg of specific Abs (GK1.5 and F23 mAbs). Primary Abs used in Western blot analyses for the detection of p56lck, p59fyn, and ZAP-70 were from Upstate Biotechnology (Lake Placid, NY). The immunoprecipitates were captured on protein A/G-agarose gel, washed two times with lysis buffer and once with PBS, and boiled for 5 min in electrophoresis buffer containing 0.1% SDS and 5% 2-ME. To detect STAT5, the lysates were precipitated with a rabbit STAT5 Ab (Santa Cruz Biotechnology) and separated by SDS-PAGE in 12% homogenous PhastGels (Amersham Pharmacia Biotech, Piscataway, NJ). To isolate the proteins bound to the TCR {zeta}-chain, the protein extracts from the plasma membrane were precipitated with a TCR {zeta}-chain mAb covalently coupled to agarose (Santa Cruz Biotechnology), the Ab precipitate was separated by SDS-PAGE in 8–25% gradient PhastGels, and electrotransferred onto polyvinylidene difluoride (PVDF) membranes in semidry conditions. The PVDF membranes were blocked with 3% BSA/PBS, and developed with RC20 anti-phosphotyrosine-HRP conjugate (BD Transduction Laboratories, Lexington, KY). To confirm the identity of phosphorylated proteins, the PVDF membranes were stripped off for 20 min with 0.1 M glycine/HCl, pH 2.8, blocked again with 3% BSA/PBS, and reprobed with specific Abs followed by incubation for 2 h with protein A-HRP conjugate (1/20,000 dilution; Sigma-Aldrich). The HRP chemiluminescent activity was measured on PVDF membranes by a Lumi-Light Western Blot Substrate kit according to the manufacturer’s instructions (Roche Diagnostic Systems). For the detection of GM1 glycosphingolipid, individual sucrose gradient fractions (5 µl per fraction) from negatively sorted CD4 TCR-HA T cells treated or not with ligands were diluted four times in Laemmli sample buffer and boiled for 5 min. Proteins were separated in 8–16% gradient polyacrylamide gels (Bio-Rad, Hercules, CA) at a 30 mA constant current. The gels were transferred to PVDF membranes (0.45 µm) at 1.5 mA/cm2 for 2 h and blocked overnight at 4°C with PBS-3% BSA. The PVDF membranes were incubated for 1 h at 37°C with cholera toxin subunit B (CTB)-HRP conjugate (1 µg/ml in PBS-1% BSA; Sigma-Aldrich), washed with PBS/0.05% Tween 20, incubated with HRP chemiluminescent substrate, and exposed onto X-OMAT Kodak films (Rochester, NY).

RT-PCR

Total RNA was isolated from 3 x 106 negatively sorted CD4 TCR-HA T cells treated or not with ligands, and rested in ligand-free medium for 2, 12, and 24 h, using a QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA) as recommended by the manufacturer. One microgram of total RNA was used to prepare first-strand cDNA using a Qiagen One Step RT-PCR kit following the manufacturer’s protocol. The specific primers for each cDNA construct are shown in Table I. A 1/10 dilution of the RT-PCR product was subjected to electrophoresis in 1.5% agarose gel and the percent of transcripts was calculated by Scion Image software analysis (Frederick, MD), based on the integration of the percent of pixels and band intensity after normalization against the corresponding {beta}-actin band in each sample.


View this table:
[in this window]
[in a new window]
  		Table I. Primers used for RT-PCR

 
Isolation of T cell plasma membrane microdomains

The plasma membrane microdomains from negatively sorted CD4 TCR-HA T cells treated or not with ligands, and rested in ligand-free medium for 2, 12, and 24 h were isolated following a modified protocol described by Cottin et al. (32). Cells (200 x 106) were washed, lysed by sonication on ice (five times of 10 s per cycle at 100 W), and centrifuged for 5 min at 800 x g. The supernatant was incubated for 1 h at 4°C with 1% Brij 58 detergent in buffer A (25 mM Tris, 150 mM NaCl, 5 mM EDTA supplemented with a mixture of protease inhibitors (Complete; Roche Diagnostic). The Brij 58 detergent preserves the association of TCR with the lipid rafts, while solubilizing the lipoglycoproteins from the plasma membrane microdomains. Some 1 ml of each cell lysates was mixed with 1 ml of 80% sucrose made in buffer A, placed at the bottom of ultracentrifuge tube, and then layered with a step gradient of 8 ml of 35% sucrose and 2 ml of 5% sucrose solution made in buffer A. Samples were centrifuged for 18 h and 4°C at 38,000 rpm in a SW41 rotor (Beckman Instruments, Fullerton, CA). Nine fractions of 1 ml each were collected from the top and dialyzed in 0.0015 M saline using Spectrapor dialysis bags (molecular mass cut-off of 1,000 kDa; Sigma-Aldrich). Fractions were concentrated by speed vacuum to 300 µl and kept frozen at -80°C until used.

ELISA

To measure the content of GM1 glycosphingolipid in the sucrose gradient fractions from negatively sorted, CD4 TCR-HA T cells treated or not with ligands and rested in ligand-free medium for 2, 12, and 24 h, some 200 µl were coated overnight at 4°C on Polysorp plates (Starwell plates; Nunc, Roskilde, Denmark) at a 1/1000 dilution in PBS buffer. The plates were blocked for 2 h at 37°C with PBS/3% BSA and incubated for 1 h at 37°C with CTB-HRP 1 µg/ml in PBS/1% BSA, then washed with PBS/0.05% Tween 20, and incubated for 15 min with 200 µl of HRP chromogen substrate (BioSource International). The reaction was stopped with 25 µl of 2 M sulfuric acid, and the OD450 was measured in a Powerwave X ELISA reader using K4 analysis software (Bio-Tek Instruments, Winooski, VT). To measure the content of TCR, CD4, p56lck, and p59fyn in the sucrose gradient fractions, a 1/10 dilution from each fraction in 0.1 M carbonate buffer, pH 9.8, was used to coat Maxisorp plates (Starwell plates; Nunc) overnight at 4°C. Plates were blocked for 2 h at 37°C with PBS-3% BSA, and then incubated overnight at 4°C with 10 µg/ml specific Abs in PBS/1% BSA. Plates were washed with PBS/0.05% Tween 20, incubated for 2 h at room temperature with protein A-HRP in PBS/1% BSA, then washed with PBS/0.05% Tween 20, and incubated for 30 min in the dark with 200 µl of chromogen substrate (BioSource International). The reaction was stopped with 25 µl of 2 M sulfuric acid, and the OD450 was measured.

EMSA

Nuclear cell lysates (7 µg of protein content) from negatively sorted, splenic CD4 T cells (20 x 106) of TCR-HA Tg mice pre-exposed to soluble ligands, and rested in ligand-free medium for 2, 12, and 24 h, were incubated for 10 min at room temperature in 20 mM HEPES, 83.3 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1.9 mM MgCl2, 0.5 mM DTT, 0.01% Triton X-100, 25 µg/ml poly(dI-dC) containing 1 ng of 32P-labeled (200,000 cpm) double-strand oligonucleotide consensus motifs for c-Myb, 5'-TACAGGCATAACGGTTCCGTAGTGA-3' (33); AP-1, 5'-TGACTCA3-'; NF-AT, 5'-GAGGAAAATTTG-3'; GATA, 5'-CACTTGATAACAGAAAGTGATAACTCT-3' (Santa Cruz Biotechnology), and cMaf, 5'-CTCATTTTCCCTTGGTTTCAGCAACTTTAACTC-3' corresponding to the -59 to -27 sequence of mouse IL-4 gene promoter (34). The binding specificity of transcription factors was determined by the ability of cold oligonucleotides to inhibit the binding of 32P-labeled oligonucleotide consensus motifs at a 1:50 ratio, as well as by their inability to bind the corresponding single base-mutated oligonucleotides. The reaction mixtures were separated in 4% nondenaturing polyacrylamide gels for 90 min at 200 V, and the protein-32P-labeled oligonucleotide complexes were visualized in dried gels exposed onto X-OMAT film at -80°C.

Confocal laser scanning microscopy (CLSM)

Negatively sorted, CD4 T cells were treated with ligands as described, rested in ligand-free medium for 2, 12, 24 h, and then resuspended in 1% BSA/PBS containing 0.05% sodium azide, stained for 30 min at 4°C with 10 µg/ml CTB-FITC conjugate, washed again, and permeabilized in CytoPerm solution (BD PharMingen). Permeabilized cells were stained for 30 min on ice with a rabbit anti-p56lck IgG (Molecular Probes, Eugene, OR), washed, and bound p56lck Abs were revealed after a 30-min incubation on ice with an anti-rabbit IgG-Alexa 594 conjugate (Molecular Probes). Cells were washed, fixed in 1% paraformaldehyde, centrifuged for 5 min on glass covers at 1200 rpm, mounted in Vectashield medium (Vector Laboratories, Burlingame, CA), and sealed with Permount solution (Fisher Scientific, Springfield, NJ). Several 0.5 µm cross-sections were analyzed by four-color CLSM in an inverted Leica microscope (Deerfield, IL) equipped with fluorescence filters for double excitation at {lambda} = 488 and 594 nm.

Phosphotyrosine kinase (PTK) assay

The in vitro protein tyrosine kinase assay was conducted in GM1high and GM1low pooled, sucrose gradient fractions from negatively sorted CD4 TCR-HA T cells treated or not with ligands, and rested in ligand-free medium for 2, 12, and 24 h. The kinase substrate was rabbit muscle enolase (Sigma-Aldrich), and the assays were conducted according to a modified protocol described by Feder and Bishop (35). Commercial rabbit muscle enolase (Sigma-Aldrich) was dialyzed against 10,000 volumes of distilled water to remove the ammonium sulfate. Prior the assay, the enolase was denatured by incubation for 10 min at 37°C with an equal volume of 100 mM acetic acid, followed by the addition of half volume of 1 M HEPES buffer, pH 7.4. GM1high and GM1low pooled fractions were precipitated with GK1.5 or 6.5.2 mAbs and the immunoprecipitates were resuspended in 50 µl of kinase buffer (50 mM Tris, pH 8, 10 mM MgCl2, 10 mM MnCl2, 1 µl of 10 µCi [{gamma}-32P]ATP, and 10 µg of acid-denatured enolase). The mixture was incubated for 5 min at 37°C and the reaction was terminated by the addition of 20 µl of 2-fold concentrated Laemmli sample buffer. The samples were boiled for 5 min and the phosphorylation products were separated by SDS-PAGE in 12.5% polyacrylamide gels. The gels were dried and exposed onto X-OMAT Kodak films at -80°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-linking of TCR and CD4 coreceptor by soluble DEF dimer at high receptor occupancy leads to Ag-specific T cell anergy

We had previously shown that the cross-linking of TCR and CD4 on resting, HA-specific CD4 T cells by a soluble dimeric HA110–120/MHC-II chimera (DEF) at low receptor occupancy (0.5–5 µg/ml/106 cells) leads to type 2 cell differentiation by a negative regulation of STAT4 signaling (30). In contrast, the incubation of resting HA-specific CD4 T cells for 4 h with this soluble DEF chimera at high receptor occupancy (50 µg/ml/106 cells) induced T cell unresponsiveness. The unresponsive cells did not proliferate and did not produce cytokines upon restimulation with HA-pulsed APCs or with plastic-immobilized 2C11 mAb (2 µg/ml) plus anti-CD28 (7D4 mAb, 0.5 µg/ml). Under the same conditions, a soluble anti-TCR V{beta}8 (F23 mAb), or anti-CD4 (GK1.5 mAb), or their equimolar combination did not induce T cell unresponsiveness (Fig. 1A). This indicated that the topology of TCR and CD4 coengagement is an important parameter that can differentially affect T cell function.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1. Induction of T cell anergy by soluble, dimeric DEF ligand. A, Proliferation and cytokine production of CD4 TCR-HA T cells exposed to DEF, or to TCR and CD4 mAb ligands at high receptor occupancy. After removal of ligands, the cells were restimulated for 3 days with HA110–120 peptide in the absence or presence of exogenous IL-2. Cell proliferation was measured by [3H]TdR in triplicate wells. Cytokine production was measured by ELISA in the cell culture supernatants after 2 days of proliferation. The mean values (picograms per milliliter) were calculated in duplicate wells (±SD for IL-2, 10.5 pg/ml; for IL-4, 14.8 pg/ml; and for IFN-{gamma}, 28 pg/ml). B, Lack of cell division of DEF anergic T cells. CFSE-labeled cells were exposed to the ligands as described, and 8 and 24 h after resting in ligand-free medium, the cells were stained with 6.5.2 clonotypic mAb-PE conjugate. The CFSE fluorescence of gated 6.5.2-positive cells was determined by FACS. Top right panel, The sequential halving CFSE concentrations corresponding to the number of cell cycle divisions. The positive control was CFSE-labeled cells stimulated with plastic-immobilized 2C11 mAb plus soluble CD28 mAb (right panels). C, Induction of complete anergy by DEF is time-dependent. The CD4 TCR-HA T cells were exposed for various intervals of time to DEF at high receptor occupancy, DEF was removed, the cells were restimulated with HA-pulsed APCs for 3 days, and the thymidine incorporation was determined as described. {diamond}, The Con A proliferative response in aliquot samples. D, DEF anergy does not affect STAT5 signaling. DEF anergic T cells and those pre-exposed or not to TCR/CD4 mAb ligands at high receptor occupancy were sorted on 6.5.2-mAb beads, stimulated or not on beads with rIL-2, then lysed on beads, and precipitated with STAT5 Ab. STAT5 phosphorylation was determined by Western blot using RC 20 anti-pTyr-HRP conjugate (upper panels). The identity of STAT5 was confirmed on stripped-off membranes reprobed with rabbit STAT5 Ab (lower panels). The results of one of the two experiments conducted are shown.

 
FACS analysis of CFSE-labeled DEF unresponsive cells showed a lack of cell division 8 and 24 h after resting the cells in ligand-free medium, while a small population among the cells pre-exposed to TCR/CD4 mAb ligands entered a single cycle of cell division. In contrast, resting CD4 TCR-HA T cells stimulated with plastic-immobilized CD3{epsilon} mAb plus anti-CD28 mAbs showed two cycles of division within 24 h (Fig. 1B). DEF unresponsiveness was restored upon the addition of rIL-2 (100 IU/ml/106 cells) to the culture, suggesting that the state of unresponsiveness was the result of anergy. A complete state of anergy could not be induced by incubation of cells for 30 min with DEF at high receptor occupancy, while incubation for 60 min reduced the index of HA responsiveness only by 50%, indicating that DEF anergy occurred by a cumulative process of negative signaling events (Fig. 1C).

DEF anergy was Ag specific because restimulation of naive CD4 T cells from the spleen of BALB/c mice (previously exposed to DEF for 4 h in vitro at high receptor occupancy) with plastic-immobilized CD3{epsilon} mAb plus CD28 mAb showed indexes of proliferation (52,450 ± 2,200 cpm) and cytokine production (IL-2, 1,150 ± 12; IL-4, 275 ± 7.8; IFN-{gamma}, 830 ± 14 pg/ml) similar to the control cells (53,240 ± 1,870 cpm; IL-2, 1,014 ± 17.5; IL-4, 258 ± 12.5; IFN-{gamma}, 865 ± 5.6 pg/ml).

DEF anergy does not affect the IL-2R-mediated STAT5 signaling pathway

Because the addition of IL-2 reversed the DEF anergy, we investigated the status of IL-2R-mediated STAT5 signaling. Phosphorylation-mediated activation of STAT5 through Janus kinase 3 subsequent to ligation of IL-2R{beta} by IL-2 leads to STAT5 recruitment to the IL-2R followed by STAT5 dimerization and translocation of STAT5 dimers into the nucleus where they bind to and activate several gene promoters, among which is the IL-2 promoter (36). DEF anergic T cells and those pre-exposed to TCR/CD4 mAb ligands for 4 h at high receptor occupancy were washed, and cells were incubated for 30 min with rIL-2 (20 IU/ml/106 cells) in ligand-free medium. The HA-specific CD4 T cells were sorted on beads coated with 6.5.2 mAb. The 6.5.2 clonotypic mAb recognizes the HA-specific (14.3d) TCR, and it does not stimulate the cells (31). The HA-specific CD4 T cells captured on 6.5.2-mAb beads were lysed on the beads under native conditions and STAT5 tyrosine phosphorylation was analyzed by Western blot. Stimulation with rIL-2 induced tyrosine phosphorylation of STAT5 in DEF anergic cells as well in those pre-exposed to mAb ligands, indicating that IL-2R-mediated STAT5 signaling was not affected by DEF anergy (Fig. 1D).

DEF anergic T cells do not enter apoptosis

Strong stimulation of resting CD4 T cells through the TCR alone leads to activation-induced apoptosis in vitro (37). Apoptosis did not occur in DEF anergic T cells, because annexin V, an early marker for the expression of phosphatidylserine on the cell surface (long before DNA fragmentation) was present in <8% of these cells as compared with those pre-exposed for 4 h to TCR/CD4 mAb ligands at high receptor occupancy (12.36%; Fig. 2A). Lack of apoptosis was consistent with the results showing reversal of anergy by restimulation with Ag in the presence of IL-2. It had been previously shown that Bcl-2, and in particular Bcl-xL mRNA expression is increased in long-term surviving cells such as Th1 memory cells (38). Semiquantitative RT-PCR showed higher Bcl-2 and Bcl-xL mRNA levels in DEF anergic cells than in those pre-exposed to TCR/CD4 mAbs or in control cells, whereas the mRNA levels of Bad and Bax antagonists were lower in DEF anergic cells (Fig. 2B). Also, 12 h after cessation of DEF anergic stimulus, the c-Myb mRNA and its protein expression were increased (Fig. 2C, upper left panel). Western blot analysis detected two elevated c-Myb isoforms in DEF anergic cells (81 and 78 kDa), and only one isoform of 78 kDa in cells pre-exposed to TCR/CD4 mAbs. Both c-Myb isoforms bound to their oligonucleotide consensus motif, as indicated by EMSA (Fig. 2C, upper right panel). The results showed that the Bcl-2 and Bcl-xL antiapoptotic signals were up-regulated by the sole interaction of the TCR and CD4 coreceptor with the DEF ligand, but not with TCR/CD4 mAb ligands. Also, the c-Myb antiapoptotic protein was differentially up-regulated, depending on the nature of ligand-mediated TCR and CD4 coligation, i.e., DEF vs TCR/CD4 mAb ligands.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 2. Lack of apoptosis, TCR, and CD4 down-modulation in DEF anergic T cells. The results of one of the two experiments conducted are shown. A, DEF anergic T cells or pre-exposed to TCR/CD4 mAb ligands as described were doubly stained with Annexin VPE and 6.5.2 clonotypic mAb-FITC conjugates, and the percent of HA-specific T cells expressing annexin V (upper right quadrants) was determined by FACS among 20,000 events. B, Semiquantitative RT-PCR of the apoptotic and antiapoptotic messages in DEF anergic T cells. The amount of Bcl-2, Bcl-xL, Bad, and Bax transcripts was expressed as percentage of pixels integrating both surface area and intensity for each band, and normalized against the corresponding {beta}-actin transcript in each sample, using Scion Image software version 4.1. Values for each amplicon (lateral tables) and the size in base pairs are indicated. C, Analysis of c-Myb expression in DEF anergic T cells. Negatively sorted, CD4 TCR-HA T cells rendered anergic by DEF dimer or pre-exposed to TCR/CD4 mAb ligands were washed and rested 12 h in ligand-free medium. Upper left panel, The Western blot analysis of c-Myb in the cell lysates. Upper right panel, The direct binding of c-Myb to its 32P-labeled oligonucleotide consensus motif, and the cold inhibition, as determined by EMSA in the total cell lysates. Arrows indicate the molecular size of c-Myb isoforms and c-Myb-oligonucleotide complexes, respectively. Lower panel, semiquantitative RT-PCR of c-Myb transcript was determined by Scion Image software, as described. An example of the integration of the percent of pixels corresponding to the surface area and intensity of the band is illustrated on the top of this panel. D, TCR and CD4 expression on DEF anergic T cells was determined by FACS analysis using 6.5.2 clonotypic mAb-FITC and CD4 (GK1.5) mAb-PE conjugates on CD4 T cells treated or not with the ligands, then rested for 18 h in ligand-free medium, washed, and incubated or not with cycloheximide. The percent of TCR and CD4 expression was calculated after subtracting the signal-to-noise background from aliquot cells stained with isotope control conjugates.

 
TCR and CD4 down-modulation does not play a role in induction of anergy by DEF

TCR or CD4 down-modulation triggered by strong and continuous cross-linking, leads to receptor unavailability on the cell surface for a subsequent challenge with Ag. The extent to which TCR-HA and CD4 down-regulation may have played in induction of DEF anergy was measured in rested cells for 2 h in ligand-free medium by FACS using 6.5.2 clonotypic mAb-FITC and GK1.5 mAb-PE conjugates. There was no significant difference in TCR-HA and CD4 expression on DEF anergic T cells (16.5 and 84% fluorescence intensity, respectively) as compared with control cells (17.2 and 88.5% fluorescence intensity, respectively) as determined 12 h after resting the cells in ligand-free medium (Fig. 2D).

To determine a possible compensatory role of de novo synthesis for the internalization of the TCR and CD4 coreceptor, we measured, by FACS, TCR and CD4 expression 16 h after resting the cells in ligand-free medium containing cycloheximide. Although DEF anergic cells showed a decrease in TCR and CD4 expression by 37.5 and 9.5%, respectively, as compared with control cells, those pre-exposed to TCR/CD4 mAbs showed a decrease by 71.8 and 43%, respectively (Fig. 2D). The results indicated that TCR or CD4 down-regulation in DEF anergic cells was not as high as in cells pre-exposed to TCR/CD4 mAbs (not rendered anergic) and, thereby, it was not crucial for induction of anergy by DEF.

DEF anergic T cells display unique CD4 and p56lck partitioning in the plasma membrane microdomains

GM1 is the major glycosphingolipid of detergent-resistant microdomains (lipid rafts) in the murine T cell plasma membrane and it is a marker for identification of lipid rafts in plasma membrane (24). We have isolated by sucrose gradient centrifugation nine fractions containing detergent-insoluble and detergent-soluble plasma microdomains from DEF anergic T cells, and from cells pre-exposed for 4 h to TCR/CD4 mAb ligands at high receptor occupancy 2, 12, and 24 h after resting the cells in ligand-free medium.

The sucrose gradient fractions were analyzed for the amount and distribution of TCR, CD4 coreceptor, p56lck, and p59fyn kinases in relation to the GM1 glycosphingolipid. Western blot analysis showed GM1 accumulation mostly in detergent-insoluble fractions 1 and 2 from DEF anergic T cells. A small amount of GM1 was also detected in detergent-soluble fractions 5–8 of DEF anergic T cells, but not in those pre-exposed to TCR/CD4 mAb ligands (Fig. 3A). In contrast, GM1 was distributed exclusively in detergent-insoluble fractions 1–4 from control cells. According to these results, we have designated fractions 1–4 as GM1high (detergent-insoluble microdomains, lipid rafts), and fractions 5–8 as GM1low (detergent-soluble microdomains).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. Redistribution of CD4-p56lck signaling modules in the plasma membrane microdomains in DEF anergic T cells. The results of one of the three identical experiments are shown. A, Distribution of GM1 glycosphingolipid in the plasma membrane microdomains isolated by sucrose gradient centrifugation (200 x 106 cells for each sample) was determined by Western blot using CTB-HRP conjugate, in the DEF anergic T cells or cells pre-exposed to TCR/CD4 mAb ligands 2 h after resting the cells in ligand-free medium. Fractions 1–4 were designated as GM1high (detergent-insoluble microdomains, lipid rafts), and fractions 5–8 as GM1low (detergent-soluble microdomains). B, Distribution of CD4 and TCR in the plasma membrane microdomains isolated by sucrose gradient centrifugation from DEF anergic T cells or cells pre-exposed to TCR/CD4 mAb ligands (200 x 106 cells) was determined by ELISA. C, Distribution of p56lck and p59fyn kinases in the plasma membrane microdomains isolated by sucrose gradient centrifugation from DEF anergic T cells or cells pre-exposed to TCR/CD4 mAb ligands (200 x 106 cells), as determined by ELISA. D, CLSM analysis of p56lck localization in the GM1 lipid rafts of DEF anergic T cells or cells pre-exposed to TCR/CD4 mAb ligands. Cells were rested for 2 h in ligand-free medium and prepared for CLSM as described. About 35 of 50 cells scored by CLSM (0.5 µm per cross-section) accumulated p56lck kinase (red color) in the plasma membrane (magnification, x400), and lacked colocalization of p56lck with GM1 (green color) in DEF anergic T cells (framed area at magnification x800). In contrast, the number of cells treated with mAb ligands that accumulated p56lck was considerably lower as compared with control cells (5 of 50 cells), but instead, they showed higher extent of colocalization with GM1 lipid rafts (41 of 50 cells). Nuclei were stained with 4',6'-diamidino-2-phenylindole (blue color).

 
Resting CD4 T cells contain >90% of the TCR and CD3{epsilon} chain in the nonlipid rafts due to their lack of palmitylation, whereas the CD4 coreceptor is palmitylated and resides mostly in the lipid rafts. ELISA showed that the amount of TCR in GM1high fractions was slightly increased in DEF anergic T cells, whereas in cells pre-exposed to TCR/CD4 mAb ligands was highly increased as compared with control cells (Fig. 3B). In contrast to the cells pre-exposed to TCR/CD4 mAb ligands, the DEF anergic cells showed significantly higher amount of CD4 in GM1low fractions. These alterations were detected 12 h after resting the anergic T cells in DEF-free medium, but they were not found in cells pre-exposed to TCR/CD4 mAbs, indicating that: 1) TCR and CD4 partitioning in the plasma membrane microdomains can be differentially altered depending on the nature of coligation, and 2) DEF-mediated displacement of CD4 from the lipid rafts correlated with the state of T cell anergy.

Displacement of the CD4-p56lck signalosome from the lipid rafts correlates with alteration of PTK activity and early TCR signaling

The p56lck and p59fyn Src kinases are critical for T cell activation. The p56lck mediates phosphorylation and recruitment of ZAP-70 kinase to the TCR’s ITAM motifs (13), while the increase in p59fyn kinase activity was associated with the anergic status of CD4 T cells (39, 40). The p56lck kinase, as well as CD4, is palmitylated in resting CD4 T cells (41) and thereby recruited to the lipid rafts where it becomes associated with the CD4 coreceptor. The p59fyn kinase is also palmitylated and is almost 70% recruited in the lipid rafts of resting CD4 T cells (42).

Two, 12, and 24 hours after resting the cells in ligand-free medium, we investigated the extent to which the displacement of the CD4 coreceptor from the plasma membrane lipid rafts of DEF anergic cells could affect the distribution and activity of p56lck, p59fyn, and ZAP-70 kinases. Like the CD4 coreceptor, p56lck kinase was displaced from the GM1high to the GM1low moieties in DEF anergic T cells, while the partitioning of p59fyn kinase remained unchanged for at least 12 h, as compared with control cells (Fig. 3C). CLSM analysis showed an accumulation of p56lck that did not colocalize with the GM1 microdomains in the plasma membrane of DEF anergic cells as compared with those pre-exposed to TCR/CD4 mAbs (Fig. 3D). This indicated that p56lck redistribution in the nonlipid rafts followed the pattern of CD4 redistribution in DEF anergic T cells. These alterations persisted only for 12 h after resting the cells in ligand-free medium.

We next compared the PTK activity in the CD4 and TCR immunoprecipitates prepared from the pool of sucrose gradient fractions 1–4 (GM1high) and 5–8 (GM1low). The results of Western blot analysis in TCR and CD4 recovered from immunoprecipitates correlated with the ELISA data on the distribution of these two receptors in the GM1high and GM1low moieties. Thus, the amount of CD4 immunoprecipitated in GM1high fractions from DEF anergic cells was 4.5 times less than in control cells, whereas in GM1low fractions was 3 times higher than in control cells, as determined by Western blot with specific Abs and computer integration by Scion Image software. In contrast, the amount of TCR immunoprecipitated in GM1high fractions from DEF anergic cells was slightly higher (1.5 times) than in control cells, whereas in GM1low fractions was roughly the same as in control cells (0.5 times lower). In contrast, the amount of CD4 immunoprecipitated in GM1high fractions from cells treated with CD4 and TCR Abs was twice as high as in control cells and almost the same in GM1low fractions, whereas the amount of TCR immunoprecipitated in GM1high fractions was 2.5 times higher than in control cells, and in GM1low fractions was 2 times lower than in control cells (data not shown). For all the experiments of PTK-receptor associated PTK activity, as well as for those on protein tyrosine phosphatase (PTP) activity, samples were normalized with respect to the protein content.

The PTK activity in CD4 immunoprecipitates from DEF anergic cells was significantly higher in the GM1low pool, whereas in cells pre-exposed to TCR/CD4 mAbs was higher in the GM1high than in the GM1low pool. The control cells showed poor PTK activity in the GM1high pool, and lack of activity in the GM1low pool (Fig. 4A). A phosphorylated band of 56 kDa identified by Western blot as p56lck was always associated with the kinase activity in the GM1low pool from DEF anergic cells, but not in those pre-exposed to TCR/CD4 mAbs or in control cells. The presence of tyrosine phosphorylated p56lck in this in vitro kinase assay was consistent with the findings reported by Bernadetta et al. (43), and it was attributed to the ability of p56lck to autophosphorylate. In addition, DEF anergic cells showed an extra band of 60 kDa in the GM1high pool identified by Western blot as p56lck, which was not tyrosine-phosphorylated as determined by Western blot (Fig. 4B), but was most likely serine-phosphorylated as previously described by Feder and Bishop (35). We also found a lack of p59fyn phosphorylation and p59fyn-related PTK activity in the CD4 immunoprecipitates of GM1high and GM1low pools from either DEF anergic cells, cells pre-exposed to TCR/CD4 mAbs, or control cells. These results indicated first that the PTK activity on the CD4 coreceptor was mostly attributed to the p56lck kinase. Secondly, higher p56lck activity in GM1low than in GM1high fractions correlated with the displacement of the CD4 coreceptor from the GM1high to GM1low fractions in DEF anergic cells. This was consistent with the ELISA and CLSM results showing accumulation of p56lck in the nonlipid rafts in these cells. These alterations persisted only for 12 h after resting the cells in ligand-free medium, which coincided with the duration of p56lck sequestration in the nonlipid rafts.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 4. TCR signaling in DEF anergic T cells. The CD4 TCR-HA cells rendered anergic by DEF or pre-exposed to TCR/CD4 mAb ligands were rested 2 h in ligand-free medium, the plasma membrane microdomains were isolated by sucrose gradient centrifugation, and the GM1high and GM1low fractions were pooled as described. GM1high and GM1low pools were precipitated with either CD4, 6.5.2 clonotypic mAb, or TCR {zeta}-chain Ab, and the immunoprecipitates were analyzed for the total PTP activity by an in vitro kinase assay and by Western blot. The results of one of three experiments are shown. A and B, PTP activity and p56lck phosphorylation associated with the CD4 coreceptor. C and D, PTP activity and phosphorylation of ZAP-70 and p59fyn associated with the HA-specific TCR. The identity of p56lck, p59fyn, and ZAP-70 after detection with RC 20 anti-pTyr mAb-HRP conjugate was confirmed on stripped-off PVDF membranes reprobed with specific Abs (B and D, lower panels). E, Phosphorylation of TCR {zeta}-chain. The p21 TCR {zeta}-chain is predominantly expressed in DEF anergic T cells.

 
The PTK activity in TCR immunoprecipitates was hardly detected in control cells (Fig. 4C). However, DEF anergic cells, but not those pre-exposed to TCR/CD4 mAbs, showed the highest PTK activity in GM1high moieties and it was mostly associated with p59fyn kinase as confirmed by Western blot (Fig. 4D). The p59fyn kinase can also catalyze the enolase substrate (35). This pattern of PTK activity persisted up to 12 h after the cessation of DEF stimulus.

Analysis of total PTP activity in the GM1high and GM1low pools against a monopeptide substrate (Glu and Tyr phosphorylated by natural kinases) at 2 and 12 h after resting the cells in ligand-free medium, using a Universal Tyrosine Phosphatase Assay kit (Takara Bio, Shiga, Japan), showed no differences between the DEF anergic T cells and those pre-exposed to the TCR/CD4 mAb ligands, i.e., 0.1 IU/µl/min and 0.25 IU/µl/min, respectively, as compared with control cells (0.18 IU/µl/min).

Shortly after productive stimulation of T cells through the TCR, the ZAP-70 kinase is recruited to the TCR’s ITAMs where it is phosphorylated by p56lck. Western blot analysis showed that ZAP-70 was inefficiently recruited to, and poorly if any at all phosphorylated on the TCR {zeta}-chain, in both GM1high and GM1low pooled fractions from DEF anergic cells (Fig. 4D). In addition, Western blot analysis conducted in immunoprecipitates from GM1high and GM1low fractions from DEF anergic cells (after normalization with respect of protein content) showed an increase in p59fyn phosphorylation on the CD3/TCR complex in the lipid rafts (GM1high pooled fractions) of DEF anergic cells (Fig. 4D), and a dominant expression of the p21 inhibitory isoform of the TCR {zeta}-chain (Fig. 4E). There was no p59fyn association with the CD4 coreceptor in either GM1high or GM1low moieties from DEF anergic cells (data not shown), but only with the TCR, as found by Western blot analysis. The ZAP-70, TCR {zeta}-chain, and p59fyn alterations were detected no later than 12 h after resting the cells in DEF-free medium (data not shown), and they were not detected in cells pre-exposed to TCR/CD4 mAbs (Fig. 4, D and E).

In assembly, the results showed that the p56lck kinase was responsible for most of the CD4-associated PTK activity and it was displaced together with the CD4 coreceptor from the lipid rafts in nonlipid raft microdomains of DEF anergic T cells. In contrast, most of the PTK activity associated with the TCR came from p59fyn kinase and it was distributed mostly in the lipid rafts. In contrast to the PTK activity, there was no significant alteration in the PTP activity in DEF anergic cells as compared with control cells, or to those treated with TCR and CD4 Abs. The altered pattern of PTK and TCR signaling persisted up to 12 h after cessation of DEF anergic stimulus, which correlated with the duration of CD4-p56lck displacement from the lipid rafts.

Th1 and Th2 transcription is down-regulated in DEF anergic T cells

Several transcription factors are instrumental for differentiation of CD4 T cells (44). AP-1 assists the IL-2 gene activation, whereas STAT4 and the recently discovered Th1 transcription factor T-box expressed in T cells (T-bet) induces secretion of IFN-{gamma}. Among the Th2 transcription factors, STAT6, cMaf, and GATA-3 are critical for IL-4 gene activation. Although NF-ATp appears to down-regulate late IL-4 production, the NF-ATc is an early positive regulator of IL-4. We have analyzed the gene expression and activity of these Th1 and Th2 transcription factors in cells rendered anergic by DEF, as compared with cells pre-exposed to TCR/CD4 mAb ligands at high receptor occupancy. Negatively sorted CD4 splenic T cells from TCR-HA transgenic mice, pre-exposed to the ligands as described, were rested for 2 and 72 h in ligand-free medium, and then restimulated for 4 h on plastic-immobilized DEF (5 µg/ml). We and others had previously shown that DEF-like chimeras induce potent activation of Ag-specific T cells when immobilized on the plastic surface (29, 45). Stimulation of T cells with plastic-immobilized DEF leaves the CD4 T cells untouched other than CD4-specific T cells for the HA110–120 peptide. The binding of AP-1, NF-AT, and cMaf, but not of GATA, to their oligonucleotide consensus motifs was drastically reduced in DEF anergic T cells up to 72 h after resting the cells in DEF-free medium. In contrast, cells pre-exposed to TCR/CD4 mAbs showed no alterations in AP-1, cMaf, and NF-AT activity, as demonstrated by EMSA (Fig. 5A). Semiquantitative RT-PCR also showed reduction of cMaf, NF-ATc, STAT4, STAT6, and T-bet, but not of GATA-3 and NF-ATp messages (Fig. 5B). In contrast, the NF-ATp mRNA level was up-regulated in DEF anergic cells. Consistent with the data on cell proliferation and cell division, these results indicate that the DEF anergic T cells were arrested in a Th0 state of differentiation (Fig. 5C).



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 5. Th1 and Th2 transcription activity in cells rendered anergic by DEF. Total cell lysates from CD4 TCR-HA cells rendered anergic by DEF or pre-exposed to TCR/CD4 mAb ligands were prepared 72 h after resting the cells in ligand-free medium, and subsequent to restimulation in DEF-coated plates, as described. The results of one of two experiments are shown. A, The binding activity of AP-1, NF-AT, cMaf, and GATA to the corresponding 32P-labeled oligonucleotides consensus motifs, and the cold inhibition with nonlabeled oligonucleotide consensus motif for each sample was determined by EMSA. Arrows indicate the position of specific oligonucleotide-protein complexes. The M12 mouse B lymphoma cells were used as negative control for cMaf and GATA-3. None of these transcription factors bound to their single-base mutated oligonucleotides (data not shown). B, Semiquantitative RT-PCR of Th1 and Th2 transcription factors. The percent of each amplicon was calculated by Scion Image software as described in the legend to Fig. 2, and is indicated in lateral tables. C, DEF anergy occurs by the arrest of differentiation in the Th0 stage.

 
DEF anergic T cells recover late and spontaneously from anergy, at the expense of low thresholds for activation-induced cell death (AICD)

We have analyzed the fate of DEF anergic T cells after spontaneous recovery from anergy, 4 days after resting the cells in DEF-free medium. These cells proliferated and secreted cytokines upon restimulation with HA-pulsed APCs in the absence of exogenous IL-2. However, the pattern of proliferation and cytokine production was quite different from that observed in cells pre-exposed to TCR/CD4 mAb ligands (Fig. 6). DEF anergic cells recovering from anergy reached the peak of proliferation 24 h after restimulation, secreted mostly IFN-{gamma}, and ceased to proliferate 48 h later. At 48 and 72 h after restimulation, the cell viability was decreased to 35 and 10%, respectively, as determined by trypan blue exclusion method. In contrast, cells pre-exposed to TCR/CD4 mAbs reached the peak of proliferation 72 h after restimulation, and secreted mainly IL-2. The viability of these cells was reduced by 58%, only 4 days after restimulation. The results demonstrated that in contrast to CD4 T cells treated with mAb ligands (that were not rendered anergic), DEF anergic T cells recovered late and spontaneously from anergy, and displayed high sensitivity to AICD upon Ag restimulation in the presence of APC-derived costimulation.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 6. Recovery from DEF anergy occurs late and spontaneously, and at the expense of low thresholds for AICD. The CD4 TCR-HA cells rendered anergic by DEF, or pre-exposed to TCR, CD4, or to a combination of TCR/CD4 mAb ligands as described, were rested for 4 days in ligand-free medium, and restimulated for another 3 days with HA-pulsed APCs. A, The HA-proliferative response was determined in triplicate wells every 24 h, for a period of 4 days by thymidine incorporation assay. B, Cytokine production was measured on day 2 of proliferation by ELISA. The mean values (picograms per milliliter) for each cytokine were in duplicate wells (±SD for IL-2, 10.5 pg/ml; for IL-4, 14.8 pg/ml; and for IFN-{gamma}, 28 pg/ml).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Soluble, dimeric peptide-MHC chimeras were shown to induce Ag-specific T cell anergy in vitro and in vivo (7, 8, 27, 28). In this study, we deciphered a mechanism underlying the Ag-specific anergy of CD4 T cells as induced by a soluble, dimeric peptide-MHC-II chimera (DEF). The anergy occurred shortly after high occupancy of the TCR and CD4 coreceptor by DEF, but not by a combination of TCR and CD4 Ab ligands, indicating that the nature of ligand-mediated TCR and CD4 cross-linking plays an important role for T cell function. We had previously ruled out the possibility of TCR masking by DEF, and thereby DEF competition for the binding of MHC-II-peptide complexes on APCs by TCR (7).

Van Rensen et al. (46) showed that induction of T cell anergy in vitro by liposomes incorporating peptide-MHC-II complexes occurred in the context of TCR down-modulation. This could be mostly related to a bystander effect due to the internalization of liposomes in T cells. We found that TCR and CD4 down-modulation was not critical for the induction of anergy by the soluble DEF dimer. A low extent of TCR and CD4 down-modulation in DEF anergic T cells was most likely due to its inability to provide a strong interaction with the receptors but rather, it provided a tickling of the receptors. The nuclear magnetic resonance data showed indeed that the soluble peptide-MHC-II molecules exert low binding avidity for the TCR as compared with Abs (47).

DEF anergy did not affect the IL-2R-mediated STAT5 signaling pathway, which argues for a lack of cross-talking between the TCR/CD4 and IL-2R signaling pathways in this anergy system. The anergic cells showed protection against apoptosis for a period of 4 days in the context of Bcl-2, Bcl-xL, and c-Myb up-regulation. The resistance to apoptosis by a mechanism of c-Myb-dependent up-regulation of Bcl-2 has been described in mouse T cell lines (48). Up-regulation of c-Myb itself requires endogenous IL-2 (49). We had previously shown that the cross-linking of the TCR and CD4 coreceptor with the soluble DEF dimer in the absence of APC-derived costimulation induces initially small amounts of endogenous IL-2 (30). This explains why c-Myb could be up-regulated in cells exposed to DEF.

Our data argue for a model of anergy, which relies on defective partitioning of signaling molecules in the lipid rafts leading to early negative signaling events in T cells. In support of this theory are previous data showing that the anergy induced by a suboptimal dose of soluble CD3 (2C11) mAb correlated with the lack of translocation of the CD3/TCR complex into the lipid rafts, followed by its accumulation in the nonlipid raft microdomains (50). According to the model of DEF anergy, low affinity interactions with the CD4 and TCR at the interface of lipid rafts were unable to induce receptor internalization during a 4-h interval, but were sufficient to induce allosteric changes in the plasma membrane microdomains, leading to the displacement of the CD4-p56lck signaling module from the lipid rafts, and its accumulation in the nonlipid raft microdomains. Sequestration of p56lck on ligation with the HIV gp120 protein on the cytoskeleton leading to TCR desensitization has been previously described (51), although a p56lck-independent CD4-mediated TCR desensitization was observed in this system (52).

The TCR content in the lipid rafts of DEF anergic cells was also slightly increased. It has been shown that the lipid raft microdomains are heterogeneously organized in terms of protein content, and that the ligand-mediated cross-linking of GM1 moieties in these microdomains facilitates an exchange of proteins (53). At a high order of cross-linking by soluble DEF dimer, a lateral diffusion of TCR residing in the marginal zone of the nonlipid rafts toward the lipid raft microdomains may have been promoted by a pseudo-cross-linking of TCR and CD4. Thus, one arm of DEF could bridge two CD4 coreceptors, while the other arm could bridge two TCRs, which explains the exclusion of the CD4-p56lck complex from the GM1 lipid rafts simultaneously with the relocation of the TCR in GM1 lipid rafts. Alternatively, the displacement of the CD4-p56lck complex from the lipid rafts may rely on signals leading to depalmitylation. The palmitate is attached to the cysteine residues near the N terminus of proteins through a reversible thioester bond. Reversible palmitylation was described for p59fyn (54), but not for CD4 and p56lck. The structural basis of these allosteric changes will require further investigation.

Furthermore, as the lipid rafts were gradually depleted of the CD4-p56lck signalosome in DEF anergic T cells, the p59fyn kinase could phosphorylate the TCR {zeta}-chain toward generation of a p21 inhibitory isoform in the lipid rafts, as observed in our experiments. Consequently, ZAP-70 kinase was poorly phosphorylated and recruited to the TCR {zeta}-chain. Increased tyrosine phosphorylation of p59fyn was previously described in anergic Th1 cells (39), and phosphorylation of the TCR {zeta}-chain by p59fyn in the absence of ZAP-70 activation was described in T cells rendered anergic by antagonist and partial-agonist peptides (55). Recently, another negative regulatory function of p59fyn was described in relation to phosphorylation-mediated activation of p50csk, a negative regulatory kinase of ZAP-70 (56). The lack of a functional TCR signalosome in DEF anergic cells explains the negative regulation of Th1 and Th2 transcription, and the "locked-in" cell differentiation in a Th0 stage. Poor AP-1 activity was previously associated with the lack of Th1 differentiation and anergy (57), whereas cMaf and NF-ATc were shown to be critical for IL-4-mediated Th2 differentiation (58, 59). It is also known that GATA-3 is important for IL-5-mediated Th2 differentiation (60), and STAT6 is required for GATA-3 activation (61). DEF anergic T cells showed a functional GATA-3, although STAT6 was deficient. This supports the recent findings showing that GATA-3 can autoactivate in T cells by a STAT6-independent pathway (62). However, "locked-in" Th2 transcription in the presence of a functional GATA-3 in DEF anergic T cells may well account for the poor activity of other Th2 transcription factors, i.e., cMaf, STAT6, and NF-ATc. In addition, the NF-ATp transcription factor, a repressor of late IL-4 production in T cells, was highly expressed in DEF anergic T cells.

Recovery of cells from DEF anergy occurred 4 days after cessation of DEF anergic stimulus at the expense of low thresholds for AICD. The increased IFN-{gamma} secretion in these cells could be the major trigger for apoptosis because it was shown that IFN-{gamma} increases the sensitivity of T cells to AICD (63). At this point, the mechanism by which DEF anergic T cells recovering from anergy restore preferentially the secretion of IFN-{gamma} remains still unknown. The cells recovering from DEF anergy were also unable to mount a Th2 response, which could be the result of up-regulation of long-lived antagonists of Th2 transcription, i.e., suppressor of cytokine signaling 3 repressor of STAT6 (64), Hox (65), or upstream stimulatory factor 2/facultative intracellular pathogen of basic helix-loop-helix zipper protein repressor of cMaf-mediated IL-4 transcription (66).

In conclusion, the results of this study showed first that DEF-mediated topology of TCR and CD4 coengagement could differentially affect T cell function. That is because, first, the cross-linking of the TCR and CD4 by DEF at low receptor occupancy polarized the cells toward a type 2 cell differentiation (30), and, second, at high receptor occupancy, only cells exposed to DEF, but not those exposed to TCR/CD4 mAb ligands, were rendered anergic. Second, an early event associated with DEF anergy was the displacement of the CD4-p56lck signalosome from the lipid rafts followed by alteration of TCR signaling, and the arrest of Th1 and Th2 transcription. Such alterations were not detected in T cells treated with TCR/CD4 mAb ligands. Third, although the DEF anergic cells recovered late and spontaneously from anergy, they were highly sensitive to AICD. According to these data, one may assume that Ag-specific ligands like DEF chimeras may provide new tools for silencing an Ag-driven autoimmune process, and may also help in searching for new mechanisms of negative regulation in T cells.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK61927 and DK61326 (to T.-D.B.) and Juvenile Diabetes Research Foundation Grant 1-2002-66 (to S.C.). Back

2 Address correspondence and reprint requests to Dr. Teodor-D. Brumeanu, Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029. E-mail address: Teodor.Brumeanu{at}mssm.edu Back

3 Abbreviations used in this paper: ZAP-70, {zeta}-associated protein of 70 kDA; ITAM, immunoreceptor tyrosine-based activation motif; HA, hemagglutinin; PVDF, polyvinylidene difluoride; CTB, cholera toxin B; CLSM, confocal laser-scanning microscopy; PTK, protein tyrosine phosphatase; PTP, protein tyrosine phosphatase; AICD, activation-induced cell death; T-bet, T-box expressed in T cells; [3H]TdR, [3H]thymidine incorporation. Back

Received for publication January 17, 2003. Accepted for publication April 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jenkins, M. K., D. M. Pardoll, J. Mizuguchi, H. Quill, R. H. Schwartz. 1987. T cell unresponsiveness in vivo and in vitro: fine specificity of induction and molecular characterization of the unresponsive state. Immunol. Rev. 95:113.[Medline]
  2. Boussiotis, V. A., J. G. Gribben, G. J. Freeman, L. M. Nadler. 1994. Blockade of the CD28 co-stimulatory pathway: a means to induce tolerance. Curr. Opin. Immunol. 6:797.[Medline]
  3. Sloan-Lancaster, J., B. D. Evavold, P. M. Allen. 1993. Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells. Nature 363:117.[Medline]
  4. Zeller, J. C., A. Panoskalktsis-Mortari, W. J. Murphy, F. W. Ruscetti, S. Narula, M. G. Roncarolo, B. R. Blazar. 1999. Induction of CD4+ T cell alloantigen-specific hyporesponsiveness by IL-10 and TGF-{beta}. J. Immunol. 163:3684.[Abstract/Free Full Text]
  5. Nagler-Anderson, C., N. H. Shi. 2001. Peripheral nonresponsiveness to orally administered soluble proteins. Crit. Rev. Immunol. 21:121.[Medline]
  6. Muller, S., L. Adorini, A. Juretic, Z. A. Nagy. 1990. Selective in vivo inhibition of T cell activation by class II MHC-binding peptides administered in soluble form. J. Immunol. 145:4006.[Abstract]
  7. Casares, S., A. Hurtado, R. McEvoy, A. Sarukhan, H. von Boehmer, T.-D. Brumeanu. 2002. Downregulation of diabetogenic CD4+ T cells by a soluble dimeric peptide-MHC class II chimera. Nat. Immunol. 3:383.[Medline]
  8. O’Herrin, S. M., J. E. Slansky, Q. Tang, M. A. Markiewicz, T. F. Gajewski, D. M. Pardoll, J. P. Schneck, J. A. Bluestone. 2001. Antigen-specific blockade of T cells in vivo using dimeric MHC peptide. J. Immunol. 167:2555.[Abstract/Free Full Text]
  9. Weiss, A., D. R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263.[Medline]
  10. Newton-Nash, D. K., P. J. Newman. 1999. A new role of platelet-endothelial cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduction. J. Immunol. 163:622.
  11. Perez-Villar, J. J., G. S. Whitney, M. A. Bowen, D. H. Hewgill, A. A. Aruffo, K. B. Kanner. 1999. CD5 negatively regulates the T-cell receptor signal transduction pathway: involvement of SH2-containing phosphotyrosine phosphatase SHP-1. Mol. Cell. Biol. 19:2903.[Abstract/Free Full Text]
  12. Bergman, M., T. Mustelin, C. Ockten, J. Partanen, N. A. Flint, K. E. Amrein, M. Autero, P. Burn, K. Alitalo. 1992. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr505 and down regulates its catalytic activity. EMBO J. 11:2919.[Medline]
  13. Weil, R., J. F. Cloutier, M. Fournel, A. Veillette. 1995. Regulation of ZAP-70 by Src family tyrosine protein kinases in an antigen-specific T-cell line. J. Biol. Chem. 270:2791.[Abstract/Free Full Text]
  14. Raab, M., C. E. Rudd. 1996. Hematopoetic cell phosphatase (HCP) regulates p56lck phosphorylation and ZAP-70 binding to T-cell receptor {zeta} chain. Biochem. Biophys. Res. Commun. 222:50.[Medline]
  15. Lupher, M. L., N. Rao, M. J. Eck, H. Band. 1999. The Cbl protooncoprotein: a negative regulator of immune receptor signal transduction. Immunol. Today 20:375.[Medline]
  16. Janes, P. W., S. C. Ley, A. I. Magee. 1999. Aggregation of lipid rafts accompanies signaling via the T-cell antigen receptor. J. Cell Biol. 147:447.[Abstract/Free Full Text]
  17. Millan, J., J. Cerny, V. Horejsi, M. A. Alonso. 1999. CD4 segregates into specific detergent-resistant T-cell membrane microdomains. Tissue Antigens 53:33.[Medline]
  18. Parolini, I., M. Sargiacomo, P. Lisanti, C. Peschle. 1996. Signal transduction and glycophosphatidylinositol-linked proteins (Lyn, Lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes. Blood 87:3783.[Abstract/Free Full Text]
  19. Janes, P. W., S. C. Ley, A. I. Magee, P. S. Kabouridis. 2000. The role of lipid rafts in T cell antigen receptor signalling. Semin. Immunol. 12:23.[Medline]
  20. van’t Hof, W., M. D. Resh. 1999. Dual fatty acylation of p59Fyn is required for association with T cell receptor {zeta} chain through phosphotyrosine-Src homology domain-2 interactions. J. Cell Biol. 145:377.[Abstract/Free Full Text]
  21. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
  22. Ohtani, Y. Y., X.-Y. Zhou, K. Toyooka, X.-G. Tai, C. S. Park, T. Hamaoka, R. Abe, K. Miyake, H. Fujiwara. 2000. Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts. J. Immunol. 164:1251.[Abstract/Free Full Text]
  23. Hope, H. R., L. I. Pike. 1996. Phosphoiniositides and phosphoinositide utilizing enzymes in detergent insoluble lipid domains. Mol. Biol. Cell 7:843.[Abstract]
  24. Simons, K., E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387:569.[Medline]
  25. Zhang, W., R. P. Trible, L. E. Samelson. 1998. LAT palmitylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T-cell activation. Immunity 9:239.[Medline]
  26. Montixi, C., C. Langlet, A.-M. Bernard, J. Thimonier, C. Dubois, M.-A. Wurbel, J. P. Chauvin, M. Pierres, H.-T. He. 1998. Engagement of T-cell receptor triggers its recruitment to low density detergent insoluble membrane domains. EMBO J. 17:5334.[Medline]
  27. Appel, H., N. P. Seth, L. Gauthier, K. Wucherpfennig. 2001. Anergy induction by dimeric TCR ligands. J. Immunol. 166:5279.[Abstract/Free Full Text]
  28. Brumeanu, T.-D., C. A. Bona, S. Casares. 2001. T cell tolerance and autoimmune diabetes. Int. Rev. Immunol. 20:301.[Medline]
  29. Casares, S., C. Bona, T.-D. Brumeanu. 1997. Engineering and characterization of a murine MHC class II-immunoglobulin chimera expressing an immunodominant CD4 T viral epitope. Protein Eng. 10:1295.[Abstract/Free Full Text]
  30. Casares, S., C. S. Zong, L. D. Radu, A. Miller, C. A. Bona, T.-D. Brumeanu. 1999. Antigen-specific signaling by a soluble, dimeric peptide/MHC Class II/Fc chimera leading to type 2 helper cell differentiation. J. Exp. Med. 190:543.[Abstract/Free Full Text]
  31. Weber, S., A. Trannecker, F. Olivery, W. V. Gerhard, K. Karjalainen. 1992. Specific low-affinity recognition of MHC complex plus peptide by soluble T-cell receptor. Nature 356:793.[Medline]
  32. Cottin, V., J. E. S. Doan, D. W. H. Riches. 2002. Restricted localization of the TNF receptor CD120a to lipid rafts: a novel role of the death domain. J. Immunol. 168:4095.[Abstract/Free Full Text]
  33. Kishi, H., Z. X. Jin, X. C. Wei, I. Nagata, I. Matsuda, S. Saito, A. Muraguchi. 2002. Cooperative binding of c-Myb and Pax-5 activates the RAG-2 promoter in immature B cells. Blood 99:576.[Abstract/Free Full Text]
  34. Ho, I. C., M. R. Hodge, J. W. Rooney, L. H. Glimcher. 1996. The proto-oncogene c-Maf is responsible for tissue specific expression of interleukin-4. Cell 85:973.[Medline]
  35. Feder, D., J. M. Bishop. 1990. Purification and enzymatic characterization of pp60csrc from human platelets. J. Biol. Chem. 265:8205.[Abstract/Free Full Text]
  36. Kagami, S., H. Nakajima, A. Suto, K. Hirose, K. Suzuki, S. Morita, L. Kato, Y. Saito, T. Kitamura, I. Iwamoto. 2001. STAT5 regulates T helper cell differentiation by several distinct mechanisms. Blood 97:2358.[Abstract/Free Full Text]
  37. Combadiere, B., C. R. Sousa, R. N. Germain, M. J. Lenardo. 1998. Selective induction of apoptosis in mature T lymphocytes by variant T cell receptor ligands. J. Exp. Med. 187:349.[Abstract/Free Full Text]
  38. Garcia, S., J. Disanto, B. Stockinger. 1999. Following the development of a CD4 T cell response in vivo: from activation to memory formation. Immunity 11:163.[Medline]
  39. Quill, H., M. P. Riley, E. A. Cho, J. E. Casnellie, J. C. Reed, T. Torigoe. 1992. Anergic Th1 cells express altered levels of the protein kinase p56lck and p59fyn. J. Immunol. 149:2887.[Abstract]
  40. Boussiotis, V. A., G. J. Freeman, A. Berezovskaya, D. J. Barber, L. M. Nadler. 1997. Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science 278:124.[Abstract/Free Full Text]
  41. Crise, B., J. K. Rose. 1992. Identification of palmitoylation sites on CD4, the human immunodeficiency virus receptor. J. Biol. Chem. 267:13593.[Abstract/Free Full Text]
  42. Alland, L., S. M. Peseckis, R. E. Atherton, L. Berthiaume, D. M. Resh. 1994. Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization. J. Biol. Chem. 269:16701.[Abstract/Free Full Text]
  43. Bernadetta, M. M., M. M. D’Elios, P. Galieni, M. Boncristiano, F. Lauria, G. Del Prete, J. L. Telford, C. T. Baldari. 1998. Expression of the T-cell-specific tyrosine kinase Lck in normal B-1 cells and in chronic lymphocytic leukemia B cells. Blood 91:3390.