Differential susceptibility of human Th1 versus T h 2 cells to induction of anergy and apoptosis by ECDI/antigen-coupled antigen-presenting cells
Arthur A. Vandenbark1,2,3,
David Barnes1,
Tom Finn1,3,
Dennis N. Bourdette1,3,
Ruth Whitham1,3,
Ian Robey1,
Johnan Kaleeba2,
Bruce F. Bebo, Jr1,3,
Steven D. Miller4,
Halina Offner1,3 and
Yuan K. Chou1,3
1 Neuroimmunology Research R & D-31, Veterans Affairs Medical Center, Portland, OR 97201, USA
2 Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201, USA
3 Department of Neurology, Oregon Health Sciences University, Portland, OR 97201, USA
4 Department of MicrobiologyImmunology, Northwestern University Medical School, Chicago, IL, USA
Correspondence to:
A. A. Vandenbark, Neuroimmunology Research R & D-31, Veterans Affairs Medical Center, Portland, OR 97201, USA
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Abstract
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Antigen-coupled antigen-presenting cells (APC) serve as potent tolerogens for inhibiting immune responses in vivo and in vitro, apparently by providing an antigen-specific signal through the TCR in the absence of co-stimulation. Although this approach has been well studied in rodents, little is known about its effects on human T cells. We evaluated the specificity and mechanisms of tolerization of human T cells in vitro using monocyte-enriched adherent cells that were pulsed with antigen and treated with the cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). Autologous antigen-coupled APC selectively tolerized T cells of the Th1 but not Th2 lineage through a mechanism that involved both antigen-specific and antigen-non-specific elements. The tolerization process was dependent on the ECDI and antigen concentration, and the coupling time, and was reflected by initial up-regulation of CD25. However, upon re-stimulation with fresh APC and antigen, tolerized Th1 cells failed to proliferate or to produce Th1 cytokine message or secreted protein, had decreased expression of CD25, CD28 and B7 and increased expression of MHC class II molecules, and demonstrated an enhanced commitment to apoptosis. Th1 cell tolerization could be prevented by adding anti-CD28 antibody, IL-2 or untreated APC at the same time as the ECDI/antigen-coupled APC, or reversed by adding anti-CD28 antibody or IL-2 upon re-stimulation with fresh APC plus antigen. Thus, the tolerizing effect of ECDI/antigen-coupled APC on human Th1 cells appears to involve a reversible anergy mechanism leading to apoptosis, whereby the targeted T cells receive full or partial activation through the TCR, without coordinate co-stimulation. These data suggest dichotomous signaling requirements for inactivating cells of the Th1 and Th2 lineages that may have important implications for treatment of Th1-mediated autoimmune or inflammatory diseases.
Keywords: altered antigen-presenting cells, human T cells, tolerance induction
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Introduction
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Induction and resolution of inflammatory T cell responses requires activation and amplification of effector cells, and their selective termination through anergy and apoptosis (14). Although events that induce antigen-specific T cell activation have been well characterized, cellular interactions leading to antigen-specific tolerance and deletion are less well understood. Peripheral tolerance may be induced by systemic administration of antigen and may involve anergy (56) or deletional mechanisms including activation-induced cell death/apoptosis (AICD/AIA) mediated by engagement of Fas or tumor necrosis receptors by their ligands (710). An additional tolerogenic approach to be explored here is incubation of T cells with antigen presenting cells (APC) that have been pulsed with antigen and chemically cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI) (11). Previous studies in rats and mice demonstrated that splenic (SPL) APC coupled with crude central nervous system tissue extracts or purified encephalitogenic proteins and peptides could inhibit induction of experimental encephalomyelitis, antigen-specific T cell proliferation and Th1-derived cytokine production (1218). Similarly, tolerance induced with syngeneic splenocytes coupled to the peripheral nerve myelin P2 protein or a synthetic peptide could inhibit experimental neuritis (1920).
Tolerance induced by antigenSPL is antigen dose dependent and is exquisitely specific for the antigen used in the coupling step (1516). In rodents, the mechanism of inhibition appears to involve both inhibition and deletion of pathogenic Th1 cells (i) by specific inactivation (i.e. anergy/apoptosis) or (ii) by stimulation of Th2 cells that may release regulatory cytokines including IL-4 and IL-10 (21). Specific inactivation could involve MHC/antigen presentation in the absence of functional co-stimulatory molecules (i.e. B7-1/2 interaction with CTLA-4/CD28) that may be altered by the ECDI treatment, in accordance with an anergy model (56). Recent studies in the Theiler's murine encephalitis virus model indicate that tolerance induced with virus-coupled splenocytes not only inhibited Th1 responses, but also promoted Th2 responses (22) and we observed a similar `split tolerance' effect in the Lewis rat experimental autoimmune encephalomyelitis (EAE) model (17). The possible expansion of regulatory Th2 cells may account for earlier reports in which splenocytes transferred from ECDI/antigen-tolerized rats could suppress EAE in naive recipients (1214).
Despite the extensive insights regarding tolerance induction in rodent T cells, little is known about the effects of ECDI-coupled APC on human T cells. The current study focuses on conditions and mechanisms of tolerance induced in vitro by ECDI/antigen-coupled APC on human Th1 cells specific for myelin basic protein (MBP), Herpes simplex virus (HSV) or tetanus toxoid (TT), or Th2 cells specific for the TCR peptide, BV5S2-3858. The results demonstrate both antigen-specific and antigen-non-specific tolerizing effects of ECDI/antigen-coupled APC on Th1 but not Th2 cells that involve a reversible anergy mechanism leading to apoptosis.
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Methods
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Human subjects
Blood donors enrolled in this study included eight patients with clinically definite relapsing or progressive multiple sclerosis (KAH, SDS, TR, JH, ESN, AL, TL and MR) and one healthy individual (EN). These donors were known to respond to MBP, HSV, TT or to the BV5S2-3858 peptide after successful vaccination in previous studies (23,24).
Antigens
MBP was extracted and purified using frozen brains supplied by the National Disease Research Interchange (Philadelphia, PA) as described previously (25). Synthetic peptides corresponding to MBP sequences 828 and 149171 were kind gifts from Dr G. A. Hashim (New York, NY). The peptide corresponding to the TCR BV5S2-3858 sequence was synthesized and purified by HPLC at the Portland VA Medical Center (24). HSV antigen was obtained from Whittaker MA Bioproducts, (Walkersville, MD) and TT from Wyeth Laboratories (Marietta, GA). TT peptides, T1 and T6, corresponding to residues 591602 (T1) and 952967 (T6), were a kind gift of Dr Michael Davey (Portland VA Medical Center).
Antigen-specific T cell clones
Antigen-specific T cell clones were selected from peripheral blood mononuclear cells (PBMC). PBMC were separated by a Ficoll-density centrifugation and cultured in medium (RPMI 1640 with 2% human pooled AB serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µg/ml penicillin G and 100 µg/ml streptomycin) at 100,000 cells/well in 96-well round-bottomed plates (Falcon, Becton Dickinson, Lincoln Park, NJ) in the presence of MBP (50 µg/ml), HSV (1/400 dilution) or TT (1/200 dilution) or BV5S2-3858 peptide (50 µg/ml) for 7 days at 37C and 5% CO2. At the end of the 7-day culture, the cells were re-stimulated with antigen presented by autologous irradiated (2500 rad) PBMC (150,000/well). After 3 days, the reactivated T cells were expanded with 5 ng/ml recombinant (rh)IL-2 (R & D Systems, Minneapolis, MN) for an additional 47 days. T cells were selected and pooled from 10 of the best growing wells to make T cell lines or were further selected into antigen-specific clones by limiting dilution as described previously (26). The clonality of selected T cell clones was demonstrated by response to a single epitope and predominant expression of a single TCR BV gene as detected by PCR.
T cell proliferation assay
The T cell proliferation assay was used to determine T cell responsiveness to various antigens. After thorough washing, 40,000 T cells from a selected T cell clone were cultured in triplicate with 150,000 autologous irradiated PBMC in a final volume of 200 µl/well with or without added antigen (10 µl/well) at optimal concentrations. The cultures were incubated for 3 days at 37°C in 5% CO2 and were pulsed with 0.5 µCi [3H]thymidine for the last 18 h. The cells were harvested on glass fiber filters and incorporated radioactivity was counted using a liquid scintillation counter, 1205 Betaplate (Wallac, Turku, Finland).
ECDI/antigen-coupled APC
A monocyte-enriched adherent cell population was collected from PBMC after 2 h adherence to the plastic surface of tissue culture dishes (Falcon), followed by 5 times washing with RPMI 1640 and removal of the cells using a cell scraper (Costar, Cambridge, MA). The cells obtained were 5070% CD11b+ as determined by flow cytometry (phycoerythrin-conjugated anti-CD11b; Becton Dickinson, Sunnyvale, CA). Collected monocytes were coupled with protein as described previously (11) using water-soluble ECDI (mol. wt 191.7; Calbiochem, La Jolla, CA) that catalyzes the formation of peptide bonds between free amino and carboxyl groups. The coupling reaction was initiated by preparing 1 ml of cell suspension containing 1 to 5x106 monocytes plus antigen in cold saline (MBP, 503000 µg/ml; MBP peptides, 50 µg/ml; TT peptides, 50 µg/ml; TCR peptides, 50 2000 µg/ml), followed by the addition of 10, 20 or 40 µl of freshly prepared 1 M ECDI (stock solution, 191 mg/ml of cold saline) into 1 ml of the cell suspension to obtain a final ECDI concentration of 1040 mM. The cell suspension was incubated for 1 h at 4°C, and the cells were washed 3 times with culture medium and maintained at 4°C until use.
Co-culture of T cells with ECDI/antigen-coupled APC
Antigen-specific T cells were collected during the IL-2 growth phase and after washing 2 times with RPMI 1640 the T cells were co-cultured with autologous ECDI/antigen-coupled APC for 18 h at 37°C in 5% CO2 at a ratio of 1:1 to 1:2 (T:APC) in a 24-well culture plate (Falcon) with 24x106 total mixed cells/well. Cultured cell mixtures were collected and applied to a Ficoll gradient, and isolated live cells were washed 3 times with culture medium, remixed with untreated monocytes (at the starting ratio) and antigen, and evaluated for proliferation, cytokine mRNA expression and cytokine secretion. In some experiments, anti-CD28 antibody (Caltag, Burlingame, CA) or rhIL-2 (R & D Systems) was added to prevent or reverse tolerance induction. The anti-CD28 antibody was either preabsorbed to the wells for 24 h at 4°C at a concentration of 20 µg/ml at pH 8.6, prior to a 3 times wash in culture medium (surface bound) or was added at the indicated concentrations in soluble form. Each experimental variable was evaluated in two separate experiments.
Cytokine mRNA expression
RNA was isolated from treated and untreated T cells by a modified method (26). Twenty thousand T cells were placed in a 96-well round-bottomed plate with freshly isolated, irradiated PBMC at 2x105/well plus antigen or medium for 48 h at 37°C in 5% CO2. The cells were harvested and immediately lysed using the RNA-STAT-60 buffer (Tel-Test, Friendswood, TX). RNA from equal numbers of T cells from each group was made into cDNA with MLV-RT (Gibco BRL, Frederick, MD), to be used as a template for the cytokine primers. The reaction was run for 34 cycles for granulocyte macrophage colony stimulating factor (GM-CSF) primers, and for 32 cycles for IFN-
, IL-5 and IL-10 primers. To normalize for cell numbers and total RNA used, the human primer pairs for the enzymatic reporter gene GAPDH were utilized in a 35 µl reaction containing 1 µl of cDNA from each sample and run for 27 cycles. The intensity of the GAPDH band signal from each sample was used to standardize the amount of cDNA to be used in subsequent reactions. All of the products were visualized in ethidium bromide-stained 1.5% agarose gels and displayed as one band of the appropriate mol. wt for each primer set. The PCR DNA oligonucleotide sequences used for the individual products were as follows: IFN-
, sense 5'-TCT TGG CTT TTC AGC TCT GCA TC-3', anti-sense 5'-GGA TGC TCT TCG ACC TTG AAA CAG-3', 474 bp product; GM-CSF, sense 5'-TGG GAG CAT GTG AAT GCC ATC CAG-3', anti-sense 5'-GAC TGG CTC CCA GCA GTC AAA GGG-3', 350 bp product; IL-5, sense 5'-TAT CCA CTC GGT GTT CAT TAC ACC-3', anti-sense 5'-GCT TCT GCA TTT GAG TTT GCT AGC-3', 510 bp product; IL-10, sense 5'-GAC TTT AAG GGT TAC TTG GGT TGC-3', anti-sense 5'-CAC TGC CTT GCT CTT ATT TTC ACA-3', 201 bp product.
Quantification of secreted cytokines
Cell culture supernatants collected on day 2 were frozen at 70°C, and later evaluated by ELISA for IL-2, IL-4 and IFN-
as described in kits purchased from PharMingen. Developed reactions were read on a Kinetic microplate reader (Molecular Devices, Sunnyvale, CA) and quantified using diluted standards.
Phenotypic analysis by FACScan
Monocyte-enriched adherent cells before and after cross-linking to ECDI or coupling with ECDI/antigen were co-stained in two replicate experiments with mouse anti-human CD11bphycoerythrin (PE) and mouse anti-human B7-1, plus goat anti-mouse IgGFITC and mouse anti-human B7-2FITC (PharMingen, San Diego, CA) or mouse anti-human HLA-DRFITC (Becton Dickinson, Sunnyvale, CA). Antigen-specific T cell clones were co-stained with mouse anti-human CD4PE, and FITC-labeled mouse anti-human CD25, CD28, B7-1, B7-2 and HLA-DR (PharMingen). The staining method used was similar to that described previously (18). Two-color fluorescence was evaluated using a FACScan (Becton Dickinson, Sunnyvale, CA).
Apoptosis detection
Apoptosis was evaluated in two replicate experiments using the Early Apoptosis Detection kit (Kamiya Biochemical, Seattle, WA). After incubation of T cells with autologous antigen-coupled APC, viable cells were recovered by Ficoll-Paque separation, washed, and stained with Annexin VFITC and propidium iodide according to the manufacturer's instructions. The cells were analyzed for two-color fluorescence using a FACScan (Becton Dickinson, Sunnyvale, CA). Early apoptotic cells were identified by staining of exposed phosphatidylserine with Annexin VFITC but not propidium iodide, and necrotic cells were identified by Annexin V staining and uptake of propidium iodide.
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Results
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Antigen-specific inhibition of Th1 but not Th2 cells by ECDI/antigen cross-linked APC
In our previous studies in rodents, antigen-pulsed APC that were cross-linked with ECDI could induce potent inhibition of T cell responses in vivo and in vitro (1718). To investigate the ability of human ECDI/antigen cross-linked APC to inhibit T cell responses to specific antigen, we titrated antigen concentration, dose of ECDI and time of the coupling reaction using autologous adherent PBMC. In initial studies, we chose two MBP-specific Th1 cell clones that were shown previously to produce IFN-
, but not IL-4, IL-5 or IL-10 (26). Monocyte-enriched PBMC were incubated with medium (sham control) or different concentrations of MBP ranging from 50 to 3000 µg for either 1 or 3 h in the presence of 10 mM ECDI, and then washed thoroughly to remove free antigen and ECDI. The ECDI/MBP-coupled APC were then incubated with the MBP-specific T cells for 18 h, washed and then re-stimulated for 3 days with fresh APC plus antigen in a standard proliferation assay. As is shown in Fig. 1
(A), the degree of inhibition was maximized with increasing doses of antigen used to pulse the APC. Moreover, a shorter coupling time of 1 h could be used with higher antigen doses. In a follow-up experiment, we evaluated the effects of ECDI concentration used for cross-linking MBP to the APC. As is shown in Fig. 1
(B), the higher the dose of ECDI used for coupling, the more pronounced the inhibition of response to MBP compared to untreated T cells or T cells incubated with sham-coupled APC. However, the coupling process itself produced an ECDI concentration-dependent inhibitory effect that was independent of MBP, resulting in progressively reduced T cell responses after exposure to sham-coupled APC.

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Fig. 1. MBP and ECDI dose-dependent inhibition of proliferation of human MBP-specific T cells pre-cultured with ECDI/MBP-coupled APC. (A) An MBP-specific T cell clone (donor EN) was pre-cultured for 18 h with autologous APC coupled with 10 mM ECDI plus saline (`sham') or with various concentrations of MBP for 1 or 3 h. (B) An MBP-specific T cell clone (donor KH) was pre-cultured with medium or with APC coupled with ECDI for 1 h at concentrations of 10, 20 or 40 mM alone or with 1000 µg/ml MBP. Treated T cells were separated on a Ficoll gradient and re-stimulated in a proliferation assay with untreated APC plus MBP. The response is presented as net c.p.m. of 3H]thymidine uptake (c.p.m. of antigen-stimulated T cells background c.p.m.). 100% response: (A) 20,000 net c.p.m.; (B) 38,000 net c.p.m. `%' represents the percent inhibition of T cell proliferation in treated versus control cultures.
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Similar experiments were carried out to titrate coupling conditions using a BV5S2-3858-specific Th2 cell clone that was shown previously to produce IL-4 and IL-10, but not IFN-
(26). Unlike the results using Th1 clones, there was no inhibition when ECDI/antigen-coupled APC were incubated with the Th2 clone, even at coupling antigen concentrations as high as 2000 µg/ml (Fig. 2A
). Moreover, at ECDI coupling concentrations
20 mM, there was no antigen-specific inhibition and no inhibition with sham coupled APC (Fig. 2B
). However, coupling with 40 mM ECDI produced a strong non-specific inhibitory effect that was not further accentuated by cross-linked antigen.

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Fig. 2. No antigen-specific inhibition of proliferation of TCR peptide-specific Th2 cells with ECDI/antigen-coupled APC. (A) A BV5S2-3858 peptide-specific Th2 clone (donor TL) was pre-cultured with APC cross-linked with 10 mM ECDI and 1002000 µg/ml BV5S2-3858 peptide for 18 h prior to re-stimulation. (B) In a separate experiment, the same BV5S2-3858 peptide-specific Th2 clone was pre-cultured with untreated APC, or APC cross-linked with 10, 20 or 40 mM ECDI alone or with 200 µg/ml BV5S2-3858 peptide for 18 h prior to re-stimulation. Treated T cells were separated on a Ficoll gradient and re-stimulated in a proliferation assay with untreated APC plus BV5S2-3858 peptide. Results are presented as net c.p.m. of [3H]thymidine uptake (c.p.m. of antigen stimulated T cells background c.p.m.). Backgrounds were (A) 2231 ± 869 (n = 5) and (B) 696 ± 399 (n = 5).
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Differential donor-dependent non-specific toxicity of ECDI cross-linking on Th1 cells
During the course of this study, we noted a variable degree of antigen-non-specific inhibition of MBP-reactive Th1 cells after incubation with `sham' ECDI-cross-linked APC versus untreated APC. Overall, there were striking differences among donors in the degree of non-specific inhibition, ranging from about 20% in donor JH to >90% in donor ESN, with an average inhibition of 57 ± 25% (Table 1
). In contrast, non-specific inhibition of TCR peptide-specific Th2 cell responses (average inhibition of 3 ± 3%) did not occur after incubation with `sham' ECDI-cross-linked APC versus untreated APC (Table 1
).
Antigen-specific T cell inhibition by ECDI/antigen-coupled APC
In addition to the antigen-non-specific component of ECDI/antigen-coupled APC described above, we sought to critically evaluate the specificity of antigen-induced tolerance. Thus, three different antigen-specific T cell clones were incubated with relevant or control ECDI/antigen-coupled autologous APC and then re-stimulated with relevant antigen (Fig. 3
). A TT-specific T cell clone selected from donor ESN, specifically responsive to TT and the TT peptide T6, but not to any other of five non-overlapping TT peptides, was significantly inhibited with ECDI/T6- but not ECDI/T1-coupled APC. Similarly, an MBP-specific clone from donor JH was significantly inhibited with ECDI/MBP- but not ECDI/HSV-coupled APC, and an MBP-149-171 peptide specific clone from donor SDS was significantly inhibited with ECDI/MBP-149171 peptide- but not ECDI/MBP-828 peptide-coupled APC. These results demonstrate that in addition to antigen-non-specific effects, ECDI/antigen-coupled APC induced T cell inhibition that was antigen-specific.

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Fig. 3. Antigen-specific T cell inhibition of proliferation responses induced by ECDI/antigen-coupled APC. (Left) A TT-selected T cell clone (donor ESN) was specific for the TT peptide T6, with the TT peptide T1 serving as an unrelated antigen. (Middle) An MBP-selected T cell line (donor JH) was specific for MBP, with HSV serving as the unrelated antigen. (Right) An MBP-selected T cell clone (donor SS) was specific for MBP peptide 149171, with the MBP peptide 128 serving as the unrelated antigen. Each T cell isolate was pre-cultured with ECDI/saline-coupled APC (control), ECDI/specific antigen-coupled APC and ECDI/unrelated antigen-coupled APC for 18 h followed by separation on Ficoll and re-stimulation with untreated APC and specific antigen in a proliferation assay. Results are presented as net c.p.m. of [3H]thymidine uptake (c.p.m. of antigen stimulated T cells background c.p.m.).
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Inhibition of Th1 cytokines with ECDI/antigen-coupled APC
In addition to proliferation responses, we evaluated the effects of ECDI/antigen tolerization on cytokine mRNA expression and cytokine secretion. As is shown in Fig. 4
(A), message for GM-CSF and IFN-
was undetectable after MBP-specific T cells were first co-cultured with ECDI/MBP-coupled APC and then re-stimulated with MBP plus untreated APC. In contrast, message for IL-5 and IL-10 was fully detectable when BV5S2-specific T cells were first co-cultured with ECDI/BV5S2-3858 peptide-coupled APC and then re-stimulated with the same peptide plus untreated APC (Fig. 4B
). These data indicate that pre-exposure to ECDI/antigen-coupled APC inhibited message production of Th1 but not Th2 cells, in accordance with proliferation data. In a separate experiment, secreted cytokines were quantified by ELISA in culture supernatants from an antigen-activated MBP-specific Th1 cell clone after pretreatment with ECDI- or ECDI/MBP-coupled APC, or no pretreatment. As is shown in Fig. 5
, pretreatment with ECDI-coupled APC markedly reduced secreted IL-2 and IFN-
levels, and pretreatment with ECDI/antigen-coupled APC essentially abrogated production of both cytokines. As expected for this Th1 clone, IL-4 levels were undetectable before and after tolerization.

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Fig. 4. Inhibition of Th1 but not Th2 cytokine message expression after tolerization with ECDI/antigen-coupled APC. (A) An MBP-specific Th1 clone (donor EN) or (B) a BV5S2-3858 peptide-reactive Th2 clone (donor TL) were pre-cultured with ECDI/antigen-coupled APC or no antigen (control) for 18 h, washed, and re-stimulated for 48 h with antigen plus fresh APC prior to RNA extraction, PCR amplification of cytokine message and visualization on agarose gels after staining with ethidium bromide.
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Induction of apoptosis by ECDI/antigen-coupled APC
One result of TCR ligation in the absence of co-stimulation is antigen-induced cell death. Thus, after treatment with ECDI/antigen-coupled autologous APC, T cells were evaluated for induction of early apoptosis and necrosis, using Annexin V and propidium iodide staining and FACS analysis. As is shown in Fig. 6
, Th1 cells committed to apoptosis/necrosis increased from a background of 4 to 27% 48 h after exposure to APC cross-linked with ECDI/saline and to 36% 48 h after the T cells encountered antigen-coupled APC. In contrast, Th2 cells were relatively resistant to apoptosis/necrosis induction, increasing from a background of 5% to only 6 and 10% respectively 48 h after exposure to ECDI-APC or ECDI/antigen-APC (Fig. 6
).

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Fig. 6. Enhanced susceptibility of Th1 cells to apoptosis induced by ECDI-coupled APC. Th1 or Th2 cells were incubated for 18 h with untreated, ECDI/saline-coupled or ECDI/antigen-coupled APC, washed, and re-stimulated for 24 or 48 h with antigen + APC prior to apoptosis detection. Coupling was carried out with 20 mM ECDI. Some 24 h cultured Th1 cells were further incubated with 10 ng/ml IL-2 for 72 h prior to apoptosis detection. Apoptosis was evaluated by FACS after staining with Annexin VFITC mAb and propidium iodide as described in Methods.
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Effects of co-activation factors on prevention and reversal of ECDI/antigen-coupled APC mediated tolerance
Our previous studies in rodents demonstrated that T cell inhibition induced by ECDI/antigen-coupled APC could be reversed by IL-2, thereby suggesting an anergy mechanism induced by engagement of the MHCantigen complex in the absence of functional co-stimulation. To determine if inhibition of human Th1 cells involved a similar mechanism, co-activation factors were added to the cultures either during or after the tolerization process to prevent or reverse tolerance induction. As is shown in Fig. 7
(A), the presence of IL-2, surface-bound but not soluble anti-CD28 mAb or untreated autologous APC prevented the inhibition of Th1 proliferation responses by ECDI- and ECDI/antigen-coupled autologous APC. To evaluate the ability of IL-2 or anti-CD28 antibody to reverse tolerance, T cells previously tolerized with ECDI/MBP-coupled APC were re-stimulated with MBP plus fresh APC with or without further addition of surface-bound anti-CD28 mAb or different concentrations of soluble anti-CD28 mAb or IL-2. As is shown in Fig. 7
(B), 20 ng/ml IL-2 completely reversed antigen-induced tolerance, whereas bound or soluble anti-CD28 mAb restored approximately two-thirds of the T cell response. Moreover, addition of IL-2 24 h after exposure to ECDI/antigen-coupled APC prevented further induction of apoptosis (Fig. 6
). Taken together, these data indicate that the mechanism of tolerance induced by ECDI/antigen-coupled APC involved both apoptosis and anergy.

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Fig. 7. Co-activation factors can prevent and reverse inhibition of Th1 responses by ECDI/saline-coupled or ECDI/antigen-coupled APC. (A) Prevention of tolerance induction in Th1 cells by co-culturing ECDI-coupled APC with plate bound but not soluble anti-CD28, IL-2 or untreated APC during the 18 h pre-culture period. Experiment 1: T cells specific for the MBP-149171 peptide (donor SS) were pre-cultured with untreated, ECDI/saline-coupled or ECDI/MBP-149171 peptide-coupled APC alone or in the presence of 20 µg/ml soluble anti-CD28, 50 ng/ml IL-2 or untreated APC. Treated T cells were separated on a Ficoll gradient and re-stimulated with fresh APC and MBP-149171 peptide in a proliferation assay (100% response = 9400 net c.p.m.). Experiment 2: T cells specific for HSV (donor EN) were pre-cultured with untreated or ECDI/HSV-coupled APC alone or in the presence of 20 µg/ml surface-bound anti-CD28 antibody. Treated T cells were separated on a Ficoll gradient and re-stimulated with fresh APC and HSV in a proliferation assay (100% response = 1900 net c.p.m.). (B) Reversal of tolerance induced in Th1 cells with ECDI-coupled APC by subsequent addition of anti-CD28 antibody or IL-2. Experiment 1: HSV-specific T cells (donor EN) pre-tolerized with ECDI/HSV-coupled APC were re-stimulated with HSV plus untreated APC in the absence or presence of 20 µg/ml surface-bound anti-CD28 antibody for 3 days and assessed for proliferation responses (100% response = 2500 net c.p.m.). Experiment 2: MBP-specific T cells (donor EN) pre-tolerized with ECDI/saline- or ECDI/MBP-coupled APC were re-stimulated with MBP plus untreated APC in the absence or presence of soluble anti-CD28 antibody or IL-2 at the indicated concentrations for 3 days and assessed for proliferation responses (100% response = 60,000 net c.p.m.).
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Expression of cell surface markers on APC and T cells
The apparent lack of functional co-stimulation suggested that the ECDI/antigen-coupled APC or responder T cells might have changes in the expression of contributing cell-surface proteins. Thus, phenotypic changes were evaluated in monocyte-enriched adherent cells after ECDI- or ECDI/antigen-coupling, and in antigen-tolerized T cells. As is shown in Table 2
, the expression of B7-1, B7-2 or HLA-DR on CD11b+ APC did not change as a result of ECDI/saline or ECDI/antigen cross-linking. However, several phenotypic changes were apparent in tolerized CD4+ Th1 cells. IL-2R (CD25) expression increased after the initial co-culture with ECDI/antigen cross-linked APC, but not with ECDI-APC alone, followed by a marked decrease in expression in both treatment groups 72 h after re-stimulation with antigen plus fresh APC (Table 3
). In contrast, non-tolerized T cells had strongly enhanced expression of IL-2R after re-stimulation. Additionally, pretreatment with either ECDI/antigen cross-linked APC or ECDI-APC followed by re-stimulation with antigen plus APC induced an increase in expression of HLA-DR, and a decrease in expression of CD28, B7-2 and possibly B7-1 (Table 3
).
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Discussion
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The observations described above provide unique evidence demonstrating that tolerization with ECDI/antigen-coupled APC differentially affects Th1 but not Th2 cells. Tolerization was a multi-step process that occurred when Th1 cells were activated in the absence of co-stimulation or ligation of IL-2R, and involved (i) an antigen-independent interaction, presumably between TCR and MHC but possibly involving other APC or T cell surface molecules (e.g. CD4), that induced partial signaling and resulted in moderate changes in T cell phenotype, proliferation response and cytokine secretion; and (ii) an antigen-dependent interaction with the TCR that induced more extensive signaling and resulted in pronounced changes that further potentiated the inhibitory effect. Antigen-specific inhibition was dependent upon the concentration of antigen and ECDI used to cross-link APC, and the time period of the coupling reaction. The inhibitory mechanism involved both apoptosis and anergy. Antigen-specific inhibition could be prevented during the tolerization step by the addition of IL-2, surface-bound anti-CD28 mAb, or untreated APC. Moreover, the tolerizing effect of exposure to ECDI/antigen-coupled APC on Th1 cells could be overcome during the re-stimulation step by either IL-2 or anti-CD28 antibody, suggesting a reversible anergy mechanism for those cells not committed to apoptosis. In contrast to Th1 cells, Th2 cells were resistant to tolerization with ECDI/antigen-coupled APC, showing neither antigen-specific nor antigen-non-specific inhibition after re-stimulation with untreated APC plus antigen, and relative resistance to apoptosis.
T cell activation usually requires at least two signals, one delivered through the TCR by the MHC/antigen complex on APC and a second co-stimulatory signal, e.g. through the CD28 molecule on the T cell that interacts with the B7 family on APC (2728) or the T cell OX-40 receptor that interacts with the APC OX-40 ligand (29). Activation induces expression of the IL-2R, and as long as IL-2 or other survival factors such as IL-4, IL-7 or IL-15 that can bind to the IL-2R common
chain are present (30), the T cell may persist for weeks or months. However, with depletion of such life-sustaining factors, most T cells undergo AICD/AIA. It is now well established that signaling through the T cell receptor in the absence of co-stimulation induces T cell anergy (6). Blockade of the CD28B-7 or OX-40ROX-40L interaction inhibits production of IL-2, and is sufficient to produce anergy (29,31) and eventually apoptosis in a majority of T cells unless IL-2 is available from another source.
It is now apparent that there are important differences in signaling pathways required for activation of Th1 versus Th2 cells. For example, early activation of Th1 but not Th2 cells involves protein tyrosine kinases Fyn and Zap-70, phospholipase C, and phosphatidyl inositol-4,5-biphosphate breakdown, whereas both cell types undergo mobilization of intracellular calcium but not Lck activation (32). In addition to differences in activation pathways, Th2 cells also have a decreased susceptibility to anergy induction and apoptosis (3336), a result also observed in our study. This resistance is not due to lack of activation or FasFasL expression, but rather may be due to an over-expression of life-sustaining Th2 cytokines (37) or protective factors such as Bcl-2 and Bcl-XL (38) or Fas-associated phospholipase (39,36) that resist induction of apoptosis.
The tolerizing signals delivered to antigen-specific Th1 clones by ECDI/antigen-coupled APC appear to be consistent with the mechanisms of anergy/apoptosis discussed above, with some degree of qualification. The ECDI cross-linking step probably constrains a wide range of cell surface molecules on the APC, including both MHC/antigen and the B-7 family of co-stimulatory molecules. The ECDI coupling reaction did not affect APC expression of HLA-DR, B7-1 or B7-2 (Table 2
) or fluorescence intensity of these molecules (not shown), indicating that conformational determinants recognized by specific mAb remained intact. However, both MHC/antigen and B7 molecules might be functionally altered by ECDI, due possibly to their decreased flexibility and mobility on the APC surface. Successful T cell activation appears to require cross-linking, aggregation and capping of the TCR, and it seems plausible that an inability of stimulatory and co-stimulatory molecules on the APC to migrate to a focal area of cell surface interaction with the T cell might produce suboptimal or disjointed signaling.
Given these considerations, it is important to consider how in the absence of specific antigen, ECDI-cross-linked APC could induce such strong inhibition of Th1 but not Th2 cells. One possibility is that the ECDI cross-linking somehow favors interaction of B-7 molecules with CTLA-4 rather than CD28. Cross-linking CTLA-4 but not CD28 on previously activated T cells can induce anergy/apoptosis (40) in the absence of IL-12 (41). However, for ECDI-coupled APC to induce inhibition and apoptosis, there would still be a TCR signaling requirement. Although specific peptide antigen was absent, it is conceivable that partial TCR engagement by cross-linked MHC II could provide enough of a signal to the TCR to trigger CTLA-4-induced apoptosis. Incubation with ECDI-coupled APC clearly induced changes in Th1 cells, including enhanced expression of HLA-DR, reduced expression of IL-2R, CD28 and B-7, and reduced secretion of IL-2 and IFN-
. Because the antigen-non-specific inhibition of Th1 cells could be negated by both IL-2 and surface-bound anti-CD28 mAb, it is likely that the inhibitory mechanism involved anergy leading eventually to apoptosis. Th2 cells were relatively resistant to the antigen-non-specific inhibitory affects of ECDI cross-linking, except at a high ECDI concentration. This resistance might well be due to growth-promoting effects of Th2 cytokines or selective induction of factors that resist apoptosis as discussed above.
Exposure of Th1 cells to ECDI/antigen-coupled APC induced a more profound antigen-specific tolerance that apparently involved the same mechanistic elements as tolerance induced without antigen. Incubation of Th1 cells with ECDI/antigen-coupled APC initially enhanced expression of the IL-2R, suggesting an early signaling event, and upon re-stimulation with antigen plus fresh APC there were marked decreases in IL-2R, CD28 and B7, and an increase in HLA-DR expression. Antigen-specific tolerance could be prevented with surface-bound anti-CD28 mAb, indicating a loss of signaling through CD28 during the interaction between the T cell and ECDI/antigen-coupled APC. Soluble anti-CD28 mAb could not prevent tolerance induction, suggesting that cross-linking of CD28 may be required for successful co-stimulation. IL-2 or untreated APC could also prevent tolerance induction, presumably by ligation of the up-regulated IL-2R or by providing alternative co-stimulatory signaling respectively. Of further interest, soluble anti-CD28 and IL-2 could reverse both antigen-specific and antigen-non-specific inhibition with ECDI/antigen-coupled APC, indicating that both effects involved a similar inhibitory mechanism and that co-stimulatory signals allowing full T cell activation could be delivered well after (
20 h) the initial altered MHC/antigen signal. Finally, treatment of Th1 cells with ECDI/antigen-coupled APC inhibited secretion of Th1 cytokines but did not enhance secretion of IL-4, thus indicating no shift towards a Th2 bias had occurred through delivery of an altered TCR/CD3-transduced signal, as has been reported during antigen presentation by T cells (42) or after `anergy' induction of Th0 cells (34).
The possibilities for utilizing ECDI/antigen cross-linked APC for in vivo human studies would appear to be limited due to the antigen-independent inhibitory effects and the need to prepare the APC tolerogens on an individual basis. However, the exquisite susceptibility of human Th1 cells to antigen-specific tolerance demonstrated in our study provides a strong rationale to develop more clinically compatible reagents with similar inhibitory properties. To this end, we have recently produced a soluble, single-chain TCR ligand, comprised of the ß1 and
1 MHC class II domains with or without bound peptide antigen (43). This genetically engineered molecule can specifically bind to antigen-specific T cells, has potent and selective inhibitory properties in vivo, and appears to deliver all or part of the antigen-specific signal through the TCR without coordinate co-stimulation of CD28, CTLA-4 or CD4. Such TCR ligands will be invaluable for dissecting the exact molecular interactions that drive the tolerization process, especially when studied in combination with various co-stimulatory agents (ligands or mAb), and represent a new class of reagents potentially useful for detection and elimination of autoreactive or inflammatory Th1 cells.
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Acknowledgments
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This work was supported by the Department of Veterans Affairs and the Nancy Davis Center Without Walls. The authors wish to thank Mrs Eva Niehaus for assistance in preparing the manuscript.
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Abbreviations
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AICD activation-induced cell death |
AIA activation-induced apoptosis |
APC antigen-presenting cells |
EAE experimental autoimmune encephalomyelitis |
ECDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide |
GM-CSF granulocyte colony stimulating factor |
HSV herpes simplex virus |
MBP myelin basic protein |
PBMC peripheral blood mononuclear cell |
TT tetanus toxoid |
TCR BV TCR ß chain variable region |
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Notes
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Transmitting editor: L. Steinman
Received 2 June 1999,
accepted 30 September 1999.
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