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,
Departments of
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Neurology,
Biochemistry and Molecular Biology, and
Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239; and
Department of Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97207
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Abstract |
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1 and 
1 domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP72–89) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP72–89 peptide-specific CD4+ T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP72–89-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP72–89 peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3
p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3
treatment produced stronger activation of these cellular events with additional activation of NF-
B and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-B or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4+ T cells. |
Introduction |
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Activation of CD4+ T cells in vivo is a multistep process initiated by coligation of the TCR and CD4 by the MHC class II/peptide complex presented on APCs as well as costimulation through additional T cell surface molecules, such as CD28 (5). Among a number of therapies for autoimmune disease that are being developed (6), two Ag-driven immunosuppression approaches of note are designed with the goal of direct disruption of normal CD4+ T cell activation. One involves modulation of T cells through the TCR with suboptimal TCR signals (altered peptide ligands (APLs)3), and the second involves modulating T cell behavior with optimal signals in the form of a variety of soluble MHC class II/peptide ligands in the absence of costimulation. APLs (7) are variant TCR-recognizing peptides that have conservative substitutions in the TCR interaction sites. After being processed and presented by APCs, APLs could act as partial TCR agonists or antagonists to block T cell activation by delivering partial (8) or inhibitory (9) signals. In contrast, soluble MHC class II/peptide ligands present native antigenic peptides in the context of MHC to T cells directly without costimulation from APCs. A variety of TCR ligands derived from MHC class II molecules have been developed and characterized (10, 11, 12, 13).
Ideally, the minimal TCR ligand could contain only the peptide and the 1 and 1 domains of the MHC molecule. Toward the long term goal of targeted Ag-driven immunosuppression of pathogenic T cells, we have developed and previously described a family of novel recombinant TCR ligands (RTLs) (14, 15, 16) that consist of the 1 and 1 domains of MHC class II molecules expressed as single exons, with and without antigenic peptides of interest genetically encoded at the 5' end (17). We previously reported that a recombinant TCR ligand (RTL201) containing the 72–89 peptide of guinea pig myelin basic protein (Gp-MBP72–89), successfully prevented and treated active and passive experimental autoimmune encephalomyelitis (EAE) (15). Amelioration of EAE was associated with a selective inhibition of the proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS. RTL200 (with rat MBP72–89 as tethered Ag peptide), which differs from RTL201 by a single methyl group (threonine instead of serine at position 80 of the MBP peptide), showed only minor clinical effects. Studies in vitro (16) showed that an RTL derived from human HLA-DR2, RTL303, induced partial T cell activation without proliferation in established Th1 cell lines specific for human MBP85–99. When used as a pretreatment agent, it showed strong inhibition of T cell proliferation induced by subsequent proper Ag stimulation. These novel RTLs have provided a platform for developing therapeutic agents for treatment of autoimmune disease. The cellular and molecular mechanisms responsible for the exquisitely selective inhibition of CD4+ T cells by RTLs, however, have not been elucidated.
Here, we report that immobilized RTL201 acts through the TCR in an Ag-specific manner, inducing partial T cell activation in an APC-free system. The partial T cell activation by RTL201 included a CD3 p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, and NFAT activation. Compared with anti-CD3 treatment, however, RTL201 could not activate NF-B or extracellular signal-regulated kinases (ERKs), and induced only a transient increase in IL-2 production. The experiments show that RTLs can work directly on T cells through a partial activation mechanism. The work provides a mechanism for the clinical efficacy of these rationally designed drugs in vivo that can be directly tested and lays the groundwork needed for continued development of these molecules for immunotherapeutic application and intervention in autoimmune disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The design, expression, and purification of RTLs (U.S. Patent 6,270,772) have been described previously (14, 15). There is a single amino acid difference between the Ag-peptide of RTL200 (rat-MBP72–89, PQKSQRTQDENPVVHF) and RTL201 (gp-MBP72–89, PQKSQRSQDENPVVHF). The residues underlined are the sole difference between the Ag-peptide sequence of RTL200 and RTL201. RTL200 was used as a negative control in some experiments. American hamster anti-mouse CD3 chain (anti-CD3, 145-2C11) was purchased from BD PharMingen (San Diego, CA). Plates were coated with 10 µg/ml of RTLs or anti-CD3 in PBS overnight 4°C. Plates were saturated after 12 h at this concentration as indicated by ELISA (data not shown), consistent with previous reports (18). Ionomycin (Iono), EGTA, U73122, U73343, xestospongin C (XeC), 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB8), and cyclosporine A (CsA) were purchased from Calbiochem (San Diego, CA).
Cells
The rat BV8S2+ T cell hybridoma C14/BW12-12A1 (A1) used in this study has been previously described. It was created by fusing an encephalitogenic LEW(RT1) T cell clone specific for Gp-MBP72–89 (19, 20) with a TCR (/)-negative thymoma, BW5147 (21). The A1 hybridoma secretes IL-2 when stimulated in the presence of APCs with whole Gp-MBP or Gp-MBP72–89 peptide.
Immunoblotting and immunoprecipitation
Primary Abs used in immunoblotting were specific for phosphorylated tyrosine (-pTyr, clone 4G10; Upstate Biotechnology, Lake Placid, NY), ZAP-70 phosphorylated at Tyr319 (New England Biolabs, Beverly, MA), and active ERKs (Promega, Madison, WI). Cells were lysed in 1% Nonidet P-40 lysis buffer, and then immunoblotting was conducted using an ECF Western blotting kit (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (16). Total protein for each sample was assayed using a detergent-compatible protein assay (Bio-Rad, Hercules, CA), and an equivalent amount of total protein was loaded in each lane of the Western blots. ZAP-70 phosphorylation was detected using whole cell lysates as previously described (22). For determining the CD3 phosphorylation pattern, immunoprecipitation was conducted with rabbit anti-mouse CD3 antiserum (provided by Dr. L. E. Samelson, National Institutes of Health, Bethesda, MD) and protein A-Sepharose (Upstate Biotechnology). The blots were then detected with clone 4G10 and the SuperSignal West Picco Kit (Pierce, Rockford, IL), exposed to Kodak Biomax light film (Eastman Kodak, Rochester, NY), and quantified using the ImageQuant 5.1 program (Amersham Pharmacia Biotech).
Calcium imaging and quantification
Cells were loaded with 0.5 M fura 2/AM in HBSS buffer (calcium and Mg2+ free; Life Technologies, Gaithersburg, MD) at room temperature for 30 min, washed, and suspended in RPMI for 30 min. When indicated, inhibitors were added to the medium 10 min before assay. The fura 2 ratio was recorded in coated or uncoated 
T dishes, by a 
Vision Image Restoration Microscopy system (Applied Precision, Issaquah, WA) focusing on the bottom of the T dishes. Fura 2 emission was detected at 510 nm after excitation by 340- and 380-nm filters. The fura 2 ratio changes in single cells were tracked and analyzed by softWoRx software (Applied Precision, Issaquah, WA).
EMSA
Whole cell extracts for NFAT and nuclear extracts for NF-B binding were prepared as previously described (23, 24). Extracts of 2–4 µg of protein were added into a final volume of 20 µl, which contained 5 µg of poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 5% glycerol, and 2 x 104 dpm of 32P-labeled consensus NFAT (CGCCC AAAGA GGAAA ATTTG TTTCA TA) or NF-B (AGTTG AGGGG ACTTT CCCAG GC) probes (New England Biolabs). Mutant probes (New England Biolabs) were used as negative controls for demonstrating specific binding. Mixtures were kept at room temperature for 20 min and were resolved by electrophoresis on 4% native polyacrylamide gels, which were then dried and exposed to Kodak Biomax MS film.
Intracellular IL-2 staining
A1 cells were split into 12-well plates (106 cells/well) and treated for 4 or 16 h at 37°C. GolgiStop (BD PharMingen) was added to the medium (0.67 µl/ml) 4 h before the end of stimulation. Cells were made permeable with a Cytofix/Cytoperm kit (BD PharMingen), stained with anti-rat IL-2 (10 µg/ml), and then PE-labeled anti-mouse IgG1 (10 µg/ml). Immunofluorescence was analyzed by FACScan (BD Biosciences, San Jose, CA) using CellQuest software.
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Results |
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and induces ZAP-70 phosphorylation
To comprehensively characterize the effect of RTL binding directly to the TCR, we first examined whether RTL treatment leads to changes in the CD3 phosphorylation pattern of A1 T cells. As shown in Fig. 1A, both immobilized RTL201 (containing Gp-MBP72–89 peptide) and anti-CD3 triggered a prominent emergence of p23 (the fully phosphorylated pattern of mouse CD3) and enhanced p21 (the partially phosphorylated pattern of mouse CD3). The p23/p21 ratio was increased 2-fold by RTL201 (from 0.25 to 0.5) and almost 4-fold by immobilized anti-CD3 (from 0.25 to 0.93). RTL200, which contains the rat MBP72–89 Ag peptide (threonine instead of serine at position 80 of the MBP peptide), consistently showed an increased level of p21, but did not have an significantly altered p23/p21 ratio compared with that in the vehicle control. We also looked at CD3 chain-associated ZAP-70, which serves as an additional indicator of signaling through the TCR/CD3 complex (25, 26). Immunoblot analysis with a primary Ab specific for Tyr319-phosphorylated ZAP-70 was used to detect the activation of ZAP-70 from whole cell lysates (Fig. 1B). RTL201 treatment significantly increased the phosphorylation levels of ZAP-70 Tyr319 within 5 min of treatment, while RTL200 induced no significant increase in ZAP-70 phosphorylation. Immobilized anti-CD3 was consistently more effective than RTL201 in raising the level of ZAP-70 phosphorylation. In summary, RTL201 increased the p23/p21 ratio of CD3 and increased ZAP-70 phosphorylation, but these primary signal transduction events induced after RTL engagement with the TCR were weaker than those induced by anti-CD3. Importantly, the effect of RTL treatment was exquisitely Ag specific, with a single amino acid substitution in the RTL-coupled Ag, as demonstrated by RTL200 treatment, showing very little effect on the p23/p21 ratio of CD3 and virtually no increase in ZAP-70 phosphorylation.
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induce differential calcium signaling
We have previously reported that human HLA-DR2-derived RTL303 triggered an Ag-specific increase in intracellular calcium in human T cell clones (16). APL-induced calcium signaling has also been previously documented (7). Here we monitored the change in intracellular calcium levels of A1 cells as a consequence of different activation treatments, using single-cell imaging and recording. As shown in Fig. 2, A and B, both immobilized RTL201 and anti-CD3 caused Fura 2/AM-loaded cells to turn bright green, indicating elevation of intracellular calcium levels. Quantitatively (Fig. 2B and Table I), the peak level of intracellular calcium induced by anti-CD3 treatment was almost 2-fold higher than that induced by RTL201 treatment (2.61 vs 1.40). Calcium activation by RTL201 was Ag specific, as no calcium elevation was observed following RTL200 treatment. The different percentages of cells activated by anti-CD3 and RTL201 (83 and 39%, respectively) are also indicative of differential early downstream consequences following treatment with the two agents. All these reagents had stimulatory effects only when immobilized on a surface. Treatment with soluble RTL201, anti-CD3, or anti-CD28 did not induce calcium elevation (data not shown).
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and the relative peak calcium concentration to a level similar to that induced by RTL201 treatment with or without EGTA present (Fig. 2C and Table I). XeC (a membrane-permeable blocker of inositol trisphosphate-mediated calcium release, 1 µM) drastically blocked calcium elevation in cells treated with either RTL201 or anti-CD3 (Fig. 2C and Table I), and EGTA plus XeC completely blocked calcium mobilization in cells stimulated with anti-CD3 (Fig. 2C and Table I). The differential effects of signaling inhibitors and calcium chelators provide a clear picture of the sources of calcium mobilized by these different reagents, demonstrating that anti-CD3 mobilized calcium from both internal and external sources, while RTL201 treatment mobilized calcium only from internal stores.
Phospholipase C
(PLC) is a common mediator between receptor stimulation and calcium mobilization (27). Pretreatment of A1 cells with the PLC inhibitor U73122 (10 µM) prevented calcium mobilization by both RTL201 and anti-CD3. U73343 (an analog of U73122 with minimal biological function, used as a negative control, 10 µM) did not block calcium mobilization (data not shown). The results showed that calcium mobilization by both RTL201 and anti-CD3 is strictly PLC dependent.
Differential activation of NFAT, NF-B, and ERKs
We next explored whether RTL201 and anti-CD3 induced differential activation of transcription factors. Using an EMSA, we monitored transcriptional activation with specific probes for NFAT and NF-B. As shown in Fig. 3A, 30-min treatment with RTL201 or anti-CD3 resulted in a marked increase in NFAT activity, although anti-CD3 treatment showed a significantly stronger effect. This difference in potency was consistent with the differences in amplitude of calcium flux observed. Treatment with Iono (a calcium ionophore; 250 µg/ml) to pump in calcium from the extracellular milieu also resulted in NFAT activation (Fig. 3A). RTL200 showed very little effect on NFAT activity. The increase in NFAT activity observed following RTL201 or anti-CD3 treatment was blocked by TMB8 (a cell-permeable calcium antagonist; 1 mM) and CsA (an inhibitor of the calcium/calmodulin-dependent phospholipase calcineurin; 2 µM) (28), indicating that NFAT was activated via a calcium/calcineurin-dependent pathway (Fig. 3B).
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led to a dramatic increase in NF-B activity, while no significant increase in NF-B activity was detected after RTL201 or RTL200 treatment. These EMSA results were further confirmed by immunoblot assay of IB phosphorylation, which releases active NF-B when it is phosphorylated or degraded (29). IB was phosphorylated after anti-CD3 treatment, but not after RTL201 or RTL200 treatment (data not shown). No IB degradation was observed at the 30 min point of our study.
Although both NFAT and NF-B are essential for T cell function and activation, other signaling factors are required for full T cell proliferation and IL-2 production. In T cells, phosphorylation of ERKs leads to the formation of the AP-1 complex (30), which is a partner of NFAT in the initialization of IL-2 mRNA transcription (31). As shown in Fig. 4A, anti-CD3 treatment (10 µg/ml) induced strong ERK phosphorylation, which peaked at 30 min and declined to the basal level by 60 min. In contrast, RTL201 (10 µg/ml) showed no significant effect on ERK phosphorylation levels. As shown in Fig. 4B, the dose-dependent induction of ERK phosphorylation following anti-CD3 treatment could be readily detected at concentrations as low as 5 µg/ml, whereas no induction of ERK phosphorylation was observed following RTL201 treatment, even at 20 µg/ml.
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RTL201 engagement with the TCR induced a subset of signal transduction events that were initiated by binding of anti-CD3 to the TCR. We employed intracellular staining to examine whether the T cell signaling induced by RTL201 induced the production of IL-2. As shown in Fig. 5A, intracellular IL-2 levels were significantly increased after 4-h treatment with immobilized RTL201, anti-CD3, or Iono/PMA, while RTL200 induced no significant change above the basal level. Notably, IL-2 production after RTL201 treatment was short-lived and returned to background levels by 16 h (Fig. 5A). This was dramatically different from the observed IL-2 production following treatment with plate-bound anti-CD3 or Iono/PMA, in which case IL-2 was still accumulating at 16 h (Fig. 5A). IL-2 production induced by RTL201 and antiCD3 was calcium dependent, as shown using TMB8 (1 mM), an intracellular calcium antagonist (Fig. 5B). IL-2 production induced by RTL201 and anti-CD3 was also PLC dependent, as shown using U73122 (10 µM), a PLC inhibitor, and its analog U73343 (10 µM) as a negative control (Fig. 5B). Thus, anti-CD3 and Iono/PMA induced a sustained increased IL-2 production that was both calcium and PLC dependent. RTL201 induced a significantly increased level of IL-2 production that was also calcium and PLC dependent, but this increase was transient, returning to basal levels by 16 h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B or ERKs, limiting specific short term effector activities that may influence the capacity of CD4+ T cells to respond to environmental cues, as required during the polarization of an immune response.
The interaction between RTL and TCR was exquisitely dependent on the covalently tethered Ag. The Gp-MBP72–89 epitope carried by RTL201 represents the dominant encephalitogenic determinant in the LEW rat, whereas the corresponding 72–89 peptide of rat MBP carried by RTL200 differs by a single conservative residue substitution (T instead of S at position 80). This seemingly minor difference in sequence has profound immunological effects in LEW rats, however, with the rat-MBP72–89 peptide possessing 
10-fold less encephalitogenic activity on a molar basis (20, 21).
Most T cells induced after immunization with Gp-MBP72–89 are RT1.B restricted, but are only weakly stimulated with Rt-MBP72–89 (32). Conversely, most T cells induced after immunization with Rt-MBP72–89 are also RT1.B restricted, but can still be stimulated efficiently with Gp-MBP72–89 (33). These findings suggest that TCR interactions are stronger with the guinea pig than with the rat epitope. This could occur if the hydroxyl side chain at position 80 of serine provided a dominant interaction with hydrogen accepting TCR residues of Gp-MBP72–89-specific T cells; in Rt-MBP72–89, the presence of the extra methyl group in threonine might limit the mobility, and hence the binding, of the hydroxyl group, thus impeding this potentially important interaction.
In previous studies we presented data demonstrating the profoundly different clinical effects of RTL201 and RTL200 on EAE. RTL201 (encoding Gp-MBP72–89) was shown to have potent suppressive and therapeutic activities for actively induced disease, substantially reducing the proliferative response of draining lymph node T cells (15). Even more striking, RTL201 completely suppressed passive EAE induced after transfer of Gp-MBP72–89-specific T cells, but had no effect on passive EAE induced with a different I-E (RT1.D)-restricted T cell line specific for a distinct encephalitogenic determinant, MBP87–99 (15). Treatment with RTL201 prevented infiltration of both CSFE-labeled donor T cells as well as host-recruited inflammatory cells into the CNS. In contrast, RTL200 (encoding Rt-MBP72–89) showed only a very mild suppression of actively and passively induced disease, a slight reduction of infiltrating cells into the CNS, and only a mild decrease in the proliferative response of draining lymph node T cells.
To begin unraveling the mechanism by which RTL201 had such profound clinical efficacy in our preclinical animal studies, we carefully documented signal transduction events within CD4+ T cells that couple RTL engagement with TCR to a biologically important effector function, increased IL-2 production. We used the A1 T cell hybridoma to carry out our studies under rigorously controlled conditions in the absence of APCs. Plate-bound RTL engagement of TCR showed very specific effects on the cells depending on which peptide Ag was covalently tethered within the RTL presentation platform. It is notable that RTLs have low affinity for TCRs, and the affinity constant (Kd) of RTL201 for a single-chain TCR construct containing BV8S2 carried by the A1 hybridoma was estimated to be 2.7–2.9 µM, as described in a recently submitted manuscript (42).
Upon TCR engagement with MHC class II on the surface of APCs, or, as documented here, after interaction with RTL, initiation of Ag-driven T cell activation can be detected as early as 5 min, beginning with tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs within the cytoplasmic domains of CD3-chain. Discrete phosphorylated forms of CD3 have been identified in T cells that reflect different discrete signaling states, including partially phosphorylated (p21) and fully phosphorylated (p23) CD3, with apparent molecular sizes of 21 and 23 kDa, respectively (8). Allen and colleagues (7) in elegant studies with APLs demonstrated that these discrete states could be correlated to biological activity (i.e., T cell activation and proliferation, downstream effector function) by quantifying the p23/p21 ratio of TCR CD3. Full TCR agonists increased the p23/p21 ratio, while antagonist and partial agonists tend to increase p21 only. CD3 phosphorylation, in turn, leads to a second discrete downstream event, ZAP-70 phosphorylation, with full agonists producing increased ZAP-70 phosphorylation, while antagonist and partial agonists cause no increase in ZAP-70 phosphorylation.
A cognate RTL presents an optimal signal 1 to the T cell through the TCR in the absence of costimulation, while APLs deliver a suboptimal signal 1 to the T cell through the TCR in the appropriate optimal costimulatory context (signal 2). While these different experimental conditions are substantial, interesting comparisons can be made that shed light on how information is processed by the TCR, as discussed further below. RTL201 induced a 2-fold increase in the p23/p21 ratio of CD3, from 0.25 to 0.5 (Fig. 1A). As would be predicted from Allen’s studies, this resulted in a dramatic increase in ZAP-70 phosphorylation (Fig. 1B). RTL200 showed a slight increase in p21 levels, but did not increase the p23/p21 ratio of CD3. Again, as would be predicted from Allen’s studies, RTL200 showed no effect on ZAP-70 phosphorylation (Fig. 1, A and B). Anti-CD3 treatment, used as a positive control in our studies, was found to markedly increase the p23/p21 ratio of CD3, from 0.25 to 0.93, and, again as predicted from Allen’s studies, resulted in a dramatic increase in ZAP-70 phosphorylation (Fig. 1, A and B).
The CD3 and ZAP-70 phosphorylation triggered by RTL treatment was further corroborated by the documentation of differential downstream calcium mobilization patterns induced by the different treatments. RTL201 triggered calcium mobilization from internal stores only (no effect of EGTA), while RTL200 had no significant effect at the level of calcium signaling (Table I). Anti-CD3, used as a positive control, induced calcium mobilization from both internal and external stores (Table I). The PLC inhibitor U73122 completely blocked calcium mobilization by both RTL201 and anti-CD3, showing that calcium mobilization by either treatment is strictly PLC dependent (Table I). Using language developed to describe APLs and their effect on the biological activity of T cells, anti-CD3 could be considered a strong TCR agonist, and RTL200 a TCR antagonist. RTL201, however, shows both similarities and differences to partial agonists. Both RTL201 and APLs bind TCR and induce partial T cell activation, including increased p21 levels (34) and calcium elevation (35, 36). However, APL partial agonists do not increase the p21/p23 ratio or result in ZAP-70 phosphorylation (8), while RTL201 does.
RTL200 had no detectable effect on transcription factor activation, while RTL201 and antiCD3 differentially induced specific activation of transcription factors. RTL201 and anti-CD3 both induced an increase in NFAT activity (Fig. 3A). Anti-CD3 treatment showed a significantly stronger effect, consistent with the different magnitude of calcium flux observed. The increase in NFAT activity observed following RTL201 or anti-CD3 treatment was blocked by TMB8 and CsA, indicating that NFAT was activated through a calcium/calcineurin-dependent pathway (Fig. 3B). RTL201 treatment had no significant effect on NF-B activity, while anti-CD3 treatment led to a dramatic increase in NF-B activity (Fig. 3C), confirmed secondarily by monitoring IB phosphorylation. IB showed significantly increased phosphorylation after antiCD3 treatment, but not after RTL201 or RTL200 treatment (data not shown). Anti-CD3 treatment also induced strong ERK phosphorylation, whereas RTL201 showed no significant effect on ERK phosphorylation levels (Fig. 4). Similar observations were made in an earlier study using human Th1 cells treated with HLA-DR2-derived RTLs. In that study Ag-specific RTL treatment triggered TCR -chain phosphorylation, increased intracellular calcium, and attenuated the level of activated phosphorylated ERK (16).
Differential modulation of calcium signaling appears to be responsible for the differences between RTL201 and anti-CD3 treatments observed at the transcriptional level, a result of mobilization of calcium from internal stores by RTL201 without triggering an influx of extracellular calcium. Studies in B cells have shown that large calcium spikes selectively activate NF-B and c-Jun N-terminal kinase, whereas NFAT is activated by a low and sustained calcium plateau (37, 38). In our study we show that the low, but sustained, intracellular calcium flux caused by RTL201 was associated with NFAT activation, but not activation of NF-B or ERKs. In contrast, mobilization of both internal and external calcium stores by anti-CD3 treatment caused high amplitude calcium peaks that were associated with NF-B and ERK activation. This activation may involve activation of PKC (39) and Ras (40), respectively, with the continuity of anti-CD3-triggered calcium flux over 15 min meeting the duration requirements of NFAT (41). Of significance, this uncoupling of the transcription factor NFAT from NF-B and ERKs during partial activation through the TCR cannot be overcome by increasing the concentration of RTL201, which is at a saturating dose for TCR response at the concentration used.
Completing the circuit from TCR binding to biological effector function, RTL201 engagement with the TCR in the absence of costimulation resulted in a transient increase in IL-2 production. Intracellular IL-2 levels were significantly increased after 4 h of RTL201 treatment, returning to background levels by 16 h (Fig. 5A). RTL200 had no effect on IL-2 production, and anti-CD3 caused a sustained increase in IL-2, still accumulating after 16 h (Fig. 5A). IL-2 production induced by RTL201 and anti-CD3 was both calcium and PLC dependent, as shown using the intracellular calcium antagonist TMB8 and the PLC inhibitor U73122 (Fig. 5B).
In conclusion, we showed in this study that RTL201 binds cognate TCR and leads directly to partial T cell activation in an APC-free system, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. Our findings provide the first molecular explanation for how these novel therapeutics may work. The unique pattern of early TCR signaling followed by a unique downstream activation pattern offers a mechanism for the clinical efficacy of these rationally designed drugs in vivo important for the continued development of these molecules for immunotherapeutic application and intervention in autoimmune disease.
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Acknowledgments |
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antiserum. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Gregory G. Burrows, Department of Neurology, UHS-46, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239. E-mail address: ggb{at}ohsu.edu
3 Abbreviations used in this paper: APL, altered peptide ligand; CsA, cyclosporine A; EAE, experimental autoimmune encephalomyelitis; ERK, extracellular signal-regulated kinase; Gp-MBP, guinea pig myelin basic protein; Iono, ionomycin; LEW, Lewis; PLC, phospholipase C; RTL, recombinant TCR ligand; XeC, xestospongin C; TMB8, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate.
Received for publication March 7, 2003. Accepted for publication June 9, 2003.
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References |
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S. Sinha, S. Subramanian, T. M. Proctor, L. J. Kaler, M. Grafe, R. Dahan, J. Huan, A. A. Vandenbark, G. G. Burrows, and H. Offner A Promising Therapeutic Approach for Multiple Sclerosis: Recombinant T-Cell Receptor Ligands Modulate Experimental Autoimmune Encephalomyelitis by Reducing Interleukin-17 Production and Inhibiting Migration of Encephalitogenic Cells into the CNS J. Neurosci., November 14, 2007; 27(46): 12531 - 12539. [Abstract] [Full Text] [PDF]
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A. P. Fontenot, T. S. Keizer, M. McCleskey, D. G. Mack, R. Meza-Romero, J. Huan, D. M. Edwards, Y. K. Chou, A. A. Vandenbark, B. Scott, et al. Recombinant HLA-DP2 Binds Beryllium and Tolerizes Beryllium-Specific Pathogenic CD4+ T Cells J. Immunol., September 15, 2006; 177(6): 3874 - 3883. [Abstract] [Full Text] [PDF]
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J. Chun and A. Prince Activation of Ca2+-Dependent Signaling by TLR2 J. Immunol., July 15, 2006; 177(2): 1330 - 1337. [Abstract] [Full Text] [PDF]
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 G. Adamus, G. G. Burrows, A. A. Vandenbark, and H. Offner Treatment of Autoimmune Anterior Uveitis with Recombinant TCR Ligands. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2555 - 2561. [Abstract] [Full Text] [PDF]
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H. Offner, S. Subramanian, C. Wang, M. Afentoulis, A. A. Vandenbark, J. Huan, and G. G. Burrows Treatment of Passive Experimental Autoimmune Encephalomyelitis in SJL Mice with a Recombinant TCR Ligand Induces IL-13 and Prevents Axonal Injury J. Immunol., September 15, 2005; 175(6): 4103 - 4111. [Abstract] [Full Text] [PDF]
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J. Huan, S. Subramanian, R. Jones, C. Rich, J. Link, J. Mooney, D. N. Bourdette, A. A. Vandenbark, G. G. Burrows, and H. Offner Monomeric Recombinant TCR Ligand Reduces Relapse Rate and Severity of Experimental Autoimmune Encephalomyelitis in SJL/J Mice through Cytokine Switch J. Immunol., April 1, 2004; 172(7): 4556 - 4566. [Abstract] [Full Text] [PDF]
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