Analysis of autoreactive T cells associated with murine collagen-induced arthritis using peptideMHC multimers
Jason C. Huang1,
Mikael Vestberg2,
Alfredo Minguela3,
Rikard Holmdahl2 and
E. Sally Ward1
1 Center for Immunology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390-9093, USA 2 Section of Medical Inflammation Research, BMC, Lund University, 22184 Lund, Sweden 3 Seccion de Inmunologia, Hospital Universitario Virgen de la Arrixaca, El Palmar, 30210 Murcia, Spain
Correspondence to: E. S. Ward; E-mail: Sally.Ward{at}Utsouthwestern.edu
Transmitting editor: M. Feldmann
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Abstract
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CD4+ T cells that recognize residues 256270 of type II collagen (CII) associated with the I-Aq (Aq) molecule play a central role in disease pathogenesis in murine collagen-induced arthritis (CIA). Disease is most efficiently induced by immunization with heterologous CII, which elicits heterologous, e.g. bovine, CII256270:I-Aq-specific T cells that only poorly cross-react with mouse CII. The self-epitope differs from heterologous CII256270 by a conservative change of glutamic acid (heterologous) to aspartic acid (mouse) at position 266 which confers a lower affinity for binding to the I-Aq molecule. To date, characterization of the nature of T cell recognition in this model has been hindered by the lack of suitable, labeled multimeric peptideMHC class II complexes. Here, we describe the biochemical properties of both recombinant bovine CII256270:I-Aq (bCII256270:I-Aq) and mouse CII256270:I-Aq (mCII256270:I-Aq) complexes, and use these as fluorescently labeled multimers (tetramers) to characterize the specificity of CII-reactive T cells. Our analyses show that an unexpectedly high percentage of bCII256270:I-Aq-specific T cells are cross-reactive with mCII256270:I-Aq. Interestingly, one T cell clone which has a relatively high avidity for binding to self-CII256270:I-Aq shows a marked increase in binding avidity at physiological temperature, indicating that this TCR has unusual thermodynamic properties. Taken together, our analyses suggest that the low affinity of mCII256270 for I-Aq may lead to a state of ignorance which can be overcome by priming CII-specific T cells with heterologous CII. This has relevance to understanding the mechanism by which CIA is induced and provides an explanation for the low arthritogenicity of mouse CII.
Keywords: autoimmunity, MHC, rheumatoid arthritis, T lymphocyte, TCR
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Introduction
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Collagen-induced arthritis (CIA) is believed to be a representative model of rheumatoid arthritis in humans (1). Arthritis can be induced in susceptible rodents and primates by injection of type II collagen (CII), and is characterized by both B and T cell responses against CII (2,3). In murine CIA, susceptibility is controlled by the MHC region (4,5) and linked to the MHC class II Ab gene, in the susceptible II-2q haplotype (46). Immunization of DBA/1 mice (which express Aq) with CII induces T cell responses that are primarily directed towards the immunodominant epitope, CII residues 256270 (core of epitope is 260267), associated with Aq (712). In addition, analogs of this peptide can modulate CIA (11), indicating that CII256270:Aq-specific T cells are centrally involved in pathogenesis. This peptide binds to the Aq molecule primarily through isoleucine (I260) and phenylalanine (F263) interactions with the P1 and P4 pockets respectively of the Aq molecule (13), whereas the major T cell contact residue is lysine (K264) that can be post-translationally modified (13,14). Interestingly, the CII-specific T cells only poorly cross-react with mouse CII (8), which differs from heterologous (bovine, chick, rat, human) CII by a glutamic acid (heterologous) to aspartic acid (murine) change at position 266. The observation that immunization with mouse CII results in a higher T cell response to heterologous CII (a heteroclitic response) has been taken as an indication that the poor cross-reactivity is due to a difference in affinity of the peptide for Aq rather than at the level of T cell recognition (8). However, at high antigen concentrations many of these CII-specific T cells also recognize mouse CII256270 (mCII256270) (15). Such potentially autoreactive, mouse CII-specific T cells are of particular interest as they most likely play a central role in the disease process.
For MHC class I-restricted T cells, multimeric MHC molecules have been widely used for the enumeration of antigen specific T cells (1618). These multimeric peptideMHC (pMHC) complexes can also be used to characterize the avidities of responding T cells (1823). However, less data are available for MHC class II- relative to MHC class I-restricted responses and this is particularly so in murine models of autoimmunity (24,25). This is most likely due to the relative instability of I-A complexes (2628), which are invariably the restricting elements associated with these models. Several laboratories, including our own, have shown that stable peptideI-A complexes can be made by covalently tethering the antigenic peptide to the I-Aß chain and stabilizing
ß chain association with acidic/basic zippers (2529). This approach can be used to generate appropriate multimers/tetramers to detect antigen-specific, autoreactive T cells in both the murine experimental autoimmune encephalomyelitis (EAE) (24) and non-obese diabetic (NOD) models (25). More recently we have shown that analogous bovine CII256270 (bCII256270):Aq complexes can be used to detect antigen-specific T cells in the draining lymph nodes of CII-immunized mice (30). To date, however, multimers comprising mCII256270:Aq complexes, which represent the autoantigen, have not been described. The use of these complexes in the current study has allowed us to directly compare recognition of the heterologous and autologous epitopes by CII-specific T cells.
Analyses of TCRpMHC interactions using soluble molecules indicate that TCRs bind to cognate ligands with relatively slow on-rates and fast off-rates (31). The low on-rate suggests that some conformational rearrangement is necessary for complex formation. Consistent with the involvement of induced fit, thermodynamic analyses have shown that TCRpMHC interactions for both MHC class I and class II systems are highly temperature dependent, and are entropically unfavorable (3234). X-ray crystallographic studies have demonstrated that the TCRpMHC interface shows poor complementarity (3538). Together with indications of conformational rearrangements for the TCR, this has led to the conclusion that TCRs are highly plastic (36,39). Plasticity of TCRpMHC interactions would be predicted to contribute to the cross-reactive nature of T cells (4044), as conformational adjustments of TCR residues might allow multiple pMHC ligands to be recognized with an appropriate affinity to result in activation of the corresponding T cell (3234). This cross-reactivity impacts many areas of T cell biology, including the activation of autoreactive T cells by molecular mimicry (43,4547).
We have recently shown in the model of murine EAE that it is possible to gain some insight into the temperature dependence of the avidities of TCRpMHC interactions by using appropriate pMHC tetramers to stain T cells at different temperatures (34). This approach can be used, at least in qualitative terms, to assess the relative plasticity of a particular TCRpMHC interaction, and we have shown correlative data between tetramer binding and thermodynamic analyses using surface plasmon resonance (34). There is a paucity of knowledge concerning the molecular details, including the plasticity, of TCRpMHC interactions in the CIA model. This is of particular interest as, in contrast to the majority of other inducible models of autoimmunity for which autoantigen is used as immunogen, heterologous CII is much more effective than autologous CII in inducing CIA (1,48). The lower efficacy of mouse CII in inducing arthritis is most likely due to weaker binding of the aspartic acid (D266)-substituted CII peptide to Aq (13). Transgenic expression of CII with glutamic acid at position 266 (E266) leads to higher exposure of CII, but also to partial T cell tolerance (7). Clearly, the degree of T cell recognition of self-CII is central for the disease process. A critical question is whether the poor cross-reactivity of heterologous CII-specific T cells with the immunodominant self-CII peptide is only due to a difference in peptideMHC binding affinity or is also caused by alteration in the disposition of the T cell contact residues of the peptide. Here, we directly address this issue.
The goal of the current study was to characterize tetrameric bCII260270:Aq and mCII260270:Aq complexes, and to use these reagents to carry out an analysis of antigen-specific T cells. We show that these tetramers can be used to stain appropriate hybridomas and responding T cells in antigen-specific, short-term lines. Further, we have used tetramer staining to analyze, in qualitative terms, the thermodynamics of TCRpMHC interactions in this disease model. Our findings indicate that cross-reactive recognition of mCII256270 by CII-specific T cells is much higher than anticipated from earlier studies in which antigen-pulsed antigen-presenting cells (APCs) were used to assess autoreactive responses (8,15). This high cross-reactivity has relevance to understanding the mechanism by which heterologous CII acts as a potent arthritogen.
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Methods
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Mice and cells
DBA/1LacJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in a pathogen-free environment in the animal facility at the University of Texas Southwestern Medical Center at Dallas. bCII256270-specific T cell hybridomas HRC.1, HRC.2, HCQ.4 and HDB.2 have been described previously (14). The 172.10 hybridoma, which is specific for the N-terminal epitope of myelin basic protein (MBP) associated with I-Au (49), was generously provided by Dr Joan Goverman. A subclone of this hybridoma (34) was used in the current study.
Antigens and antibodies
Bovine CII was purchased from Chondrex (Redmond, WA). The peptides bCII256270, mCII256270 and mCII254274 were synthesized at the Peptide Synthesis Unit of the Howard Hughes Medical Institute, UT Southwestern Medical Center (Dallas, TX). Phycoerythrin (PE)-labeled anti-TCR Cß (H57-597) was purchased from PharMingen (San Diego, CA). FITC-labeled anti-CD8 (RM2201) and allophycocyanin- labeled anti-CD4 (RM2505) were purchased from Caltag (Burlingame, CA). 7-Aminoactinomycin D and cytochalasin D were purchased from Sigma (St Louis, MO).
Generation of peptide:Aq tetramers
The constructs used for the expression of recombinant Aq molecules were derived from previously described constructs used to generate bCII260270:Aq complexes (28). The leader sequences of
and ß chains were replaced by a signal peptide sequence derived from honey bee melittin. A KpnI site and a flexible linker (GSGSGSS) sequence were inserted between the honey bee melittin signal peptide (50) and the ßq chain sequence. The KpnI site was used for insertion of the corresponding sequences of bCII256270, mCII256270 or myelin oligodendrocyte protein (MOG) 7990 peptide [the latter peptide binds to Aq (51) and was used to generate a control tetramer]. The
and ß chains were truncated before the transmembrane region, and sequences encoding acidic and basic zippers were inserted as described (28). In addition, the ß chain also encodes a biotinylation signal peptide following the basic zipper. Both
and ß chains were tagged with C-terminal polyhistidine tags and cloned into the dual baculovirus expression vector pAcUW51 (PharMingen, San Diego, CA). The recombinant plasmids were co-transfected with Baculogold DNA (PharMingen) into Sf9 cells, and recombinant viruses plaque purified and used to make high-titer virus stocks. HIGH FIVE cells (Invitrogen, Carlsbad, CA) were infected with virus stocks and supernatants from 62- to 64-h cultures harvested. Recombinant protein was purified using Ni-NTA2+ affinity columns. Following overnight dialysis against PBS, recombinant CII256270:Aq complexes were further purified using a second affinity column made by coupling Y-3P to Protein GSepharose [Y-3P recognizes Aq in a conformational dependent way (52)]. Recombinant proteins were dialyzed into biotinylation buffer (20 mM TrisHCl, 50 mM NaCl, pH 8.0) overnight. Biotin ligase (Avidity, Denver, CO) was added to a final concentration of 10 µg/ml and the reaction was carried out at room temperature for 1620 h. Following biotinylation, free biotin was removed by extensive dialysis. Tetrameric peptide:Aq complexes were generated by adding PE-labeled Extravidin (Sigma, St Louis, MO) to the recombinant protein at a molar ratio of 1:4.
Maintenance of T cell hybridomas
Bovine CII-specific T cell hybridomas (HRC.1, HRC.2, HCQ.4 and HDB.2) and 172.10 hybridoma cells were maintained in complete DMEM: DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Life Technologies), penicillin/streptomycin (100 U/ml; Life Technologies), non-essential amino acids (0.1 mM; Life Technologies), sodium pyruvate (1 mM; Life Technologies) and 2-mercaptoethanol (55 µM; Life Technologies).
Production of short-term T cell lines
Male DBA/1LacJ mice (812 weeks old) were immunized intradermally with 100 µg bovine CII in complete Freunds adjuvant (Sigma) at the tail base. Ten days following immunization, splenocytes and lymph node cells were extracted and expanded in vitro with 50 µg/ml bovine CII for 3 days. Following separation with NycoPrep 1.077A (NycoMed Pharma, Oslo, Norway), the cells were cultured in IL-2 (80 U/ml) complete DMEM for 57 days. Thereafter, the T cells were re-stimulated with irradiated (2500 rad), syngeneic spleen cells and 50 µg/ml bovine CII for 3 days, and then IL-2 for 57 days. A short-term line was established by in vitro expansion 4 times before staining with peptide:Aq tetramers and other antibodies. This short-term line was maintained in complete DMEM.
Tetramer staining and flow cytometric analysis
Staining with peptide:Aq tetramers was carried out in the presence of 10 µg/ml anti-CD3
(145-2C11) at 37°C for 30 min as described (34) unless otherwise indicated. For the analysis of the effect of temperature on staining, TCRs of hybridoma cells were either pre-clustered by incubation with 10 µg/ml anti-CD3
(145-2C11) at 37°C for 30 min or untreated. Following this, cells were treated with 100 µM cytochalasin D at room temperature for 5 min and then incubated with bCII256270:Aq tetramers at 12, 25 or 37°C for 1 h. Levels of tetramer staining were normalized for TCR expression using PE-labeled anti-TCR ß chain antibody, H57-597, as described (24,34). Multi-color staining of short-term T cell lines was performed in two steps. Cells were pre-incubated with allophycocyanin-labeled anti-CD3
(145-2C11) for 30 min at 37°C. Following a wash in PBS, cells were incubated with PE-labeled tetrameric bCII-, mCII- or MOG-Aq complexes and PerCP-labeled anti-CD4 at 12°C for 2 h. Stained cells were analyzed with a FACSCalibur (Becton Dickinson) and data analyzed using WinMDI (Scripps Research Institute).
T cell activation assays
T cell hybridomas were stimulated with serially diluted plate-bound anti-CD3
(145-2C11), bCII256270:Aq and mCII256270:Aq as described (28,53). For some assays, T cell hybridomas were incubated with Aq-expressing splenocytes (from DBA/1 mice) pulsed with bCII256270 or mCII256270. In all stimulation assays, cells were stimulated in duplicates for 24 h at 37°C and IL-2 levels in culture supernatants quantitated by IL-2 ELISA (53). To account for differences in TCR expression levels, normalized IL-2 levels were obtained by dividing the optical density at 450 nm (OD450) by the OD450 corresponding to responses to immobilized anti-CD3
(54,55).
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Results
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Expression and SDS stability of the Aq molecules
Recombinant Aq molecules covalently tethered to mouse or bovine CII256270 were expressed and purified from baculovirus-infected HIGH FIVE cells in yields similar to those reported for the N-terminal epitope of MBP tethered to Au (24). We first analyzed the ability of the CII256270 complexes to form SDS-resistant, compact dimers (56) (Fig. 1). SDSPAGE analysis comparing the recombinant bCII256270:Aq and mCII256270:Aq demonstrates that bCII256270:Aq complexes form compact dimers, whereas mCII256270:Aq complexes do not. This is consistent with the observation that substitution of residue 266 by aspartic acid results in a lower affinity of CII256270 for Aq (13). However, the lack of compact dimer formation does not appear to negatively affect the stability of the recombinant mCII256270:Aq complexes during storage and this is most likely due to the covalent tethering of the peptide. Similarly, MBP1-11:Au complexes which do not form SDS-resistant dimers are also stable for many months [(24) and unpublished data], indicating that SDS resistance is not a reliable indicator of the storage stability of recombinant peptide:I-A complexes.

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Fig. 1. 12% SDSPAGE analysis of recombinant peptide:Aq molecules, stained with Coomassie brilliant blue: B, samples were heated to 100°C in 2% SDS sample buffer for 5 min before loading; NB, samples were kept at room temperature in 2% SDS sample buffer for 5 min before loading. Sizes (in kDa) of mol. wt standards are shown on the left margin.
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Binding of tetramers to cognate T cells and analysis of responsiveness
Recombinant CII256270:Aq complexes were site specifically biotinylated and incubated with PEExtravidin to generate tetramers. A series of T cell hybridomas, specific for the heterologous CII256270 epitope and with poor cross-reactivity in vitro for mouse CII (8), was used for analyzing the tetramers. The tetramers show specific binding to CII256270:Aq-specific T cell hybridomas and, unexpectedly, this is also seen for mCII256270:Aq complexes (Fig. 2A). Interestingly, HDB.2 hybridoma cells consistently stain to higher levels with mCII256270:Aq than bCII256270:Aq complexes, whereas the reverse is true for HCQ.4 cells (Fig. 2A). Although absolute avidities/affinities cannot be derived from the current analyses, this demonstrates that the relative avidities of the two hybridomas for these complexes are distinct. It also shows that TCR recognition, and not only MHC binding, is affected by the amino acid difference at position 266.


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Fig. 2. Staining of the T cell hybridomas and short-term T cell lines with fluorescently labeled CII:Aq tetramers. (A) T cell hybridomas were incubated with ExtravidinPE labeled bCII256270:Aq (bCII), mCII256270:Aq (mCII) or PE-labeled H57-597 (TCR, anti-Cß) at 37°C as described in Methods. Histogram plots for the staining are shown. Filled histograms represent fluorescence levels of T cell hybridomas incubated with ExtravidinPE only. (B) Tetramer staining of in vitro stimulated, CII-specific T cells following four rounds of re-stimulation/resting using 50 µg/ml bovine CII or 80 U/ml IL-2. Cells were incubated with allophycocyanin-labeled anti-CD3, PE-labeled bCII, mCII or MOG tetramer, and PerCP-labeled anti-CD4. Percentage of tetramer+ CD4 or CD3 cells is shown in the right upper quadrant. Data are representative of five (A) or two (B) independent experiments.
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Consistent with the high degree of cross-reactivity of the CII-specific hybridomas, similar percentages of CD4+ T cells are stained with bCII256270:Aq and mCII256270:Aq complexes following the generation of short-term CII-specific T cell lines from bovine CII-immunized mice (Fig. 2B). For these analyses, tetramer staining was carried out at 12°C, as we have found that this yields improved staining relative to staining at more physiological temperatures. This is consistent with studies indicating that, in general, TCRpMHC affinity increases with decreasing temperature (3234). The data shown in Fig. 2(B) indicate that the level of cross-reactivity of bovine CII for autologous CII may be higher than originally suggested (8,15). As a specificity control in these experiments (Fig. 2B), background levels of staining are seen with complexes made by covalently tethering MOG residues 7990, which bind tightly to Aq (51), in an analogous construct to that used for the CII peptides.
In a number of different antigen systems, tetramer staining has been used to assess the avidity of TCRpMHC interactions (1823). We therefore quantitated the tetramer staining levels following normalization for TCR expression levels and compared these levels with responsiveness (functional avidity) to bCII256270 or mCII256270 peptide-pulsed APCs (Fig. 3). As the TCR expression levels differ for each hybridoma, and are particularly low for HDB.2 cells, it was also necessary to normalize the T cell responses to antigen using anti-CD3
stimulation (54,55). For bCII256270:Aq responses, there is a good correlation between the responses of the hybridomas to peptide-pulsed APCs and tetramer staining levels (Fig. 3A and C). In addition, the responses to plate bound bCII256270:Aq complexes correlate well with binding by the tetrameric complexes (Fig. 3A and E). However, there is a marked difference in responsiveness of the hybridomas to plate-bound mCII256270:Aq complexes and mCII256270-pulsed APCs (Fig. 3D and F). Although the hybridomas respond to recombinant complexes in a pattern that correlates well with mCII256270:Aq tetramer staining levels (Fig 3A and F), they are unresponsive to mCII256270-pulsed APCs (Fig. 3D). Similar results were obtained for a longer variant of the peptide, mCII254274 (data not shown). This unresponsiveness to peptide-loaded APCs is consistent with earlier data (8) and suggests that the low affinity of mCII256270 for Aq which results in poor presentation (13) can be overcome by covalently tethering the peptide to the Aßq chain. Consistent with the low affinity of the mouse CII peptide for I-Aq, the corresponding complexes do not form compact dimers (Fig. 1). Direct comparisons (on a per nanogram basis) of the stimulatory properties of mCII256270:Aq versus bCII256270:Aq complexes therefore cannot be made, as it is possible, for example, that the relative stabilities to immobilization on plastic could vary.
Temperature dependence of the TCRpMHC interactions
Recent studies have shown that TCRpMHC interactions are highly temperature dependent, with the affinity increasing with a decrease in temperature (3234). This temperature dependence has led to the concept that TCRpMHC interactions occur via an induced fit mechanism involving highly unfavorable entropic forces. We have previously shown that tetramers comprising MBP111 covalently tethered to I-Au bind to higher levels at lower temperatures (12°C) than at 37°C to T cell hybridomas bearing TCRs of known affinity (34). This higher level of binding at lower temperatures was shown to be consistent with analyses using soluble TCRs and pMHC complexes in surface plasmon resonance studies (34). Thus, tetramer staining can provide insight into the temperature dependence of the corresponding TCRpMHC interaction, which in turn relates to the thermodynamics of complex formation. We therefore compared the binding of bCII256270:Aq tetramers at 12, 25 and 37°C to hybridomas HDB.2, HCQ.4, HRC.1 and HRC.2 (Fig. 4). These experiments were initially carried out using a pre-incubation of the cells with the anti-CD3
antibody, 145-2C11, to induce TCR aggregation on the cell membrane. This pre-clustering was done in an attempt to exclude temperature effects on TCR mobility, which in turn would be expected to affect the tetramer staining levels. Following clustering with anti-CD3
at 37°C, cells were treated with cytochalasin D to block tetramer-induced internalization and incubated with tetramer at different temperatures (Fig. 4A). The concentration of tetramer used (15 µg/ml) in these analyses was not saturating (Fig. 3B). HRC.1 and HRC.2 behave very similarly, and therefore data only for HRC.1 is shown. The HRC.1 and HCQ.4 hybridomas show greater (normalized) staining at 12 and 25 than at 37°C, whereas the reverse is observed for the HDB.2 hybridoma. Similar results were seen for the HDB.2 hybridoma with mCII256270:Aq tetramers (data not shown), indicating that this temperature dependence is not a unique property of the HDB.2 TCR interaction with bCII256270:Aq. A decrease in affinity with temperature in other systems has been interpreted to indicate that the corresponding TCRpMHC interaction involves induced fit and is entropically unfavorable, i.e. plastic (3234). Our data suggest that for three of four of the TCRpMHC interactions analyzed here, this is also the case. In contrast, the HDB.2 TCRpMHC interaction appears to be thermodynamically distinct.
The tetramer staining levels were also analyzed in the absence of anti-CD3 treatment (Fig. 4B). Several differences in the results become apparent. First, the staining levels of HRC.1 and HCQ.4 relative to those of HDB.2 become much lower, although the pattern of temperature dependence for HCQ.4 is the same. Anti-CD3
pretreatment reduces TCR (H75-597) staining levels to similar extents for the hybridomas analyzed (data not shown). Thus, it is improbable that this pretreatment results in the differential effects of the staining procedures on the HCQ.4 and HDB.2 hybridomas. Second, at 37°C, HRC.1 cells show slightly higher levels of tetramer staining than at 12 and 25°C. These differences might relate to differences in mobility of the TCRs of distinct hybridomas in the membrane that in turn might affect the temperature dependence of tetramer staining. For this reason, we predict that by pre-clustering the TCR with anti-CD3
antibody, we obtain a more accurate representation of the nature of the corresponding TCRpMHC interaction. Nevertheless, the marked increase in staining of the HDB.2 TCR at 37°C is apparent under all of the staining conditions used.
Sequences of the complementarity-determining region (CDRs) of the TCRs
The unusual temperature dependence of the HDB.2 TCRpMHC interactions prompted us to analyze the sequences of the CDRs of the corresponding TCRs (14). For both TCR
and ß chains, the lengths and amino acid composition of CDR1 and CDR2 are similar for the HDB.2 TCR and at least two of the three other TCRs (Fig. 5). However, CDR3
of the HDB.2 TCR is one residue shorter relative to the other three TCRs. It is possible that this may contribute to the apparent decreased flexibility of this TCR, although additional biophysical/structural studies are needed to confirm this. In contrast, CDR3ß of HDB.2 is at least two residues longer than that of the other TCRs and also contains two central glycines, suggesting that this region may be more flexible.

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Fig. 5. Comparison of sequences of CDRs for the TCRs of the T cell hybridomas HRC.1, HRC.2, HDB.2 and HCQ.4 (14).
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Discussion
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In the current study we describe the production and characterization of tetrameric pMHC complexes comprising bovine or mouse CII256270 bound to the MHC class II molecule, Aq. Much evidence supports the idea that CD4+ T cells are involved in pathogenesis in murine CIA (3,5760). In addition, and of direct relevance to the current analyses, multiple studies indicate that bCII256270 and mCII256270 are highly immunodominant following immunization of mice with heterologous CII (712). bCII256270 differs from mCII256270 by a glutamic to aspartic acid change at position 266 and the epitopes are therefore closely related. Substitution of position 266 with alanine or aspartic acid (to generate the self-peptide) leads to unresponsiveness of all T cell hybridomas analyzed (HDB.2, HRC.1, HRC.2 and HCQ.4) by peptide-pulsed APCs (8,9). To date it has not been possible to distinguish whether this effect is due to differences in TCR recognition or peptideMHC association. This question is of central importance to understand the behavior of CII-specific T cells in vivo; if self-CII256270 (or an equivalent overlapping epitope) is efficiently presented, the T cells would be subjected to tolerance induction, whereas, if not, ignorance would be predicted which could explain the relative inefficiency of inducing arthritis with mouse CII (1,48). By covalently tethering mCII256270 to Aq in recombinant complexes to generate high-density antigen complexes, we have obtained a clear answer to this question. We demonstrate that inefficient presentation of this peptide rather than poor TCR recognition most likely leads to low arthritogenicity. Indeed, we find that essentially all CII256270-specific T cells can recognize both heterologous and self-CII256270 bound to Aq. An alternative explanation for our observations is that the recombinant mCII256270:Aq complexes are antigenically distinct from complexes formed by mCII256270-pulsed APCs. Although we cannot exclude this possibility, it is highly improbable that the hybridomas analyzed here would all be equally sensitive to such a potential difference and that these effects would not be seen for bCII256270:Aq complexes.
Our results provide an explanation for a number of previous observations. The original finding that immunization with mouse CII induces a stronger T cell response to rat CII rather than mouse CII, i.e. a heteroclitic response (8), was correctly interpreted based on earlier findings on T cell recognition of cytochrome c (61). The observation that many T cell hybridomas raised against bovine CII cross-react with only very high concentrations of the mouse peptide in vitro can be explained by the fact that high peptide concentrations may compensate for low-affinity binding to the MHC molecule. It follows from this observation that T cell recognition, and thereby arthritis pathogenesis, is most likely different between H-2q mice immunized with mouse CII (48) and mutated mouse collagen (MMC) mice (i.e. mice expressing glutamic acid at position 266 of CII) immunized with rat CII (7). In normal H-2q mice immunized with mouse CII, the low frequency of arthritis is best explained by T cell ignorance, whereas partial T cell tolerance appears to operate in the MMC mice. A similar situation to that in the MMC mouse probably also occurs in mice humanized for both human CII and DR4, in which T cells are tolerized more efficiently to the aglycosylated, CII259-273 peptide compared with the glycosylated, modified forms of the peptide (62).
Analysis of responding T cells from H-2q mice immunized with bovine CII indicates that following re-stimulation in vitro, a significant proportion (
40%) of T cells are CII256270:Aq specific. However, a large proportion is not recognized by either of the recombinant bCII256270:Aq or mCII256270:Aq complexes. A probable explanation for this is that a high proportion of CII:Aq-specific T cells only recognizes glycosylated variants of the CII256270 peptide (14), whereas the recombinant bCII256270:Aq complexes represent only the aglycosylated form of the epitope. In this context, expression of recombinant CII in insect cells results in poor hydroxylation of lysine residues (63) and this post-translational modification is needed for glycosylation of CII256270 (14). To analyze CII-specific T cells that are directed towards glycoforms of the immunodominant CII256270 peptide, it will be necessary to make empty Aq that can be subsequently loaded with distinct glycopeptides to analyze glycopeptide-specific cells; however, given the instability of empty I-A or I-A with weakly bound peptides (24), this may not be feasible. Still, however, T cells directed towards aglycosylated forms of the CII peptide play an important regulatory role in the disease as recently demonstrated in experiments showing that expression of only aglycosylated collagen in the skin can protect from arthritis (30) and with vaccination studies using aglycosylated peptides (11). Interestingly, T cell tolerance seems to operate more efficiently to the unmodified form of the peptide and it is possible that position 266 is exposed differently to glycopeptide specific T cells as a subset of such clones is dependent on a glutamic acid at this position for recognition (64).
We have also used the recombinant CII256270:Aq tetramers to analyze the avidities of the CII-specific hybridomas. One of the TCRs (HDB.2) specific for the aglycosylated CII256270 peptide apparently has a high avidity for antigen and cross-reacts strongly with mCII256270:Aq. The high avidity of the HDB.2 TCR for antigen, together with the cross-reactive recognition of autoantigen, might be representative of a class of TCRs that are borne by highly pathogenic T cells. In this context, in both the NOD and EAE models, high-avidity recognition of autoantigen correlates with disease activity (23,65).
The use of bCII256270:Aq tetramers to analyze the temperature dependence of the TCRpMHC interactions corresponding to HDB.2, HRC.1, HRC.2 and HCQ.4 hybridomas indicates some interesting differences in behavior. The HRC.1, HRC.2 and HCQ.4 hybridomas all show greater binding of multimer at lower temperature (12 and 25 versus 37°C), consistent with the avidity decrease with temperature that is observed for other TCRpMHC interactions in distinct antigen recognition systems (3234). This temperature dependence of affinity/avidity is related to the unfavorable entropic terms that to date have appeared to be a representative feature of TCRpMHC interactions (3234) and has provided support for the concept that TCRs are highly plastic (36). However, here we show that the HDB.2 TCR shows a reverse temperature dependence, i.e. higher avidity at 37 than at 12°C. This suggests that the HDB.2 TCR might interact with cognate ligand in an unusual way that may not involve the same degree of plasticity as that described for other TCRs (3234). The unusual temperature dependence of this TCRpMHC interaction has prompted us to carry out a comparative analysis of the CDR sequences of the TCRs of the hybridomas (Fig. 5). There are not marked differences in sequences or length of the CDR1 and CDR2 loops of the HDB.2 TCR relative to at least a subset of the other TCRs, whereas there are differences in CDR3 lengths for both
and ß chains of this TCR. For example, the HDB.2 CDR3
is one residue shorter than that of the other three TCRs and it is therefore possible that it is more constrained, which in turn might lead to lower conformational flexibility. In contrast, comparison of the CDR3ß of the HDB.2 TCR indicates that it is at least two residues longer than the CDR3ß of the other TCRs. In addition to the greater CDR3ß length, the presence of two central glycines in the center of CDR3ß would suggest that this loop is highly flexible, which would be inconsistent with a less flexible TCR. Taking into consideration the unusual temperature dependence of the HDB.2 TCRpMHC interaction, analysis of the CDR3 sequences therefore leads to the suggestion that the HDB.2 V
domain may play a dominant role in the corresponding TCRpMHC interaction, with the CDR3ß loop/Vß domain making more limited contacts with antigen. This would be analogous to the dominance of V
domain contacts in other TCRpMHC interactions [(3538), reviewed in (39)]. However, in the absence of structural/biophysical data, the TCR docking configuration and loop flexibility obviously cannot be predicted with any degree of certainty. Quantitative analysis of the thermodynamics and molecular details of this TCRpMHC interaction will be an area of future investigation.
In summary, we have used recombinant bCII256270 and mCII256270 multimers to characterize the properties of CII-specific T cells associated with murine CIA. Unexpectedly, we have found that these T cells are highly cross-reactive and therefore potentially autoreactive in vivo. We have shown that the TCRpMHC interaction in this model can be accompanied by unusual temperature dependence, suggesting that decreasing affinity with temperature increase is not a universal feature of TCRpMHC complex formation. Thus, the multimers can be used to evaluate several features of T cell recognition in this system. Future studies will be focused on using them to evaluate the factors, which at the level of the TCRpMHC interaction, lead to pathogenesis.
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Acknowledgements
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We thank Silvia Pastor for assistance with flow cytometric analysis and Mihail Firan for assistance with the generation of expression plasmids. This work was supported by an Arthritis Foundation Research Grant (E. S. W), the National Institutes of Health (RO1 AI42949; E. S. W.) and the Swedish Medical Research Council (R. H). A. M. was supported by FIS (FIS-00/5030 and FIS-01/5037).
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Abbreviations
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APCantigen-presenting cell
CIItype II collagen
CDRcomplementarity-determining region
CIAcollagen-induced arthritis
EAEexperimental autoimmune encephalomyelitis
MBPmyelin basic protein
MMCmutated mouse collagen
MOGmyelin oligodendrocyte glycoprotein
NODnon-obese diabetes
PEphycoerythrin
pMHCpeptideMHC
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