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The Journal of Immunology, 2006, 176: 1062-1071.
Copyright © 2006 by The American Association of Immunologists

I-Ep-Bound Self-Peptides: Identification, Characterization, and Role in Alloreactivity1

Nathan J. Felix*, Anish Suri*, James J. Walters{dagger}, Stephen Horvath*, Michael L. Gross{dagger} and Paul M. Allen2,*

* Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; and {dagger} Department of Chemistry, Washington University, St. Louis, MO 63130


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell recognition of peptide/allogeneic MHC complexes is a major cause of transplant rejection. Both the presented self-peptides and the MHC molecules are involved; however, the molecular basis for alloreactivity and the contribution of self-peptides are still poorly defined. The murine 2.102 T cell is specific for hemoglobin(64–76)/I-Ek and is alloreactive to I-Ep. The natural self-peptide/I-Ep complex recognized by 2.102 remains unknown. In this study, we characterized the peptides that are naturally processed and presented by I-Ep and used this information to define the binding motif for the murine I-Ep class II molecule. Interestingly, we found that the P9 anchor residue preferred by I-Ep is quite distinct from the residues preferred by other I-E molecules, although the P1 anchor residue is conserved. A degree of specificity for the alloresponse was shown by the lack of stimulation of 2.102 T cells by 19 different identified self-peptides. The binding motif was used to search the mouse genome for candidate 2.102 reactive allopeptides that contain strong P1 and P9 anchor residues and possess previously identified allowable TCR contact residues. Two potential allopeptides were identified, but only one of these peptides, G protein-coupled receptor 128, was able to stimulate 2.102 T cells. Thus, the G protein-coupled receptor 128 peptide represents a candidate allopeptide that is specifically recognized by 2.102 T cells bound to I-Ep and was identified using bioinformatics. These studies highlight the specific involvement of self-peptides in alloreactivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Alloreactivity is the ability of a T cell to recognize foreign MHC molecules to which the T cell had never been exposed, and occurs in a relatively high frequency of unprimed T cells (1). The study of alloreactivity led to the identification of the MHC as a whole and to the recognition of class I and class II gene products, and the concept has been an important component of immunology ever since (2, 3). Alloreactivity is observed in vivo as graft rejection or graft-vs-host disease and in vitro by a MLR. Alloresponses against class I and class II molecules are readily detectable, and both are important clinically. Alloreactivity evolves from the ability of the {alpha}{beta} TCR to recognize a foreign MHC molecule, and self-peptides bound to the MHC molecule are an integral part of this recognition. There were several studies strongly implicating self-peptides in alloreactivity (4, 5, 6), and direct proof was obtained by Sherman and colleagues (7) for an H-2Kb alloresponse. Most studies of alloreactivity focused on class I restricted responses because of the available MHC mutants (Kbm series) and the cell lines that possessed defined defects in the class I presentation pathway (8, 9, 10). Class II alloresponses were also described, and several of them were explored in detail (11, 12, 13, 14, 15). The precise role of self-peptides in the alloresponse of an individual T cell has not yet been determined.

The 2.102 T cell is specific for Hb3/I-Ek and has been used extensively in our study of altered peptide ligands and partial T cell activation (16, 17, 18). We discovered that the 2.102 T cells (hybridomas, clones, and TCR transgenic T cells) are alloreactive to I-Ep molecules and characterized this recognition event (19, 20). Evidence of an important role for self-peptides in this alloresponse is the inability of CHO cells transfected with I-Ep molecules to stimulate 2.102 T cells. The class II molecules in CHO cells do not access the endocytic pathway, and, thus, the cells do not express any appreciable levels of self-peptides. Essentially, nothing was known about I-Ep molecules, including any self-peptides. Therefore, we generated an allomimic peptide, termed EPM, using an I-Ep {beta}-galactosidase epitope and a sequential combinatorial peptide technique. EPM presented on I-Ep strongly stimulated 2.102 T cells, demonstrating a key role of the peptide in this alloresponse. We then explored the recognition of EPM by 2.102 T cells and found that it was more degenerate than that observed for Hb/I-Ek, especially at the P5 TCR contact residue. To examine the contribution of the I-Ep residues, we recapitulated the alloreactivity of I-Ep by mutation of the well-characterized I-Ek molecule. Six of the 20 differences between I-Ek and I-Ep are located on the {beta}-chain {alpha} helix, and 14 are on the {beta}-strands of the peptide-binding groove. We generated a mutant I-Ek molecule, I-Ek{Delta}6, in which the six residues on the {beta}-chain {alpha} helix were substituted by those present in I-Ep, and reconstituted the 2.102 alloreactivity. We showed that these I-Ep helical residues were responsible for the broadened peptide recognition. From these studies, we were able to identify and characterize the 2.102 alloresponse against I-Ep and demonstrate the critical involvement of peptides.

In this study we explored the role of self-peptides in I-Ep alloreactivity. To this end, we used capillary HPLC coupled via electrospray ionization to tandem mass spectrometry (MS/MS) to identify and sequence self-peptides naturally bound and presented by I-Ep. This information was used to define the I-Ep binding motif and identify potential alloligands. We found that the I-Ep binding motif is distinct from that of the other I-E molecules. In addition, we used the I-Ep binding motif to search the mouse genome database for self-peptides that possessed allowable TCR contact residues and that might stimulate 2.102. Two potential allopeptides were identified, one of which, G protein-coupled receptor 128 (GPR128), strongly stimulated 2.102 T cells. Analysis of the stimulatory GPR128 peptide revealed that residues beyond the P9 anchor residue are essential to the recognition of GPR128, which contrasts with 2.102 reactivity to Hb(64–76) or EPM. This finding suggests that either there is a slight shift in how the 2.102 TCR contacts the GPR128/I-Ep complex or that the P10 and P11 residues alter the conformation of the GPR128 peptide in the MHC. Through the combined use of MS/MS and bioinformatics to identify self-peptides, a single candidate allopeptide was identified, thus demonstrating a degree of specificity in the alloresponse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6J (B6, H-2b) and B6.AK-H2k/FlaE (hereafter referred to as B6.K) mice were purchased from The Jackson Laboratory, and BALB/c (H-2d) mice were obtained from Charles River Laboratories. B6.P (H-2p) mice were generated by backcrossing B10.P mice (originally obtained from D. Shreffler’s colony) to the C57BL/6J line for six generations and intercrossing to produce the B6.P strain. All mice were bred and housed under specific pathogen-free conditions in the animal facility at the Washington University Medical Center.

Cell lines and mAbs

The 2.102 T cell hybridoma, CH27-Ep and CHO-Ep cells have been described previously (18, 19, 20). The 2.102 and CH27-Ep cell lines were cultured in RPMI 1640 containing 10% FCS (HyClone), whereas CHO-Ep cells were cultured in MEM-{alpha} containing 5% FCS. Media were supplemented with 2 mM GlutaMAX (Invitrogen Life Technologies), 5 x 10–5 M 2-ME, and 50 µg/ml gentamicin. To generate a cell line that expresses a high level of I-Ep, we fused LPS-activated spleen cells from B6.P mice with the class II negative M12.C3 B cell line. This approach has been successfully used in previous studies with similar cells. The fused cells were sorted once for high expression of I-Ep and cloned. Individual clones were tested for the ability to stimulate 2.102 T cells and for expression of high levels of I-Ep. The B6P.C3 cell line was selected based on its stable expression of high levels of I-Ep and its ability to strongly stimulate the 2.102 T cell. B6P.C3 cells were cultured in DMEM containing 7% FCS, 25 mM HEPES, 0.1 mM nonessential amino acids, and the above-listed supplements. The 14.4.4s mAb, specific for the I-E {alpha}-chain, was obtained from the American Type Culture Collection. The mAb 2B6.B8, specific for the I-Ep {beta}-chain, was described previously (19).

Purification of peptides from I-Ep

Peptides were purified from I-Ep class II molecules following an established protocol (21, 22). Briefly, B6P.C3 cells (3–5 x 109) were lysed in PBS containing the detergents octanoyl-N-methylglucamide (MEGA 8) and nonanoyl-N-methlyglucamide (MEGA 9) (40 mM each) and protease inhibitors. After incubation at 4°C for 1 h, the lysate was cleared by centrifugation at 10,000 x g for 30 min. I-Ep class II molecules were purified by using the mAb 2B6.B8 coupled to cyanogen bromide-activated Sepharose beads. Beads were collected on a disposable chromatography column and washed extensively with PBS and MilliQ H2O. I-Ep-peptide complexes were eluted with 0.1% trifluoroacetic acid, and peptides were separated from the individual MHC chains by centrifugation through a Centriprep YM-10 membrane (Millipore).

Liquid chromatography (LC)-MS

MHC peptide extracts from B6P.C3 and CH27-Ep lines were analyzed by on-line reverse phase (RP) LC-MS and MS/MS and/or by off-line strong cation exchange LC followed by on-line RP-LC-MS/MS (two-dimensional LC-MS). When two-dimensional LC was used, off-line strong cation exchange was performed. Samples were resuspended in solvent C (30% CH3CN, 0.1% formic acid (FA)). Ten microliters was injected using an Ultra-Plus II LC with flow split to 5 µl/min and a polysulfoethyl column (15 cm x 320 µm, Microtech Scientific). The gradient was from 0% solvent D (30% CH3CN, 0.1% FA, and 1 M ammonium acetate) to 25% solvent D at 35 min, 50% solvent D at 50 min, and 100% solvent D at 65 min. Ten 7-min fractions were collected, dried down, and resuspended in 10 µl of solvent A (3% CH3CN and 0.1% FA). Five microliters was injected for on-line RP-LC electrospray ionization-MS as described below, and product ion spectra were obtained by using data-dependent MS/MS scanning.

For the on-line reverse phase portion, two LC-MS systems were used. For both systems 5 µl of a sample was loaded onto a silica capillary column with a PicoFritTM tip (New Objective) packed in-house with C18 RP material (0.075 x 100 mm, 5-µm, 300-Å; Delta-Pak, Waters). The gradient was from 0% solvent B (97% CH3CN, 3% H2O, and 0.1% FA) to 50% solvent B over 70 min.

For the LCQ Deca ion trap system the gradient was pumped using a Waters CapLC, and eluent flow was ~5 µl/min and split before the column at a ~1:25 ratio to maintain a flow rate at the tip of 260 nl/min. Flow was directed into the entrance of the heated capillary of an LCQ Deca quadrupole ion trap mass spectrometer (Thermo-Electron) equipped with a custom-built nanospray source. For MS, the scan range was m/z 600-1400 in the profile mode. For MS/MS, the scan range was from 30% of the m/z of the parent ion to m/z 2000. The isotope clusters of parent ions were dynamically selected and isolated with a 2.5 m/z window. The collision energy was set to 28% of the maximum, which is ~5 eV. For detection of peptides, the mass spectrometer selected peptides based on their signal intensity. In such experiments, the first and second most abundant precursor ions were selected for MS/MS analysis.

For the linear ion trap LTQ-Fourier transform system the gradient flow was generated by an Eksigent Technologies nanoLC-1D system at a splitless flow rate of 260 nl/min and directed into an LTQ-Fourier transform (Thermo Electron) using a PicoView PV500 (New Objective) nanospray source. The MS parameters were similar to those of the LCQ Deca system; however, the relative collision energy was 30%, and the eight most abundant ions were selected for MS/MS analysis.

All product-ion spectra were analyzed, and peptide sequences were determined automatically using MASCOT software (Matrix Science). All of the automatically determined sequences were manually verified against the experimental product ion spectra.

Peptide synthesis

Peptides were synthesized using standard Fmoc chemistry on a Rainin Symphony/Multiplex multiple peptide synthesizer (Protein Technologies). Peptides were purified by reverse phase HPLC on a C18 column (Vydac), and molecular mass and purity were confirmed by MALDI mass spectrometry (Washington University Mass Spectrometry Facility).

T cell hybridoma response

Activation of the 2.102 hybridoma was detected by assaying for the production of IL-2 in the tissue culture supernatant. The 2.102 T cell hybridoma cells (1 x 105) were cultured for 24 h with CHO-Ep APCs (2 x 103) and a range of peptide concentrations. Supernatants (100 µl) were removed and added to the IL-2 dependent CTLL-2 line (5 x 103) and cultured for 48 h. CTLL-2 cells were pulsed with 0.4 µCi of [3H]thymidine for the last 18–24 h. Quantitation of IL-2 production was determined by measuring [3H]thymidine incorporation.

Limiting dilution analysis (LDA)

B6 and B6.K spleen and lymph node cells were depleted of class II-positive cells using MACS beads and columns (Miltenyi Biotec) in accordance with the manufacturer’s protocol. Following depletion, the percentage of CD4 and CD8 single-positive cells was determined by FACS analysis. Various concentrations of B6 or B6.K lymphocytes were cultured with 5 x 105 irradiated B6.P spleen cells (2,000 rads) in 96-well U-bottom plates in medium containing 50 U/ml IL-2. After seven days, growth-positive wells were expanded into four wells under the same conditions. After an additional 7 days, the specificity of the cells was determined in a proliferation assay against irradiated B6.P and syngeneic spleen cells in the absence of exogenous IL-2. T cell clones with thymidine incorporation >250 cpm and >2-fold above the control values were considered to be Ag-specific. To determine the frequency of alloresponders specific for I-Ep, T cell responses were blocked with the anti-I-Ep Ab 2B6.B8 (10 µg/well). The frequency of alloreactive T cells was determined according to the maximum likelihood method (23) with two previously described modifications to the formula (24).

Flow cytometric analysis

Samples were stained according to established protocols in PBS containing 0.5% BSA and 0.02% NaN3. Cells were incubated at 4°C for 1 h with directly labeled or biotinylated primary Abs, followed by streptavidin-FITC secondary reagent, if needed. Cells were analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences).

Peptide binding assays

Peptide binding was measured by competitive ELISA essentially as described elsewhere (25, 26). Purified I-Ep MHC class II molecules (1 µM) were incubated with 300 nM biotinylated reference peptide (biotin-6-aminohexanoic acid-TTRVNHATPSAAYA) and various concentrations of competitor peptide in 0.1 M citrate/phosphate buffer (pH 5.0) with 0.2% Nonidet P-40 and protease inhibitors. After 48–72 h at 37°C, peptide/I-Ep complexes were captured on Immulon II 96-well microtiter plates coated with the 14.4.4s Ab. Biotinylated-peptide/I-Ep complexes were detected by streptavidin-HRP dependent color change. Peptide binding is expressed as IC50, the amount of peptide needed to inhibit binding of the reference peptide by 50%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Frequency of allo-I-Ep responses

The H-2p MHC haplotype, expressed in P/J, C3H.NB, B10.P, and B6.P mice, was studied immunologically in a limited manner previously, mostly involving the I-Ap molecule in autoimmune diseases (27, 28, 29, 30). To put our finding of the 2.102 I-Ep alloreactivity into a broader immunological context, we wanted to determine the alloprecursor frequency specific for naive T cells against I-Ep. We performed LDA using two different responder populations to examine the number of T cell precursors capable of recognizing I-Ep. First, B6.K (H-2k) T cells were tested for the ability to respond to cells from B6.P mice. The B6.K responder strain was chosen because it is of the same genetic background as B6.P, thus eliminating any minor H differences. In addition, B6 (H-2b) T cells were also used as responders. Because B6 mice do not express I-E molecules, the T cells in these animals are neither positively nor negatively selected for reactivity to I-E, potentially affecting the level of alloreactivity.

LDA established an overall alloreactive precursor frequency of one in 1547 for B6.K T cells and one in 1645 for B6 T cells (Table I). These values are within the range that was reported for other alloreactivities against only an H-2 difference. By comparison, when both major and minor H differences were present (B6 vs BALB/c) we observed a precursor frequency of one in 950 (data not shown). The portion of the H-2p response that was specific for I-Ep was determined by using an I-Ep-specific blocking Ab. Approximately one in 4500 B6.K T cells and one in 9800 B6 T cells are capable of recognizing I-Ep. Interestingly, mice expressing an I-E molecule have a modest increase in the frequency of I-E-specific alloreactive T cells. These studies establish that alloreactivity to I-Ep represents a conventional and strong primary immune response.


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  		Table I. I-EP-specific allo-T cell precursor frequencya

 
Isolation and identification of peptides from I-Ep

To define the 2.102 allorecognition of I-Ep at a molecular level, we needed to characterize the self-peptides bound to I-Ep. The I-Ep peptide-binding motif has not been established, and nothing is known about the self-peptides presented by I-Ep. To facilitate the isolation of the naturally processed and presented peptides selected by I-Ep, we generated a cell line (B6P.C3) that expresses a high level of I-Ep on its cell surface (Fig. 1A). Importantly, this line strongly stimulates 2.102 T cells (Fig. 1B), confirming the presence of the alloepitope(s) on the B6P.C3 cell line.



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  		FIGURE 1. B6P.C3 cells express high levels of I-Ep and stimulate 2.102 alloreactivity. A, B6P.C3 cells were stained with an I-Ep {beta}-chain-specific mAb or isotype control. B, Increasing numbers of B6P.C3 APCs were used to stimulate the 2.102 T cell hybrid. IL-2 production by activated 2.102 T cells was measured with the CTLL-2 indicator cell line.

 
We identified naturally processed and presented peptides selected by I-Ep and defined the I-Ep peptide binding motif by using an approach involving MS that we (J. J. Walters and A. Suri) have successfully used to characterize the peptides bound to I-Ad and I-Ag7 molecules (21, 22). Peptides were eluted from purified I-Ep molecules, separated by capillary HPLC, introduced into the mass spectrometer via electrospray ionization, and sequenced using MS/MS. We isolated and identified a total of 295 peptides (Table II presents some of the data; other data not shown). Similar to peptides eluted from other class II molecules, the peptides isolated from I-Ep ranged from 8 to 34 amino acids in length, with most falling between 13 to 20 residues (21, 22, 31, 32, 33, 34, 35, 36, 37, 38). The 295 peptides were found in 120 different families. A peptide family consists of a series of peptides that contain the same nine-amino acid core sequence and vary by the lengths of their N- and C-terminal flanking residues. The peptides were derived primarily from proteins of the plasma membrane, vesicular, and cytosolic compartments. To verify the ability of these peptides to bind I-Ep, 35 peptides were synthesized, purified, and tested for their binding to I-Ep. Of these, 13 peptides bound strongly with an IC50 of ≤10 µM, whereas six others bound more weakly, with an IC50 between 11 and 100 µM. The remaining peptides had an IC50 of >100 µM (i.e., showed no detectable binding in the competitive assay) and were excluded from further analysis. It is possible that these peptides are contaminants, as was observed in previous studies (21, 22), or that they require the presence of chaperones (e.g., H2-DM) for proper loading onto I-Ep. This demonstrates the importance of verifying a peptide’s ability to bind by testing in a direct binding assay. Thus, we have successfully identified endogenous peptides bound to I-Ep, and these peptides are a "data base" that can be used to characterize the I-Ep binding motif.


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  		Table II. Naturally processed and presented peptides isolated from the B6P.C3 cell linea

 
Identification of the I-Ep binding motif

To establish the I-Ep peptide binding motif we initially made use of the well-characterized I-Ek molecule and its binding motif (32, 34, 37, 39), given the overall similarity between these two MHC alleles (40). Sequence comparison of the I-Ep and I-Ek {beta}-chains suggests that the polymorphic differences between the two class II molecules will have the largest impact on the P9 pocket and perhaps a smaller impact on the P6 and P4 pockets (41, 42). Given that I-Ep and I-Ek have identical I-E{alpha}-chains, the P1 pocket is completely conserved between the two molecules. Based on this conserved P1, we determined a preliminary I-Ep binding register that places Ile, Leu, or Val as the P1 anchor. Importantly, almost every peptide we identified had at least one of these residues near its amino terminus that could serve as the P1 anchor. To ensure the accuracy of our I-Ep binding motif analysis and eliminate the impact of nonbinding peptides, we limited our analysis to only those peptides that were determined to bind I-Ep. As a result, the binding motif may under-represent the occurrence of certain residues at individual anchor positions. Alignment of the peptides capable of binding I-Ep (IC50, <100 µM) according to a conserved P1 also suggested that the P9 anchor of I-Ep would be distinct from the P9 anchor of the other I-E molecules.

The dominant binding register for the peptides isolated from I-Ep was determined by individually replacing each potential anchor residue with Lys and measuring its effect on binding (Table III). Substitution of Lys for the wild-type P9 residue decreased peptide binding between 8- and 30-fold compared with the wild-type peptides. Interestingly, the P9 pocket of I-Ep predominantly prefers Gln, but Ala, Thr, and Pro are also commonly found (Fig. 2). This is unlike I-Ek and the other I-E molecules, which prefer Lys or Arg at P9. In fact, our Lys scan demonstrated that I-Ep disfavors Lys at P9.


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  		Table III. Lysine mapping of the peptide-binding register

 


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  		FIGURE 2. Preferred anchor residues for I-Ep were determined by Lys mapping to identify the primary and secondary anchor residues. The naturally presented peptides isolated from I-Ep and shown to bind I-Ep by direct binding assay were then aligned according to this defined binding register. The percentage of occurrence of each amino acid is plotted for this subset of peptides.

 
The P6 pocket of I-Ep accommodates mainly nonpolar aliphatic amino acids (Val, Pro, Ala, Ile, and Leu) but also accepts small polar residues (Ser and Thr) and may occasionally accommodate a charged residue (Fig. 2). By contrast, the P4 pocket prefers small polar residues (Thr and Ser) and small aliphatic residues (mainly Ala). The P4 pocket appears to contribute significantly to peptide binding, because Lys substitution at this position abolished binding for all tested peptides. The Lys mapping approach validated the initial assumption that P1 is conserved between I-Ep and I-Ek.

A similar analysis of I-Ek-bound self-peptides revealed that no peptides were found to bind both I-Ek and I-Ep (our manuscript in preparation). These results demonstrate that the allocomplex recognized by 2.102 will differ from the Hb(64–76)/I-Ek complex both by the {alpha} helical differences between I-Ep and I-Ek and by the unique spectrum of peptides displayed by I-Ep. In fact, any I-Ep specific alloresponse has the potential to be directed against both of these distinguishing features of I-Ep.

Testing the role of self-peptides in alloreactivity

Having identified the set of self-peptides naturally bound to I-Ep, we wanted to ascertain the contribution that these peptides made in the 2.102 alloresponse. Our previous data implied that self-peptides make a significant contribution to the alloreactivity, but we never investigated the frequency of peptides that can make this contribution. To this end, we synthesized and tested a set of 19 different I-Ep peptides for their ability to stimulate 2.102 T cells by using CHO-Ep as APC. None of the peptides stimulated the 2.102 T cells, even at concentrations up to 100 µM (data not shown). Therefore, the peptide(s) involved in the 2.102 I-Ep alloresponse make a significant contribution to the recognition surface. In one proposed model for alloreactivity, the role of the self-peptide is merely to stabilize MHC molecules and not to contact the TCR directly (1, 43, 44). Our data strongly argue against that model; 19 tested self-peptides all stably bind to I-Ep molecules, but none stimulate 2.102 T cells.

Identification of a 2.102 reactive candidate allopeptide

To identify potential 2.102 alloreactive peptide(s), we considered two different approaches. Purifying MHC-bound peptides, testing the peptides for their T cell stimulatory activity, and then identifying the peptides by MS has been a successful approach for class I peptides (45, 46, 47, 48, 49). However, for class II-restricted peptides this approach is much more difficult because the T cell assays are less sensitive, the availability of APCs lacking self-peptides is limited, and the length of the bound peptides is heterogeneous. In fact, to date no unknown peptide bound to a class II molecule possessing a specific T cell reactivity has been identified. With the recently available complete mouse genome and our I-Ep peptide-binding motif we chose to pursue a bioinformatics approach to identify candidate allopeptides. We searched the National Center for Biotechnology Information (NCBI) protein database for potential 2.102 reactive allopeptides. The search sequence was based on the core motif of EPM with specific substitutions at P1 (Val, Ile, or Leu), P5 (Trp or Asn), and P9 (Ser or Gln) (Fig. 3A). Each possible combination of substitutions at P1, P5, and P9 was used to search the NCBI murine protein database. Each search returned nearly 100 sequences, with many sequences appearing in more than one search. The resulting sequences were evaluated for their likelihood to bind to I-Ep based on strong P1 and P9 anchor residues and for the overall similarity of their TCR contact residues to EPM. However, the only required conserved TCR contact was at P5 (Trp or Asn). We synthesized two peptides that satisfied these criteria and tested them for the ability to stimulate the 2.102 T cells when presented by CHO-Ep cells. Significantly, a peptide derived from the GPR128 protein strongly stimulated the 2.102 hybrid (Fig. 3B). The GPR128 protein is broadly expressed and it is highly expressed in the spleen. However, the level of GPR128 expression in APCs is not known. Therefore, GPR128 represents a natural allopeptide candidate but may not be the actual allopeptide recognized by 2.102. The other peptide, derived from RNA PAF53 (PAF53), did not stimulate 2.102 T cells (Fig. 3B).



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  		FIGURE 3. The GPR128 peptide is recognized by the 2.102 TCR. A, Two potential allopeptides were identified by their TCR contact residues and by their likelihood to bind to I-Ep. B, The GPR128 peptide stimulates the 2.102 T cell hybrid, whereas RNA PAF53 does not. EPM, an epitope mimic known to stimulate 2.102, is included as a control. ND, not determined.

 
Analysis of the GPR128 sequence revealed two key features: it is very hydrophobic in nature and is highly homologous to EPM, the allomimic peptide we had generated previously (20). Initial attempts to purify the synthesized GPR128 peptide were unsuccessful because of this peptide’s extreme hydrophobicity. To verify that the GRP128 peptide could stimulate the 2.102 hybrid, we re-synthesized the GPR128 peptide as the original 15-mer peptide and as 13-mer and 9-mer peptides (Fig. 4A). In addition, we also synthesized two GPR128 variants that contained the sequence GKK at the N-terminal region of the peptide, which is known to increase solubility (P. M. Allen, unpublished observation). The substitution of GKK for the wild-type residues at P-4 through P-2 is not expected to influence TCR recognition but should significantly improve solubility.



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  		FIGURE 4. A soluble GPR128 GKK analog strongly stimulates the 2.102 T cell hybrid. A, Various truncations of GPR128 and their more soluble GKK variants were synthesized to confirm the ability of the GPR128 peptide to stimulate the 2.102 TCR. B, Both the GPR128 15-mer peptide and its GKK analog stimulate the 2.102 T cell hybrid. C, The GPR128 13-mer peptide does not stimulate the 2.102 TCR, whereas its more soluble analog stimulates 2.102 weakly. The GPR128 9-mer peptide is not recognized by 2.102.

 
The unpurified GPR128 15-mer peptide stimulated the 2.102 T cell hybrid when presented in the context of I-Ep (Fig. 4B). Importantly, MALDI of the unpurified peptide demonstrated that the expected full length GPR128 peptide was the major product of our synthesis ([M + H + Na]+ ion of m/z 1755.1; data not shown). However, we were unable to identify reverse-phase HPLC conditions that permitted purification of the wild-type GPR128 peptide. By contrast, both GPR128 GKK peptides were soluble and easily purified. Significantly, the purified GPR128 GKK peptide strongly stimulated 2.102 (Fig. 4B). These results establish GPR128 as a candidate allopeptide recognized by the 2.102 TCR and validate this bioinformatics approach.

Specificity in the recognition of alloreactive peptides

We also synthesized and tested the wild-type and GKK analog 13-mer peptides that lack the C-terminal Ala and Thr at positions P10 and P11. Surprisingly, the 13-mer GKK analog only weakly stimulated the 2.102 hybrid (Fig. 4C). Consistent with the results of the GKK 13-mer analog, the unpurified wild-type GPR128 13-mer peptide failed to stimulate the 2.102 T cell hybrid (Fig. 4C). These results show that the 2.102 TCR is influenced by the P10 and P11 residues of GPR128, which are not critical for 2.102 recognition of Hb/I-Ek. For other T cells, C-terminal extensions were shown to be critical for their conventional reactivity (50).

The failure of PAF53 to stimulate 2.102 cannot be explained by an inability to bind I-Ep, because it bound I-Ep well (Fig. 3a). Nonstimulatory PAF53 differs from GPR128 at P2 and P7 (Fig. 3A). Although PAF53 also differs at P5 (Asn) compared with GPR128 (Trp), this difference is unlikely to influence negatively 2.102 recognition. We previously determined that at P5 Asn is equivalent to Trp in its ability to stimulate 2.102 (N. J. Felix, unpublished observation). Thus, despite the overall similarity of the two candidate allopeptides, 2.102 has a degree of specificity for the allopeptide it recognizes.

Having identified GRP128 as a potential source of a 2.102 allopeptide, we reexamined our MS data of I-Ep eluted peptides to find any evidence that this epitope is naturally processed and presented. We were unable to identify any species with a mass that would match the GPR128 allopeptide in the MS analysis. This, however, is not unexpected given the hydrophobic nature of this peptide and our difficulty in purifying it using conventional reverse-phase HPLC. It is likely that the natural peptide either never dissolved or was not eluted from the column. To determine whether reactivity to GPR128 is a feature of a broad alloresponse, we tested the ability of naive B6.K T cells to proliferate in response to the GPR128 GKK 15-mer peptide presented by CHO-Ep cells. Although we were unable to detect a proliferative response to GPR128 by this assay (data not shown), this failure does not indicate that a response to GPR128 cannot occur. Instead, it may reflect a low precursor frequency of alloreactive T cells capable of recognizing the GPR128/I-Ep complex. Although we cannot definitively identify GPR128 as the allopeptide recognized by the 2.102 TCR, GPR128 represents a potential source of a 2.102 reactive allopeptide, and the specificity of its recognition reveals the critical role peptides play in alloreactivity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous work has established that self-peptides play an important role in direct recognition of allogeneic MHC (1, 7, 46, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59). In only a few cases has peptide-independent allorecognition been demonstrated for class I, and these cases are the exception (60, 61, 62). However, the extent of specificity for the bound peptide has not been firmly established, especially for class II responses. In this study we report the identification of GPR128, a candidate allopeptide recognized by the 2.102 TCR, and demonstrate the specificity of the recognition by 2.102 of allogeneic I-Ep. Our results suggest that 2.102 is only capable of recognizing a limited number of self-peptide/I-Ep complexes and support the theory that allorecognition is a relatively peptide-specific event.

The high frequency of alloreactive T cell precursors has been attributed to the high density of self-peptide/allo-MHC complexes on allogeneic cells and/or decreased specificity for the bound peptide (1, 43, 44). Various methods have been used to determine the precursor frequency of alloreactive T cells (e.g., LDA, ELISPOT, tetramer staining, and CFSE dilution) with diverse results (ranging between <0.0001 and 10%) (63, 64, 65, 66, 67, 68). Consensus is lacking, because ultimately each method lacks sufficient sensitivity for a comprehensive answer. However, it is still useful to determine the precursor frequency of a novel alloresponse by comparison to a proven response. We found that the B6 and B6.K anti-H-2p precursor frequency approached the precursor frequency of the strongly allo-B6 anti-BALB/c combination (~60% of the response). In addition, ~30% of the B6.K and 16% of the B6 anti-H-2p alloresponse was specific for I-Ep, indicting that alloreactivity to H-2p and I-Ep represents a vigorous and abundant primary response.

MHC-bound self-peptides are an important component of the 2.102 alloresponse. We found that I-Ep prefers Gln at P9, whereas other I-E molecules (e.g., I-Ek) have a strong preference for Lys or Arg (32, 34, 37, 39, 41, 42). An examination of the P9 pocket in the high resolution I-Ek crystal structure provides a potential explanation for the change in specificity at P9 in I-Ep molecules (41). In I-Ek the P9 pocket is a narrow hydrophobic tunnel with Glu{beta}9 at its base that forms a salt bridge with the Lys or Arg side chain of the peptide. The Glu{beta}9 residue is conserved in I-Ep, but there are three substitutions in residues forming or near the P9 pocket that could significantly affect the nature of the pocket. The Leu{beta}8 and Tyr{beta}10 residues are replaced with Met and Gln residues respectively. These nonconservative changes would likely cause a repositioning in the Glu{beta}9 residue, thereby precluding it from forming a salt-bridge with the peptide Lys or Arg side chain. Additionally, the nonconservative Asn to Tyr substitution at {beta}37 could also affect the nature of the P9 pocket. These changes to the P9 pocket, separately or together, could easily account for the change in the anchor residue preference in I-Ek to I-Ep from a Lys to a Gln.

Because both the I-Ep {beta}-chain {alpha} helical differences and the unique self-peptides presented by I-Ep can contribute to alloreactivity, we examined the degree to which 2.102 was specific for a particular self-peptide/allo-MHC complex. This question was addressed for class I-restricted responses by testing RP-HPLC fractions of class I-bound peptides (45, 46, 47, 48, 49). However, the abundance of individual peptides in RP-HPLC fractions is generally below the nanomolar or picomolar range and, therefore, may not be abundant enough to detect in class II restricted in vitro assays. We synthesized and directly tested 15% of the identified self-peptide families (19 of 120 families) for the ability to stimulate 2.102 T cells. These peptides were identified by MS/MS analysis because their molecular ions were among the most abundant ions detected and, therefore, represent the some of the most abundant peptides presented by I-Ep. Yet none of them stimulated the 2.102 T cell hybridoma. These results argue against the peptide-dependent model by demonstrating that the 2.102 TCR is not widely peptide degenerate. The results also begin to reveal peptide specificity by demonstrating that 2.102 does not recognize some of the most common peptides presented by I-Ep.

Peptide specificity was further examined by pursuing a bioinformatics approach to identify natural allopeptides. This approach identified two candidate peptides, one of which, GPR128, stimulated 2.102 T cells. Both GPR128 and RNA PAF53 have significant similarity to the artificial allomimic EPM. Previous analysis of 2.102 specificity for EPM suggested there was significant degeneracy at P5 (20). Similar analysis of peptide libraries with various peptide pools at P2, P3, P7, or P8 also implied a degree of degeneracy at these residues; however, this possibility was not examined in detail. RNA PAF53 differs from EPM at P1, P2, P5, P7, and P9. However, P1 and P9 are strong I-Ep anchor residues, and the Val at P1 is even shared with the stimulatory GPR128 peptide. In addition, 2.102 recognizes Asn at P5 as strongly as it does Trp. Therefore, the major TCR contact residue differences between EPM and PAF53 occur at only P2 and P7. By comparison, the major differences between GPR128 and EPM occur at P4 and P7. Although there are clear differences in the TCR contact residues for GPR128 and the nonstimulatory RNA PAF53, it is not immediately apparent why GPR128 is active. In fact, 2.102 responds very differently to three relatively similar peptides (GPR128, PAF53, and EPM). This observation is illustrated by the discovery that the GPR128 13-mer peptide fails to stimulate 2.102 cells. The 2.102 TCR is not dependent upon the P10 and P11 residues of Hb or EPM, but these residues are clearly involved in the allorecognition of the GPR128/I-Ep complex. This finding implies that for 2.102 allorecognition of GPR128/I-Ep there might be a slight shift in the orientation of the TCR bound to the peptide-MHC. Alternatively, these residues may alter the conformation of the GPR128 peptide bound to I-Ep. Therefore, despite the modest similarity of the GPR128 and PAF53 core peptides, there are also residues laying outside the peptide-binding groove that play a critical role in allorecognition of GPR128. Although this feature is not unique to allorecognition, it provides another mechanism by which peptide recognition can be altered in an alloresponse (50).

It is clear from these studies that 2.102 has a degree of specificity for the bound peptide. However, the extent of 2.102 peptide specificity is still unknown for alloresponses as well as for conventional responses (69, 70). The contribution of the I-Ep {alpha} helical residues to alloreactivity was explored previously (20). In these studies, mutating the I-Ek {alpha} helical residues to those of I-Ep increased 2.102 degeneracy for Hb(64–76) at P5. In addition, these mutations altered fine specificity but did not increase degeneracy at P3. Although {alpha} helical polymorphisms allow broadened recognition of Hb(64–76) at the P5 TCR contact residue, the degree of degeneracy promoted by {alpha} helical interactions has not been extrapolated to the entire peptide by a similar analysis with simultaneous substitutions at multiple TCR contact residues. Clearly, MHC polymorphisms can alter the fine specificity of alloreactive T cells for the bound peptide, and these effects can either increase or decrease specificity at individual TCR contact residues. However, our analysis of two candidate allopeptides demonstrates that the {alpha} helical polymorphisms present on I-Ep do not eliminate the role of the bound peptide and that increased degeneracy at a single TCR contact does not, in this case, result in excessive peptide degeneracy. Although a combined tandem MS and bioinformatics approach identified a single self-peptide capable of eliciting 2.102 allorecognition, it is premature to suggest that GPR128 represents the only peptide/I-Ep complex recognized by 2.102. Given the presence of polymorphic differences between I-Ep and I-Ek and the presence of numerous peptides in the murine proteome with similarity to the EPM core motif, as well as the discovery that peptide residues outside the binding grove can contribute to 2.102 allorecognition, it seems likely 2.102 may recognize additional as yet unidentified self-peptides. However, the specificity demonstrated by 2.102 in these studies suggests that any additional self-peptide/I-Ep complexes it recognizes will be limited in number.

It has been suggested that a high degree of degeneracy for peptide exists among some alloreactive T cells (1, 43, 59). Whether there exist multiple types of alloreactive T cells that vary in the extent of their peptide specificity remains controversial. Our results suggest that the 2.102 TCR is relatively peptide-specific, not excessively peptide-degenerate. Additional experiments to define the fine specificity of 2.102 for GPR128 and to exhaustively identify any potential additional allopeptides recognized by 2.102 will help determine the degree to which the polymorphic {alpha} helical regions of I-Ep substitute for extensive contacts with the bound peptide. Improvements in sensitivity, speed, and specificity will undoubtedly make the use of MS/MS more effective in identifying unknown peptides with specific reactivities from class II molecules. This will be necessary to determine whether self-peptides with substantially divergent sequences can stimulate 2.102 T cells. In addition, our results demonstrate the effectiveness of bioinformatics-based searches when the binding motif and acceptable T cell contact residues are known.


   Acknowledgments
 
We thank Dr. Peter Jensen for the binding assay protocol, Dr. Emil Unanue for support and suggestions, Dr. Ted Hansen for critical review of the manuscript, Darren Kreamalmeyer for maintenance of the mouse colony, and Jerri Smith for assisting in the preparation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The mass spectrometry was supported by National Centers for Research Resources, National Institutes of Health Grant 2P41RR00954. Back

2 Address correspondence and reprint requests to Dr. Paul M. Allen, Washington University School of Medicine, Department of Pathology and Immunology, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110. E-mail address: pallen{at}wustl.edu Back

3 Abbreviations used in this paper: Hb, hemoglobin; CHO, Chinese hamster ovary; FA, formic acid; GPR128, G protein-coupled receptor 128; LC, liquid chromatography; LCQ, LC quadrupole; LDA, limiting dilution analysis; MS, mass spectrometry; MS/MS, tandem MS; RP, reverse phase. Back

Received for publication August 4, 2005. Accepted for publication November 2, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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