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Identification of a Novel HLA-B60-Restricted T Cell Epitope of the Minor Histocompatibility Antigen HA-1 Locus¹

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

	Abstract

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The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1^H and HA-1^R, with a single amino acid difference. Whereas the immunogenicity of the HA-1^R allele has not yet been shown, the nonameric HA-1^H peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1^H or HA-1^R associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1^H/R peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1^H/R peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVL^H/_RDDL and decameric KECVL^H/_RDDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1^H and HA-1^R peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1^R peptide did not induce CTLs that recognize the natural HLA-B60/HA-1^R ligand. In contrast, dendritic cells pulsed with the nonameric HA-1^H peptide induced IFN--secreting T cells specific for the natural HLA-B60/HA-1^H ligand in three HLA-B60⁺ HA-1^RR individuals, demonstrating the immunogenicity of the HLA-B60/HA-1^H ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.

	Introduction

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Differences in minor histocompatibility Ags (mHags)³ between HLA identical stem cell (SC) donor and recipient may lead to alloimmune T cell responses after SC transplantation (SCT). The mHag-specific T cells are key players in both the graft-vs-host disease and the graft-vs-leukemia reaction (1). It is important to understand the basis of the mHag-induced alloimmune responses for both latter graft-vs-host reactivities. Now that mHags can be characterized, information on issues such as mHag processing and presentation will give insight into their potential clinical relevance. With the molecular identification of the autosomally encoded mHag HA-1, it was shown for the first time that a human mHag is encoded by a locus with two alleles with a single amino acid difference (2). The difference between the HA-1 alleles comprises a histidine (H) to an arginine (R) substitution. Although nonameric peptides of both the HA-1^H and the HA-1^R alleles bind to HLA-A2, the HA-1^R allele fails to be expressed at the cell surface. In contrast, the HA-1^H peptide is presented at the cell surface and is known to induce HLA-A2-restricted CTLs both in vivo and in vitro (3, 4). Because of its hemopoietic system-restricted expression, the mHag HA-1 can be used for the treatment of relapsed leukemia after HLA-identical, HA-1-mismatched SCT (4, 5).

Since the HA-1-specific immunotherapy is currently restricted to HLA-A2-positive patients, we have investigated whether the HA-1^H/R polymorphic region contains peptides that can be presented by other HLA molecules. We analyzed the binding capacities of HA-1 polymorphic peptides to nine HLA-A and -B molecules that have a frequency of >10% in the Caucasian population. Since cognate T cell epitopes of mHags do not always confirm the predicted MHC class I binding motifs (6, 7), all nonameric HA-1^H/R peptides (n = 18) were tested for binding to these frequent HLA alleles. The peptide binding analyses were extended with two decameric HA-1^H/R peptides that contained binding motives for HLA-A3 and with five nonameric/decameric peptides that were predicted to bind to HLA-B14 or -B60. After the binding studies, cellular processing was executed by in vitro proteasome digestion of 29-aa-long HA-1^H and HA-1^R peptides. To enlarge the patient population for HA-1-specific immunotherapy, the HLA-B60 binding peptides were analyzed for their in vitro immunizing potential. Peptide-loaded dendritic cells (DCs) were used to induce T cell responses from healthy individuals.

	Materials and Methods

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HA-1 peptides

HA-1^H and HA-1^R peptides were synthesized using an automated multiple peptide synthesizer (Syro II; Multisyntech, Witten, Germany) according to the known HA-1 amino acid sequence (2). The purity of the peptides was 90%. The peptides were dissolved in DMSO, diluted in 0.9% NaCl, and stored at -20°C until use.

Prediction of HLA peptide binding

The polymorphic HA-1^H and HA-1^R regions were screened with the HLA-peptide binding prediction software of BIMAS (BioInformatics and Molecular Analysis Section, National Institutes of Health, Bethesda, MD; http://bimas.dcrt.nih.gov./) for octameric, nonameric, or decameric HA-1 peptides capable to bind to HLA class I molecules. The selection of peptide candidates was made by comparison of the computed scores with that of the HLA-A2-restricted HA-1^H CTL epitope with the amino acid sequence VLHDDLLEA (score of 79.6). This score corresponds to the estimated half-time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Five HA-1^H/R peptides with scores ranging from 32 (intermediate binding score) to 176 (strong binding score) were selected to assay for binding to the relevant HLA class I molecules. The predicted HLA class I/HA-1^H/R peptide associations and their computed binding scores are presented in Table I. In addition, we selected two decameric HA-1^H/R peptides that contained anchor residues for binding to HLA-A3, but were not predicted by the BIMAS software.

We used the competition-based HLA peptide binding assay as described previously, with some modifications (8). Briefly, HLA-typed EBV-transformed B cell lines (EBV-LCLs) were washed with PBS, kept on ice for 5 min, and treated with an ice-cold 0.132 M citric acid/0.062 M Na₂HPO₄·2H₂O elution buffer for 90 s (8). The pH of the elution buffer was optimized for each HLA molecule to enable maximal elution of HLA-bound peptides (J. H. Kessler, B. Mommaas, I. Huijbers, D. Vissers, W. E. Benckhuijsen, R. Offringa, T. Mutis, E. Goulmy, C. J. M. Melief, S. H. van der Burg, and J. W. Drijfhout, manuscript in preparation). Immediately after mild acidic treatment, the cells were washed with 12 ml IMDM (BioWhittaker, Verviers, Belgium) containing 2% FCS and resuspended in IMDM containing 2% FCS/1.5 µg/ml ₂-microglobulin (Sigma-Aldrich, St. Louis, MO). Acid-treated EBV-LCLs (4 x 10⁴) were then incubated in 96-well V-bottom plates (Costar, Cambridge, MA) with fluorescent-labeled reference peptide (25 µl/well; final concentration, 150 nM) mixed with serial dilutions of competitor (test) peptides (25 µl/well; final concentrations, 100–0.78 µM) in a total volume of 150 µl. All reference peptides were deduced from previously reported peptides that show strong binding to the respective HLA class I molecules (9). After incubation for 24 h at 4°C, the cells were washed twice with 100 µl/well PBS/1% FCS and fixed with 0.5% paraformaldehyde in PBS. The mean fluorescence expressed by the cells was determined by a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). The percent inhibition of HLA binding of the fluorescent reference peptide is calculated with the formula: % inhibition = 1 - [(mean fluorescence in the presence of competitor peptide - mean background fluorescence)/(mean fluorescence in the absence of competitor peptide - mean background fluorescence)] x 100%. The relative binding affinity of the peptides is expressed as the IC₅₀.

Proteasomal cleavage of the HA-1 polymorphic region

Twenty-nine-amino acid-long HA-1^H and HA-1^R peptides were purified to 95% by reverse phase HPLC. Peptides (10 µg/ml) were incubated with 20S proteasomes isolated from EBV-LCLs for 15, 30, and 45 min as described previously (10, 11, 12). The proteolysis products were analyzed by tandem mass spectrometry as previously described (13).

DC culture

Monocyte-derived DCs were generated from healthy individuals by culturing peripheral blood-derived CD14⁺ monocytes with 1000 U/ml IL-4 (Genzyme, Cambridge, MA) and 800 U/ml GM-CSF (donated by Dr. S. Osanto, Leiden University Medical Center, Leiden, The Netherlands) for 6 days as described previously (14). On day 6 the DCs were maturated by culturing on irradiated (750 Gy) CD40 ligand-transfected mouse fibroblasts at a DC to fibroblast ratio of 2:1 or by adding 50% of monocyte-conditioned medium (14). Mature DCs were pulsed with HA-1 peptides for 2 h at 37°C in Aim-V medium (Invitrogen, Breda, The Netherlands) before their use as stimulator cells.

In vitro induction of HLA-B60/HA-1-specific T cell responses

Peptide-pulsed DCs were cocultured with autologous PBMC at a DC to PBMC ratio of 1:10 in IMDM, 10% human serum supplemented with 1 U/ml IL-2 (Cetus, Emeryville, CA), and 1 U/ml IL-12 (R&D Systems, Minneapolis, MN). On day 5, 20 U/ml IL-2 was added. On day 7, the T cell lines (TCL) were depleted of CD4⁺ cells using immunomagnetic beads (Dynal Biotech, Oslo, Norway) and were restimulated with irradiated (150 Gy) peptide-pulsed mature DCs (DC:T cell ratio, 1:10) or with irradiated (150 Gy) peptide-pulsed monocytes (monocyte:T ratio, 1:3). Twenty-four and 96 h after restimulation, medium containing 20 U/ml IL-2 was added. TCL were subsequently restimulated every 7 days and were tested for HA-1-specific activity in IFN- ELISPOT assays (15) before each restimulation.

	Results

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Effective binding of nonameric and decameric HA-1^H and HA-1^R peptides to HLA-B60

Three categories of HLA molecules were selected for the peptide binding assays: those molecules with a frequency of >10% in the Caucasian population, those with binding motifs, and those that were predicted to bind nonameric/decameric HA-1^H/R peptides. All nonameric HA-1^H and HA-1^R peptides (n = 18) were tested for binding to the so-called frequent HLA class I molecules HLA-A1, -A2, -A3, -A11, -A24, -B7, -B8, -B35, and -B62. The peptide analysis was extended with two decameric HA-1^H/R peptides with a binding motif for HLA-A3 and with five nonameric/decameric peptides predicted to bind to either HLA-B14 or -B60 (Table I). The HLA-A1, -A11, -A24, -B7, -B8, -B14, -B35, and -B62 molecules did not bind nonameric HA-1^H/R peptides despite the predictions of BIMAS software for intermediate to strong binding of peptide ECVLRDDLL to HLA-B8 or to -B14 (table I). The decameric HA-1^H/R peptides VL^H/_RDDLLEAR showed weak to intermediate binding to HLA-A3 molecules with IC₅₀ values of 15.6 and 37.5 µM, respectively (Fig. 1). In agreement with the prediction obtained using BIMAS software, the nonameric and decameric HA-1^H/R peptides KECVLHDDL, KECVLRDDL, KECVLHDDLL, and KECVLRDDLL showed strong binding to HLA-B60 molecules with very low IC₅₀ values of 5.3, 3.9, 1.0, and 1.6 µM, respectively (Fig. 2). As expected, the original HLA-A2/HA-1^H CTL epitope, also predicted by the BIMAS software, displayed binding to HLA-A2 with an IC₅₀ of 6.4 µM (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Binding of HA-1H/R peptides to HLA-A3. The results are expressed as the percent inhibition of HLA binding of the 150 nM fluorescent reference peptide by the indicated peptides added at serial dilutions (see Materials and Methods). Curves were fitted by nonlinear regression and one-site binding equation. The IC50 value of the HLA-A3 binder-positive control peptide KQSSKALQR (9 ) was 9.4 µM.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Efficient binding of HA-1H/R peptides to HLA-B60. The results are expressed as the percent inhibition of HLA binding of the 150 nM reference peptide by the indicated peptides added at serial dilutions (see Materials and Methods). Curves were fitted by nonlinear regression and one-site binding equation. The IC50 value of the HLA-B60 binder-positive control peptide KESTLHLVL (9 ) was 1.1 µM.

The stability of the HLA-B60/HA-1^H/R peptide binding was addressed by testing for the HLA peptide binding capacities at 4 and 25°C. HLA-A2/HA-1^H/R peptide binding stability was analyzed in parallel for comparison. Increasing the temperature from 4 to 25°C did not affect the strong binding of decameric HA-1^H/R peptides to HLA-B60 (Fig. 3A). Less binding was observed with the nonameric HA-1^H/R peptides to HLA-B60 (Fig. 3B), which was comparable to the nonameric HA-1^H peptide to HLA-A2 (Fig. 3C). Increasing the temperature from 4 to 25°C further decreased the intermediate binding of the nonameric HA-1^R peptide to HLA-A2 (Fig. 3C). Thus, the binding of both HA-1^H and HA-1^R peptides to HLA-B60 was stable and not temperature sensitive.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Stable binding of nonameric and decameric HA-1H/R peptides to HLA-B60. The nonameric and decameric HA-1H/R peptides were tested for binding to HLA-B60 (A and B) and HLA-A2 (C) at the indicated temperatures. The results are expressed as the percent inhibition of HLA binding of the reference peptide. Curves were fitted by nonlinear regression and one-site binding equation.

Twenty-nine-amino acid-long HA-1^H/R peptides were subjected to in vitro digestion with EBV-LCL-derived 20S immunoproteasomes. Within a time frame of 15 min, major peptide fragments were cleaved at the COOH termini of both nonameric and decameric HLA-B60 binding HA-1^H/R peptides. The latter cleavage products contained the intact HLA-B60 binding sequences with three to five additional amino acid residues at the N termini for the HA-1^H and HA-1^R peptides as demonstrated in Tables II and III, respectively. Thus, both HA-1^H and HA-1^R products can be effectively cleaved by proteasomes to generate the precursors of the peptides that bind to HLA-B60.

To test the immunogenicity of both the HA-1^H and HA-1^R peptides in the context of HLA-B60, PBMCs from three HLA-B60⁺ HA-1^RR and from two HLA-B60⁺ HA-1^HH healthy individuals were stimulated with autologous DCs pulsed with the nonameric HA-1^H or HA-1^R peptide, respectively. After two or three rounds of stimulation, the two TCLs induced with the HA-1^R peptide contained significant numbers of IFN--producing T cells that recognized HA-1^R peptide-pulsed HLA-B60-transfected T2 cells. Nevertheless, neither TCL induced with HA-1^R peptide produced IFN- upon stimulation with EBV-LCLs that express the natural ligand HLA-B60/HA-1^R (data not shown). On the contrary, all three TCL induced with the HA-1^H peptide contained, besides HA-1 nonspecific T cells, a significant number of T cells that produced IFN- not only upon stimulation with HA-1^H peptide-pulsed, HLA-B60-transfected T2 cells, but also upon stimulation with EBV-LCLs that express the natural HLA-B60/HA-1^H ligand (Fig. 4).

View larger version (17K): [in this window] [in a new window]   FIGURE 4. T cell recognition of HLA-B60/HA-1H ligand. TCLs secreting IFN- in response to the target cells are indicated. The EBV-LCLs (HLA-B60/HA-1RR and HLA-B60/HA-1HR) are derived from HLA-identical, but HA-1-non-identical siblings. The number of IFN- spots per 105 cells is expressed on the y-axis. The SEM was <5%.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In search of novel T cell epitopes in the HA-1^H/R polymorphic region we studied the binding of polymorphic HA-1 peptides to 11 HLA class I molecules and analyzed the proteasomal cleavage sites in the HA-1^H/R polypeptides. These analyses suggested novel interactions of both alleles of the mHag HA-1 locus with HLA-B60 molecules. Both nonameric and decameric HA-1^H/R peptides effectively bind to HLA-B60. In vitro proteasomal analysis showed cleavage at the COOH termini of HLA-B60 binding peptides, indicating proper intracellular processing. Functional analyses revealed the immunogenicity of HLA-B60/HA-1^H ligand, but as yet not of the HLA-B60/HA-1^R ligand.

Both nonameric and decameric HA-1^H/R peptides show strong binding to HLA-B60, with IC₅₀ values between 1.6 and 5.3 µM. These HLA binding levels are similar to or higher than those of the immunogenic HLA-A2/HA-1^H CTL epitope and of other reported T cell epitopes measured in similar assays (8, 16). Furthermore, we compared the stability of the HLA-B60/HA-1^H/R with HLA-A2/HA-1^H/R peptide interactions by increasing the temperature of the binding assays. These assays reveal that, unlike the HLA-A2/HA-1^R peptide interaction, the HLA-B60/HA1^H/R and HLA-A2/HA-1^H interactions are stable. The stability of HLA-B60/HA-1^H/R interactions was confirmed in separate experiments using fluorescent HA-1^H/R peptides (data not shown). Thus, both HA-1^H and HA-1^R peptides can efficiently interact with HLA-B60, which is an important biochemical feature of strongly immunogenic T cell epitopes (16). This actually predicts immunogenicity of both HA-1^H and HA-1^R locus products in association with HLA-B60.

The HLA peptide binding is preceded by intracellular processing of cellular proteins. In the endoplasmic reticulum, proteasomally cleaved peptides can undergo NH₂-terminal trimming by aminopeptidases (17). COOH-terminal trimming in the endoplasmic reticulum has not been demonstrated. The proper generation of the correct COOH terminus by an early major cleavage site by proteasomes is thus a key event for efficient epitope generation as demonstrated by recent studies (18, 19, 20, 21). In our in vitro cleavage studies, the correct COOH termini of HLA-B60 binding sequences of both HA-1^H and HA-1^R alleles were generated within 15 min. These peptide fragments contained the intact HLA-B60 binding sequences. The exact sequences of the HLA-B60 binding peptides were not present as proteasomal degradation products. Some additional cleavage sites within the putative T cell epitopes were also observed. Nonetheless, the successful generation of HLA-B60/HA-1^H-specific T cells demonstrates the proper cleavage of the HLA-B60 binding HA-1^H peptides by cellular Ag processing machinery. Our results confirm recent reports of the importance of generation of the exact COOH terminus for the intracellular generation of T cell epitopes. Once the relevant peptides are generated by proteasomal cleavage, other cleavage sites found within the epitope do not prevent generation of immunogenic T cell epitopes expressed on the cell surface (13). Based on these results, one may expect that the HA-1^R peptides are also expressed on the cell surface in association with HLA-B60. However, the TCL generated against the HA-1^R peptide-loaded HLA-B60⁺ stimulator cells recognized only peptide-pulsed stimulator cells, not the stimulator cells that naturally express HLA-B60/HA-1^R ligand. It is possible that these TCL may have low affinity to the natural HLA-B60/HA-1^R ligand. Alternatively, the naturally expressed HLA-B60/HA-1^H complex may act as an altered peptide ligand to antagonize the high avidity HLA-B60/HA-1^R-specific T cells. In murine models it was shown that in vivo expression of altered peptide ligand as a natural MHC/peptide complex can antagonize mature T cells specific for the agonistic ligand (22). The antagonistic complex may also cause negative selection of the high avidity T cells in the thymus (23, 24). Thus, both mechanisms may result in failure of detection of HLA-B60/HA-1^R-specific T cells in the periphery. However, it may also be possible that, similar to the HLA-A2/HA-1^R ligand, the HA-1^R peptide is not expressed on the cell surface. HLA-B60 HA-1^R peptide elution experiments are currently underway, but to date have not shown the presence of the HLA-B60/HA-1^R peptide. Moreover, we will apply a more efficient CTL induction protocol and use HA-1-transduced DCs instead of peptide-pulsed DCs as APC. cDNA-transduced DCs result in natural processing and constitutive expression of the mHag HA-1, which is more effective than peptide-pulsed DCs in the generation of HA-1-specific CTLs (25).

In conclusion, our analyses reveal a novel HLA-B60-restricted HA-1^H-specific T cell epitope. Herewith, the HA-1-specific immunotherapy for relapsed leukemia can be extended to patients who receive HLA-B60-matched, HA-1^H-mismatched SCT.

	Acknowledgments

 
We thank Drs. E. Spierings and S. H. van der Burg for reading of the manuscript.

	Footnotes

 
¹ This work was supported in part by grants from the Leiden University Medical Center, the Dutch Cancer Society, and the J. A. Cohen Institute for Radiopathology and Radiation Protection.

² Address correspondence and reprint requests to Dr. Bregje Mommaas, Department of Immunohematology and Blood Transfusion, Building 1, E3-Q, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail address: b.mommaas{at}lumc.nl

³ Abbreviations used in this paper: mHag, minor histocompatibility Ag; CVIA, computer-assisted video image analysis; DC, dendritic cell; EBV-LCL, EBV-transformed B cell line; HS, human serum; SC, stem cell; SCT, SC transplantation; TCL, T cell line.<./>

Received for publication January 9, 2002. Accepted for publication July 18, 2002.

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