A Common Minimal Motif for the Ligands of HLA-B*27 Class I Molecules

CD8+ T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.


Introduction
The proteolytic degradation of newly synthesized pathogen proteins in the cytosol through the combined actions of proteasomes and different peptidases continuously generates peptides of typically 8 to 11 residues long, and these fragments were translocated to the endoplasmic reticulum (ER) lumen through a transporter associated with antigen-processing (TAP) molecules. These short peptides are subsequently assembled with nascent human leukocyte antigen (HLA) class I heavy chain and b 2 -microglobulin molecules [1]. This assembly likely occurs through the interaction of the lateral chains of anchor residues at position 2 (P2) and the C-terminus (PV) of the antigenic peptide [2,3], and these chains are inserted deeply into specific pockets of the antigen recognition groove of the HLA class I molecule [4,5]. The stable HLA/peptide complexes are eventually exported to the cell membrane and presented for cytotoxic T lymphocyte (CTL) recognition [6]. The detection of pathogen peptides by specific T cell receptors results in the killing of pathogen-infected cells.
HLA class I is the largest polymorphic biological system described. More than 7,000 HLA class I alleles have been identified to date (Immuno Polymorphism Database, http://www. ebi.ac.uk/ipd), and classic HLA serologies have been largely divided into complex HLA gene families with increasing numbers of expressed protein subtypes. For example, to date, HLA-B*27 (a well-studied HLA class I family) comprises at least 100 different alleles. Although the presence of Arg at P2 is necessary for HLA-B*27 ligands (SYFPEITHI Database [3]), only a partial overlapping of the peptide repertoire has been observed in different HLA-B*27 subtypes [7]. Individual HLA-B*27 subtypes could present or not specific ArgP2-containing peptides, or the same ligand could bind to different HLA-B*27 subtypes with a broad range of affinity values [8]. Thus, the existence of HLA-B*27 ligands with additional binding motifs for presentation by all or most of the different HLA-B*27 subtypes remains unknown. To address this question, the binding affinity of a homogeneous set of nine naturally processed viral HLA-B*2705 ligands with different sequences, identified using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of Human respiratory syncytial virus (HRSV)-infected cells [9], was examined using seven phylogenetically and functionally different major HLA-B*27 subtypes [10,11]. This analysis revealed a common minimal peptide motif for efficient binding to different HLA-B*27 subtypes.

Synthetic peptides
The peptides were synthesized in a peptide synthesizer (model 433A; Applied Biosystems, Foster City, CA) and subsequently purified through reversed-phase HPLC. The molecular mass of the peptides was established using MALDI-TOF MS, and the peptide composition was determined through mLC-MS/MS.

HLA/Peptide Stability Assays
The synthetic peptide CMV pp65 294-302 (VAFTSHEHF, HLA-C*012-restricted) [18] was used as a negative control in complex stability assays. In addition, for some HLA-B*27 subtypes, the Flu NP peptide (SRYWAIRTR, HLA-B27-restricted) [19] was used as a positive control. The transfected RMA-S B*27 cell lines were incubated at 26uC for 16 h to promote the expression of empty HLA class I molecules (without antigenic peptide) at the cell membrane, as these molecules are stable at 26uC but not at 37uC. The cells were washed and incubated for 2 h at 26uC with various concentrations of peptide in medium without fetal bovine serum. The cells were maintained at 37uC for an additional 2 h to facilitate the internalization of empty MHC class I molecules. Subsequently, the cells were collected for flow cytometry to discriminate between bound or unbound peptides. MHC expression was measured using 100 ml of hybridoma culture supernatant containing the ME1 (anti-HLA-B27) mAb and the secondary antibody as previously described [20]. The data were acquired using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using BD FACSDiva software version 6 (BD Bioscience). The cells alone exhibited peak fluorescence intensities similar to the background staining observed with secondary Ab alone. The fluorescence index was calculated as the ratio of the mean peak channel fluorescence of the sample to that of the control incubated without peptide. Peptide binding was expressed as the EC 50 , the molar concentration of the peptide at 50% of the maximum fluorescence obtained at a concentration range of 0.01-200 mM.

Statistical analysis
Unpaired Student's t test was used to analyze statistical significance. P values,0.05 were considered significant.
Basic, but not nonpolar, C-terminal residues reduce binding to the B*2704 molecule of viral B*2705-restricted ligands In the binding groove, the B*2704 subtype differs from the B*2705 molecule, reflecting two amino acid changes at residues 77 (Asp to Ser) and 152 (Val to Glu) located in the F and E pockets, respectively ( Figure S1). The analysis of peptide binding to HLA-B*2704 in transfected RMA-S cells showed almost no effect with the five HRSV synthetic peptides harboring nonpolar amino acids     Tables 1 and 2). Thus, nonpolar PV residues are necessary, but not sufficient, for efficient binding to the B*2701 class I molecule.
Hydrophobic C-terminal residues, such as Leu or Ile, preserve the binding to the B*2702 molecule of HRSV B*2705-restricted ligands The B*2702 subtype differs from the B*2705 molecule, reflecting three amino acid changes at positions 77 (Asp to Asn), 80 (Thr to Ile) and 81 (Asp to Tyr), located in the F pocket ( Figure  S1). Only the two HRSV peptides with Leu/Ile PV residues, M [169][170][171][172][173][174][175][176][177] , similarly stabilized the binding to both B*2705 and B*2702 molecules (Figure 7 and Tables 1 and 2). In contrast, the binding affinity to B*2702 was moderately decreased for the NP 184-194 peptide, and strongly reduced for the other six viral ligands (Tables 1 and 2). Thus, hydrophobic PV residues, such as Leu/Ile, serve as additional auxiliary anchor motifs for B*2702-binding.
A common minimal motif for ligands of the seven different HLA-B*27 subtypes Table 3 summarizes the HLA-B*27-binding patterns of the different HRSV B*2705-restricted ligands used in this study (Table  S1). Most of the viral ligands bound to various HLA-B*27 subtypes, despite amino acid differences in relevant residues that contribute to the antigen binding site; however, only two of these ligands (NP [184][185][186][187][188][189][190][191][192][193][194] , and NP 195-205 ) efficiently bound to all seven HLA-B*27 subtypes studied. Both ligands contain basic amino acids in the N-terminus and a large and hydrophobic lateral chain in the C-terminal residues. This structure establishes the Arg/Lys-Arg-X n -Ile/Met sequence as the common minimal peptide motif for efficient binding to seven different HLA-B*27 subtypes. To test this prediction three different peptides, HIV gp160 500-508 [21], MV F 438-446 [22], and Influenza virus PB1 238-246 [23], harboring the identified minimal peptide motif (basic residue at P1 and large hydrophobic aliphatic residue at PV), were studied. Interestingly, MHC/peptide complex stability assays showed efficient binding of these synthetic peptides to all seven HLA-B*27 subtypes tested (Tables 2 and 3). In addition, a synthetic peptide with only the common minimal peptide motif (RRAAAAAAAI) efficiently bound to all seven HLA-B*27 subtypes analyzed (Tables 2 and  3). Conversely, the exchange for Ala of the Met residue at CV position in both NP [184][185][186][187][188][189][190][191][192][193][194] peptides abolished the interaction with B*2701 subtype (Table 4). Also, this monosubstitution significantly decreased the binding of A11-NP 195-205 peptide to B*2702 subtype (Table 4). Moreover the exchange for Ala of the Arg residue at P1 position in NP 184-194 peptide considerably decreased the binding to B*2703 subtype (Table 4).
Compared with the affinity of the nine HRSV ligands for B*2705, 7 ligands exhibited decreased affinity for B*2701 and B*2702, 6 ligands showed decreased affinity for B*2703, 4 ligands showed reduced affinity for B*2704 and B*2706, and only 3 ligands exhibited decreased affinity for B*2709 (summarized in Table 3). These data do not correlate with either the amino acid differences between subtypes or the individual interactions in the respective pockets, indicating compensatory effects of changes in

Discussion
Using large scale mass spectrometry analysis, an extensive knowledge of HLA-B*2705 ligandome (with approximately 2,000 peptides identified) has been reported [7,24]. In contrast, few endogenous natural ligands have been identified in other HLA-B*27 subtypes: 32, 49, 38 and 50 from B*2703, B*2704, B*2706 and B*2709, respectively, and only 8 and 15 from B*2701 and B*2701, respectively (summarized in [7] and SYFPEITHI database [3]). Except for the ArgP2 residue, no additional anchor or auxiliary anchor motifs were identified in these studies. Thus, in the present study the Arg/Lys-Arg-X n -Ile/Leu/Met sequence was identified as the minimal common peptide motif for efficient binding to the seven major, phylogenetically ( Figure S2  B*2704 [26], B*2705 [10], and B*2709 [27] subtypes at the clonal level to assess T cell epitope sharing among HLA-B*27 subtypes have been previously described. These studies defined the functional relationship as B*2705.B*2709.B*2703.B*2702. B*2701.B*2704.B*2706, reflecting amino acid differences between subtypes. However, in contrast to self-restricted pathogen recognition in normal cellular immune responses, the allogenic anti-B*27 CTL clones recognized both polymorphic allo-MHC residues and self-derived peptides [14]. Thus, the direct contribution of these polymorphic HLA-B*27 residues to the CTL epitope could disguise both cross-presenting antigen peptide properties and the degree of functional relationship between the different HLA-B*27 subtypes. As the antigen processing and presentation machinery is similar, if not identical, in all cell lines expressing the different HLA-B*27 subtypes, the nine HLA-B*2705-restricted ligands endogenously processed and presented in the HRSVinfected cells might also be presented by the diverse HLA-B*27 subtypes according to affinity. Thus, the functional relationship between subtypes established with the HRSV ligands through affinity assays in the current report could be a better approximation to determine the antigen peptide-presenting properties of HLA-B*27 class I molecules, and this information could be applicable to the rational design of vaccines. Few studies have analyzed antigen binding and/or presentation of the same viral ligands to different HLA-B27 subtypes. Four were tested, and only the EBNA3C 258-266 ligand tended to be immunodominant and was recognized in the context of all three B27 subtypes studied, whereas the LMP 236-244 ligand was only recognized associated to B*2704 [28]. In contrast, in another study, the LMP 236-244 ligand was recognized by one of four CTL clones in the context of five HLA-B*27 (B*2702, B*2704, B*2705, B*2706 and B*2709) subtypes analyzed [29]. In addition two ligands, HIV gag 265-279 (KRWIILGNKIVRMYC) and Flu NP 380-393 (ELRSRYWAIRTRSG), were presented by both B*2702 and B*2705 subtypes [30]. Only one of these six viral epitopes harbors the minimal peptide motif for the efficient binding described in the current study, and this peptide was restricted by the three HLA-B27 subtypes analyzed (B*2702, B*2704 and B*2705). However, only an endogenous ligand derived from human histone H3.3 has been previously described for binding to B*2701 [13], B*2702 [31], B*2703 [32], B*2704 [33], B*2705 [34], B*2706 [33] and B*2709 [16] subtypes. This ligand, containing the RRYQKSTEL sequence, also harbors a basic amino acid in the N-terminal residue and a large and hydrophobic lateral chain in C-terminal residue, consistent with the motif defined in the present study.
Thus, studies examining different HLA class I families and supertypes are needed to determine the conserved anchor or auxiliary motifs common to these HLA clusters and validate new bioinformatics tools for the functional clustering of MHC molecules [35]. These data are also relevant for the identification of antiviral cytotoxic T lymphocyte responses and vaccine development.  Table 3. Summary of the relative affinity of HRSV ligands for different HLA-B27 subtypes.  Supporting Information Figure S1 Scheme of the polymorphisms in each HLA-B*27 subtype. (PDF) Figure S2 Phylogenetic tree of the HLA-B*27 subtypes [36,37]. (PDF)