Classical major histocompatibility complex (MHC) molecules are divided into two groups: class I and class II [1]. Each contains large numbers of alleles, all of which bind antigen for cell surface presentation to T cells. MHC class I molecules comprise a single polymorphic chain coupled to a single conserved protein: β2 microglobulin. In contrast, MHC class II molecules comprise two polymorphic subunits: an α and a β chain. A notable difference between the classes is the open-ended peptide-binding groove of MHC class II molecules compared to the closed binding site of MHC class I. This feature, which has been observed in many X-ray crystallographic structures, has been used to explain the observed differences in peptide length accommodated by the two classes [2–4]. Class I molecules typically accommodate peptides of eight to ten residues, although instances of longer peptide binding have now been reported [5].

In contrast, MHC class II molecules can accommodate much longer peptides. Stern et al. investigated an influenza-derived peptide complexed with HLA-DR1; they saw the peptide bound in an extended conformation with five binding pockets, which engaged peptide side chains, while the flanking regions extended out of the groove [3]. Since MHC class II molecules have an open-ended binding groove, they do not, in general, restrict the length of bound peptides. The length of peptides occupying the groove can vary considerably as a consequence of semi-stochastic proteolytic degradation [6].

Several studies have shown the influence of residues outside the main nonameric core on binding and subsequent T cell recognition. Residues outside the nonameric binding region but within an extended binding groove have been called peptide-flanking residues (PFRs) [7]. The importance of PFRs for T cell recognition has also been investigated. It is widely accepted that these flanking regions can contribute to T cell mediated pMHC recognition [8]. Arnold and colleagues showed that certain T cell responses were completely dependent on residues at peptide positions P-1 and P11. Similar findings were reported by Stepniak et al for immunostimulatory HLA-DQ2 binding peptides derived from gliadin and glutenin proteins [9]. The findings of both authors can be partly explained by the results of X-ray crystallographic studies of peptide-MHC class II-TCR trimolecular complexes [10, 11]. These reports indicate that the CDR3 regions of the TCR α and β chains tend to locate over the p5 residue in the binding groove allowing the TCR to interact with the PFRs. It has also been shown that the properties of residues in the PFRs can influence T cell recognition, with residues capable of forming salt-bridges or hydrogen bonding with the TCR being most favoured [8].

Much circumstantial if anecdotal evidence suggests that PFRs contribute strongly to peptide-MHC stability. They can provide an increased measurable affinity of peptide for the MHC binding groove, in particular the residue at position P-1 [12]. Nelson et al. reported that the effect of increased stability was greater for a peptide length increase of six amino acids compared to four. They also found the effect to differ depending on which terminus was elongated. Moreover, a previous study by Srinivasan et al showed increased affinity resulting from the peptide elongation of a single peptide [13]. How such peptide elongations might increase affinity remains unclear. Although it is theoretically possible PFRs interact with the MHC molecules outside the groove, this has yet to be shown experimentally, nor has it been seen in X-ray crystallographic structures.

We have sought to elucidate this phenomenon further, by examining the relationship between peptide length and MHC class II affinity. We devised custom algorithms and applied them to data available in the AntiJen database [14] to firstly ascertain whether peptide length can impact on affinity of a peptide for an MHC class II molecule. This was assessed by searching the database for instances of peptide elongation and determining the reported effect on MHC class II affinity. Following this, we examined whether there was a demonstrable limit to the effect of peptide length on MHC class II affinity and subsequently determined whether any effects on affinity were peptide terminus specific. Our analysis indicated the positive impact of peptide length on affinity. We also identified an optimal length for peptide-MHC class II binding and demonstrated that the effects on peptide affinity are terminus independent.

While MHC class II molecules and their ligands are well characterised, the impact of peptide length on MHC affinity is not well understood. While several anecdotal observations have purported to show that peptide length may influence affinity we have for the first time systematically quantified this relationship using in silico analysis. Using novel computational analyses, we exploit the wealth of manually-curated quantitative binding data available within the AntiJen database [14].

When attempting to derive a relationship between sequence length and observed MHC affinity, one is limited by several factors. One such factor is the presence of different binding determinants in most sequences that could significantly bias any simple length-affinity correlation. Thus, if one examines the affinity of two sequences of different length and low sequence similarity, one cannot ascertain if an altered affinity is due to differences in the binding determinant or to changes in sequence length. To isolate such a relationship, one must normalise all factors (such as MHC allele, experimental conditions, and peptide sequence) except for sequence length before analysing the affect on affinity. To that end, we identified examples of affinity measurements, for a single sequence and an elongated equivalent, which were performed under the same experimental conditions. The benefits of this approach were twofold. Firstly, inter-laboratory variation in affinity measurements would be excluded. Secondly, bias from variations in the binding determinant would also be reduced if not eliminated, thus removing bias from intra- but not inter-sequence comparisons. Bias from inter-sequence comparisons is unavoidable. We optimised for the required task in order to avoid representing bias as effect.

The data presented in Figure 2 allowed us to examine the limits of the effect with respect to final peptide length. By examining the log fold-change in affinity for MHC class II as the length of the peptide increases it can clearly be seen that the impact on affinity lessens. The regression plot also shows that at approximately 19 amino acids the effect of peptide elongation on affinity becomes zero. Elongating peptides beyond this point would – in the majority of cases – result in a diminished affinity. This seems logical: if no such ceiling existed for the positive effect of peptide length then sequence length alone would be the key determinant of peptide-MHC affinity. This discovery is also strengthened by the findings of Lippolis et al. who eluted and sequenced peptides from HLA-DR*0401 and found that the most abundant species within a family of nested peptides were between 14 and 21 residues in length [15]. Our findings do not however suggest that a single optimal length would exist for all peptides for all MHC class II alleles, and indeed the relative heterogeneity of peptides binding to MHC class II molecules is well reported [15–17]. However, this approximation to an optimal length is consistent for a large number of peptide sequences and a large number of MHC class II alleles.

Many explanations for this length ceiling present themselves. It may be that as peptides become very long they are no longer able to form favourable interactions with the MHC or TCR. Alternatively, when MHC class II binding peptides attain a certain length they may form secondary or tertiary structures outside the binding site that are less compatible with MHC class II binding. An exception to this rule may be the 33-amino acid peptide initially described by Shan et al [18]. It binds to HLA-DQ2 and stimulates disease associated T cells. Despite its unusual length, this peptide retains its MHC class II binding abilities as its proline rich nature helps it to form a type II polyproline helix. This is the conformation normally seen in MHC class II binding grooves [3].

Our results clearly imply a general correlation between increasing MHC binding affinity and increasing peptide length. It is not peculiar to a restricted subset of peptides. Sercarz and Maverakis posited that longer peptides have a greater binding affinity for MHC class II [19]; this assertion was based on observations from a few articles, e.g. the study by Srinivasan et al [13]. However, to the best of our knowledge, ours is the first study to test this hypothesis systematically for several MHC class II alleles. The results of our study are compelling: across 1279 identified peptide elongation events for peptides binding to 19 distinctive MHC class II alleles, affinity predominately increased. It is important to note, however, that the examined peptides are more representative of the HLA-DR species than either HLA-DP or HLA-DQ, and this may result from an apparent publication bias in favour of HLA-DR which will be reflected in the AntiJen database.

Our findings have clear implications for experimental immunology and vaccinology, as well as computational immunology; and these implications are many. Increased affinity in vivo may result in increased reactivity to recently degraded peptides. Presumably, recently degraded peptides may be less completely digested and – as a result of increased length – have an increased affinity for MHC class II relative to shorter more fully degraded peptides. Such an effect would, in theory, increase the likelihood of T cells recognising more recently endocytosed peptides rather than a background of self-peptides. Additionally, the ability of exopeptidases to digest termini of peptides within the MHC class II binding cleft [6] may effectively reduce their MHC class II affinity. Thus, peptides susceptible to digestion by exopeptidases may prove to be less immunogenic in vitro. The field of subunit vaccine design may be able to augment vaccine efficacy by increasing peptide length or eliminating potential proteolytic cleavage sites, which may result in an inability of normal proteolytic enzymes to digest peptides to shorter equivalents with reduced immunogenicity.

Using an in silico approach we were able to define a relationship between peptide length and MHC class II affinity. The results of this study demonstrate that, within limits, elongation of a peptide is likely to result in a greater affinity for MHC class II molecules. It was also possible to show that the optimal length for peptide-MHC affinity was approximately 18–20 amino acids. Additionally, it was also possible to demonstrate that the effects are consistent regardless of which terminus was elongated. Given the well-characterised importance of peptide-MHC interaction to the cellular immune response, it is likely that the findings of this study will prove of significance in vaccine design and the epitopic basis of immunological disease.