Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

1Highly polymorphic class I molecules encoded by the major histocompatibility complex (MHC) are crucial in the adaptive immune response to viruses and some intracellular bacteria, binding peptides inside the cell and presenting them on the cell surface to CD8 T lymphocytes. The impact of the MHC in response to human immunodeficiency virus (HIV) is well recognized, with class I alleles like HLA-B*35:01 leading to rapid onset of AIDS while HLA-B*57:01 and HLA-B*27:05 confer long-term non-progression. Various explanations for these associations have been suggested, including antigen presentation of a particularly effective peptide or of a number of peptides to cytotoxic CD8 T cells or recognition (primarily) independent of peptide by natural killer (NK) cells. Recently, the level of cell surface expression of HLA-C alleles correlated with CD8 T cell cytotoxicity has been proposed as one important basis for control. Understanding how cell expression level might impact on disease resistance is complicated in humans due to the presence of three class I loci, so we began by studying a simpler animal system before examining human class I alleles.

2Long ago, we reported that the relative expression level of MHC class I molecules on the surface of chicken red blood cells as assessed by flow cytometry varies significantly, with cells of the MHC haplotype B21 approximately ten-fold lower than B4, B12, B15, and B19. This finding was of interest because the level of cell surface expression is inversely correlated with the reported levels of MHC-determined resistance to Marek's disease, an economically important disease caused by the oncogenic herpesvirus, Marek's disease virus (MDV). Decades of investigation identified B21 (and other haplotypes like B2, B6, and B14) as generally conferring resistance and B19 (and other haplotypes such as B4, B12, and B15) as generally conferring susceptibility (reviewed in ). On this basis, we proposed that MHC-determined resistance to Marek's disease could be due to the cell surface expression polymorphism of class I molecules.

3Compared to humans, the chicken MHC is relatively simple, with two classical class I genes BF1 and BF2 that flank the genes for the transporter associated with antigen presentation (TAP). The TAP transporter pumps peptides from the cytoplasm to the lumen of the endoplasmic reticulum for loading nascent class I molecules, and in typical mammals is functionally monomorphic, pumping a wide variety of peptides for all members of the polymorphic class I multigene family. In contrast, chicken TAP genes are highly polymorphic, with each haplotype encoding a TAP molecule with a peptide-translocation specificity matching the peptide-binding specificity of the dominantly expressed class I molecule encoded by the BF2 gene, with the BF1 gene expressed poorly or not at all.

4In chickens, the peptide-binding specificity of the dominantly expressed class I molecule can determine resistance to infectious pathogens as well as responses to vaccines. The peptide motifs from B4, B12, B15, and B19 class I molecules are if anything more fastidious than human and mouse motifs, with only one or two amino acids found at the positions of anchor residues, with the motifs explaining the immune response to infection and vaccination. The fastidious binding of these molecules could be easily understood from wire models of the binding site, in which charged and hydrophobic residues were found in appropriate places to interact in a simple manner with the anchor residues of the bound peptides. This view was confirmed from the crystal structure of the dominantly expressed class I molecule BF2*0401 from the B4 haplotype, with positive-charged residues in a narrow groove allowing only certain anchor residues from the peptide to be accommodated. In contrast, much less peptide material was isolated from B21 cells, with many amino acids found in every peptide position. Crystal structures demonstrated that the dominantly expressed class I molecule BF2*2101 remodels the binding site to accommodate two peptides with completely different sequences, including the anchor residues. Thus, the lack of a clear peptide motif could be explained as promiscuous peptide binding due to the remodelling of the peptide-binding site.

5In this paper, we find that two other haplotypes known to confer resistance to Marek's disease also have low cell surface expression and promiscuous peptide motifs, and examine the structural basis for promiscuous binding in three low expressing molecules. We show that the same relationship between cell surface expression and peptide-binding repertoire is found for four human class I molecules, associated with progression to AIDS. Finally, we propose how promiscuous peptide binding might confer resistance to some pathogens, what the basis for the cell surface expression polymorphism might be, and how these properties may relate to different strategies for resistance to pathogens.

6We reported that the relative level of class I molecules on the surface of erythrocytes is ten-fold lower for adult chickens of the B21 MHC haplotype compared to the B4, B12, B15, and B19 haplotypes, as assessed by flow cytometry with two different monoclonal antibodies (mAb). Extending this work, we examined another cell type, spleen lymphocytes, with a quantitative flow cytometric assay using a mAb to chicken heavy chain. We found a similar rank order for these and other haplotypes (Figure 1), with the difference between the highest and lowest ranging up to fourfold over many experiments. The B2, B14, and B21 haplotypes can be considered as ‘low-expressing haplotypes’, while B4, B12, B15, and B19 haplotypes are ‘high-expressing haplotypes’, with low-expression correlating historically with resistance to Marek's disease.

7For the high-expressing haplotypes B4, B12, B15, and B19, the dominantly expressed class I molecules all have definite peptide motifs, with two or three peptide positions each bearing one or two chemically similar residues. In contrast, much less peptide material was isolated from low expressing B21 cells, with pool sequences showing many amino acids in every peptide position. Extending this work, we sequenced pools of peptides from erythrocytes and leukocytes of chickens with B2 and B14 haplotypes and found that they also showed many amino acids in each position, mostly with very different chemical characteristics (Figure 2A). Sequencing of single peptides from B2, B14, and B21 haplotypes confirmed this sequence diversity, with no obvious anchor positions bearing one or two residues with similar chemical characteristics (Figure 2B).

8Thus, the chicken class I molecules from high-expressing haplotypes have clear peptide motifs, at least as fastidious as such motifs described in humans and mice. In contrast, the class I molecules from low-expressing haplotypes have no obvious peptide motifs, with promiscuous binding unlike what has been described in mammals. To confirm that these properties result in different numbers of distinct peptides on the cell surface, we isolated class I molecules from equal numbers of cells from the B19 cell line 265L and the B21 cell line AVOL-1, and analysed the bound peptides by mass spectrometry. Despite the B19 line having twice as many class I molecules on the cell surface as the B21 line as assessed by quantitative flow cytometry, there were only one third as many distinct peptides identified by mass spectrometry (Figure 3). Thus, a promiscuous class I molecule can and does bind a greater variety of peptides that appear on the surface of the cells, in comparison to a fastidious molecule.

9Unlike high-expressing haplotypes, the wire models of the dominantly expressed class I of the B21 haplotype, BF2*2101, gave no clue as to which peptides might bind (Figure 2C). Crystal structures, based on heavy chain and β2-microglobulin (β2m) expressed in bacteria and refolded with synthetic peptides, showed that BF2*2101 remodels the binding site to accommodate two peptides with completely different sequences, including the anchor residues. Small residues bordering the groove lead to a large cavity in the middle of the groove, within which Asp24 and Arg9 can move, creating different configurations, as illustrated for the 11mer peptide GHAEEYGAETL and the 10mer peptide REVDEQLLSV (Figure 4A,B). The peptide position P2 His of the 11mer interacts with the Asp24, while the Pc-2 Glu interacts with the Arg9 (and Pc Leu fits in a hydrophobic pocket at the end of the groove). The 10mer remodels the binding site, so that the P2 Glu interacts with Asp24 that also interacts with Arg9 in a so-called charge transfer mechanism, creating a hydrophobic pocket that accommodates Pc-2 Leu (and Pc Val fits in the hydrophobic pocket).

10Additional peptides eluted from MHC molecules of B21 cells (Figure 2B) show that other amino acids at P2 and Pc-2 can be accommodated by the critical Asp24 and Arg9 within the large central cavity in the binding site. In fact, the 10mer TNPESKVFYL binds BF2*2101 in similar way to the 10mer REVDEQLLSV (Table 1, Figure 4C), with P2 Asn binding the Asp24 aided by charge transfer with Arg9, and with the rearrangement of Arg9 permitting the accommodation of Pc-2 Phe, just as for Pc-2 Leu in the previous structure.

11However, the two structures with the 10mers TAGQEDYDRL and TAGQSNYDRL display a completely different mode of binding (Table 1, Figure 4D,E), for which the P2 Ala does not interact with the MHC molecule at all (and therefore is not an anchor residue), with Arg9 interacting as a bridge between Asp24 and Pc-2 Asp. A further mode of binding is shown by the 10mer YELDEKFDRL (Table 1, Figure 4F), for which both anchor residues at positions P2 and Pc-2 are acidic. In this structure, the P2 Glu interacts with both Asp24 and Arg9, and Pc-2 Asp interacts with Arg9. These various modes of binding are presumably just a few out of many and illustrate the promiscuous binding of BF2*2101, unlike anything seen for mammalian class I molecules.

12Similar to B21, the peptides isolated from cells of the low expressing haplotypes B2 and B14 have no obvious motifs. Pool sequences show no position at which only one or two chemically similar amino acids are present as anchor residues (Figure 2A). Individual peptides have no classic pattern of anchor residues, but some features are discernable (Figure 2B).

13The B2 peptides fall into two groups. One small group of peptides has P2 Arg and mostly P1 Arg and Pc Tyr (Figure 2B), much like the dominantly expressed class I molecule of the high-expressing B15 haplotype, BF2*1501. These three peptides are likely to have been isolated from the poorly expressed molecule encoded by the minor gene of the B2 haplotype, BF1*0201, the wire model of which (Figure 2C) looks very similar to BF2*1501. Size exclusion chromatography (SEC) of heavy chain and β2m expressed in bacteria and refolded with peptide showed that BF1*0201 but not BF2*0201 binds these peptides (Figure 5).

14All of the other B2 peptides have generally smaller hydrophobic amino acids at P2 and somewhat larger hydrophobic amino acids at Pc (Figure 2B). Crystal structures with two of these peptides (Table 1, Figure 6A,B), YPYLGPNTL and VIFPAKSL, show that Pro and Ile at peptide position P2 and Leu at Pc bind shallow hydrophobic pockets (Figure 6A,B), similar to the way in which anchor residues bind pockets B and F in many mammalian class I molecules. This mode of binding is completely different from BF2*2101 that remodels the binding site, despite the fact that BF2*0201 shares Asp24 and Arg9 with BF2*2101. In these BF2*0201 structures, Arg9 interacts mostly with peptide main chain atoms and Asp24 interacts with Tyr43 (residue 45 being the equivalent position in human class I molecules), a residue that differs between BF2*0201 and BF2*2101, ultimately leading to a more hydrophobic pocket B to accommodate P2 residues (Figure 6A,B).

15The B14 peptides all share two features, generally a medium to large hydrophobic amino acid at P2 and one or more basic amino acids at the end of the peptide (Figure 2B). A wire model of BF2*1401 shows small hydrophobic and polar residues in and around the beginning of the groove and reveals many acidic residues at the end of the groove, well placed to interact with the basic residues at the peptide C-terminus (Figure 2C). The crystal structure of peptide SWFRKPMTR bound to BF2*1401 shows that Trp at position P2 interacts with hydrophobic surfaces in pocket B, while Arg at Pc interacts with Asp73(human position 74), Asp76(77) and Asp113(116) in pocket F (Table 1, Figure 6C). Both these pockets are much deeper in BF2*1401 compared to BF2*0201, which explains the larger anchor residues found in the B14 peptides compared to most B2 peptides. The differences in pocket sizes are primarily due to difference in size between Val43(45) and Tyr43(45) for pocket B and between Asp113(116) and Met113(116) for pocket F, which are supported by a network of hydrogen bonds from Thr24 and Gln9 in BF2*1401 and from Asp24 and Arg9 in BF2*0201 (Figure 6).

16These data show that the three class I molecules with lower expression on the cell surface all bind a wide range of peptides with no obvious peptide motif, although the mechanism by which the promiscuous binding is achieved is different for each molecule. One feature that may be important is the width of the peptide-binding groove (Figure 7), which is the narrowest in the high expressing BF2*0401 molecule and the widest in the low expressing BF2*2101.

17The structural analysis of BF2*2101 showed that this low expressing class I molecule achieves promiscuous binding by remodelling the peptide binding site in a way never described for a mammalian class I molecule. However, analysis of BF2*0201 and BF2*1401 shows promiscuous binding by chicken molecules using anchor positions into pockets B and F, much like many mammalian classical class I molecules. In order to determine whether the inverse correlation between cell surface expression level and peptide binding promiscuity is a feature just of chickens or is indicative of a more fundamental property, we examined some human class I alleles.

18Only a few studies have explored the extent of the peptide-binding repertoire of different human class I molecules. One of these studies reported the predicted peptide-binding repertoires for four human class I alleles, finding a rank hierarchy from the extremely fastidious HLA-B*57:01 to HLA-B*27:05 to HLA-B*07:02 to the highly promiscuous HLA-B*35:01, that correlated directly with progression from HIV infection to AIDS. The rank hierarchy of these alleles is the same as that determined by binding of peptide libraries.

19We identified two mAbs reported to react with all four HLA-B alleles (along with HLA-C alleles, which are poorly expressed on blood cells), but not with certain HLA-A alleles. Volunteers with the proper combinations of homozygous HLA-A and HLA-B alleles were recruited from a large group of bone marrow donors (Table 2), and the cell surface expression levels on their blood lymphocytes and monocytes were examined by quantitative flow cytometry. The two mAb (Tu149 and B1.23.2) reacted with all four HLA-B alleles and gave similar (but not identical) results for both lymphocytes and monocytes, with a rank hierarchy in cell surface expression ranging from HLA-B*57:01 as the highest to HLA-B*27:05 to HLA-B*07:02 to HLA-B*35:01 as the lowest (Figure 8A–D).

20One potential concern with these data was that the difference in expression level of the HLA-B alleles might actually be due to differences in HLA-C levels, which are known to vary between alleles. However, inspection shows that there is no correlation of expression levels reported for the HLA-C alleles found for the individual donors and the expression level of the mAb binding (Table 2), if anything the reverse with the low expressing HLA-B*35:01 donors all having HLA-C alleles reported to have high expression.

21Another concern was that the difference in expression level of the HLA-B alleles actually reflects differences in affinity of binding by the two mAb. The fact that both antibodies gave similar but non-identical results suggests that they do not recognize exactly the same epitopes and therefore are unlikely by chance to vary similarly in affinity. We tested whether the two antibodies bound independently and found that B1.23.2 inhibited both itself and Tu149 very well, while Tu149 inhibited both itself and B1.23.2 much less well (Figure 9). These results suggest that B1.23.2 has a much higher affinity than Tu149 and that their epitopes overlap. We also utilized a third antibody (22E-1) that reacts with the HLA-B*57:01 and HLA-B*27:05 but not HLA-B*07:02 and HLA-B*35:01 (nor with any HLA-C allele), and therefore must recognize a different epitope. We found the same relationship of expression level for HLA-B*57:01 and HLA-B*27:05 as with the other two antibodies (Figure 8E,F).

22Thus, in humans the high expressing class I molecules have fastidious peptide binding, and the low expressing molecules have promiscuous binding. We conclude that the correlation between peptide-binding repertoire and expression level is the same in chickens and humans and is indicative of a fundamental property of class I molecules.

23In this article, we make three major points. First, we extend previous work showing that the chicken BF2*2101 achieves promiscuity by remodelling the peptide binding site in a way unknown for mammals, but we find that two other low expressing chicken class I molecules achieve promiscuity with the same pockets typically used in mammals. By extension, these latter results provide a potential molecular explanation for recent reports that human class I molecules vary in peptide repertoire and other reports that they vary in cell surface expression level. Second, we show that peptide-binding repertoire and level of cell surface expression are inversely correlated in both chickens and humans, indicating that this relationship is a fundamental property of class I molecules. Finally, we find that there is a clear association with resistance to infectious diseases, with the low expressing promiscuous class I molecules associated with resistance to Marek's disease in chickens but associated with faster progression to AIDS in humans. We discuss each of these points in turn.

24The first point is the structural and mechanistic basis for differences in peptide repertoire of chicken class I molecules. In this article, we confirm and extend the finding that BF2*2101 binds 10mer and 11mer peptides with three anchor positions, with many different amino acids with different chemical characteristics, mostly charged and polar at positions P2 and Pc-2 (and hydrophobic at Pc), which is unlike any mammalian molecule described thus far. In contrast, we find that BF2*0201 and BF2*1401 bind 8mer and 9mer peptides with two anchor residues (much like typical mammalian class I molecules), each with a variety of amino acids at P2 and Pc.

25One question is why BF2*2101 and BF2*0201 bind in such different ways, given that both have Asp24 and Arg9 in their binding sites. In part, this may be due to the difference in residues lining the peptide-binding groove. In BF2*2101, small residues like Gly68(human position 69), Ser69(70), Ser97(99), and Gly152(155) create a big cavity in the centre of the binding groove, allowing Asp24 and Arg9 to move. In BF2*0201 and BF2*1401, the two Ser residues are replaced by the large residues Asn69(70) and Tyr97(99), creating a constricted groove much more like narrow grooves found in high expressing fastidious molecules such as BF2*0401. It is also possible that the length of peptides is determined at least in part by TAP specificity. Together, the narrower groove and the shorter peptides ensure that Asp24 and Arg9 in BF2*0201 (as well as Thr24 and Gln9 in BF2*1401) cannot reach the P2 and Pc-2 positions, and thus they interact (if at all) with the main chain atoms of the peptide.

26Another question is how chicken class I molecules BF2*0201 and BF2*1401 compare to human molecules like HLA-A2, all of which bind peptides with hydrophobic anchor residues. HLA-A2 has one of the widest peptide repertoires among human class I molecules, as assessed by peptide-binding studies. The anchor residues for HLA-A2 fit into narrow and specific-binding pockets, which allow almost only P2 Met and Leu for pocket B and Pc Val and Leu for pocket F. Although Leu and Val in particular are relatively common in proteins (so that the number of peptides bearing these amino acids should be relatively high), there is a much larger number of hydrophobic amino acids that are accommodated by the shallower pockets in both BF2*0201 and BF2*1401. Hence, it seems likely that the peptide repertoire of the most promiscuous chicken class I molecules will be greater than the most promiscuous human class I molecules.

27Finally, are there obvious mechanisms by which differences in peptide repertoire arise? In chickens, the peptides presented depend on the peptide-binding specificities of the class I molecule, but also on the peptide-translocation specificities of the TAPs and perhaps also on peptide-editing by tapasin. The genes for chicken TAP and tapasin have at least as many alleles as class I genes. On this basis, we propose that the difference in peptide repertoire (and in cell surface expression level) between chicken class I alleles is determined at the level of peptide loading and editing, likely to take place mostly in the peptide-loading complex. It might not be immediately apparent whether such an explanation could account for the differences in peptide repertoire and cell surface expression of human class I molecules, since human TAPs and tapasin are functionally monomorphic. However, a recent paper suggests that the assembly and cell surface expression of different HLA-B alleles is determined by interaction with the near invariant tapasin molecule, based on variation in the certain HLA-B residues. Indeed, it is striking that the rank order of expression level that we find for the four HLA-B alleles tested is exactly the same as the rank order of tapasin dependence found in this recent paper.

28The second important point for discussion is why the cell surface expression would be inversely correlated with peptide-binding repertoire. A proximal explanation might be that the biochemical mechanism of peptide loading, as mentioned in the paragraph above, leads to this relationship. Another explanation, similar to the suggestion that multiple MHC molecules lead to a greater chance of autoimmunity, is that promiscuous class I molecules confer more resistance to pathogens but also lead to greater autoimmunity compared to fastidious class I molecules.

29We favour a third possibility, prompted by the experimental and supporting theoretical evidence for the notion that a greater number of MHC loci would lead to greater level of negative selection in the thymus. In a similar way, one might expect that class I molecules presenting a lesser variety of peptides would negatively select fewer T cell clones, but class I molecules presenting a greater variety of peptides would negatively select many more T cells and create a pauperized T cell repertoire. Therefore, we propose that the peptide-binding repertoire and the cell surface expression levels are inversely correlated precisely to allow similar numbers of T cell clones to survive negative selection and be available in the periphery. In this view, low expressing and promiscuous class I molecules might present many more peptides but each individual peptide would be present on the surface at a much lower level, so that T cells would escape the negative selection that might have occurred at a higher concentration of a particular MHC-peptide complex. In essence, we propose that peptide-binding repertoires and cell surface expression levels have evolved to optimize peripheral T cell responses. Many papers have examined naïve CD8 T cell repertoire in humans and mice (for example,;; ); such analyses with high and low expressing haplotypes would be informative.

30The third important point for discussion is the fact that the inverse correlation between peptide binding repertoire and cell surface expression level also correlates with resistance and susceptibility to certain infectious pathogens. One important question is how it might work.

31In chickens, these phenomena are correlated with MHC-determined resistance to historic Marek's disease. Many genetic loci contribute to resistance to Marek's disease, but historically the B locus (containing the MHC) was by far the most important, and under strong selection by MDV. Here, we show that the low expressing promiscuous class I molecules are associated with resistance to Marek's disease, while the high expressing fastidious class I molecules are associated with susceptibility. A consequence of promiscuous binding is that these low expressing class I molecules present a larger repertoire of peptides which should activate a wider range of T cell clones, which may be beneficial in the immune response to certain pathogens. We propose that this breadth of antigen presentation leads to a breadth of T cell response that is the basis of MHC-determined resistance to Marek's disease. Conversely, a narrow T cell response to Marek's disease would be the basis for MHC-determined susceptibility, and there is already evidence for a limited repertoire of CD8 T cell clones infiltrating tumours in susceptible B19 chickens. Parenthetically, if true this model suggests that the narrowing of CD8 T cell clonality characteristic of responses in humans and mice may not be a feature of responses involving promiscuous class I molecules in chickens.

32For our example in humans, however, we find the opposite disease association. We find that the low expressing promiscuous HLA-B alleles are associated with rapid progression from HIV infection to AIDS, while the high expressing fastidious HLA-B alleles are found in non-progressors. The correlation with peptide binding repertoire (as assessed by prediction) of HLA-B alleles had already been noted, but the relationship with cell surface expression level has not. The controller alleles HLA-B*57:01 and HLA-B*27:05 are known to bind and present particular viral peptides that are both protective and difficult for the virus to change without a loss in fitness. Thus, particular class I molecules may be selected because their fastidious motif presents a particularly protective peptide.

33Is there an obvious evolutionary basis for differences in peptide-binding repertoire? The fact that the fastidious HLA-B*57:01 confers resistance to AIDS while the promiscuous BF2*2101 confers resistance to Marek's disease may be no paradox, in that breadth of peptide presentation may be an appropriate response to some pathogens, while presentation of a particular peptide may be more suitable for other pathogens. Viewed from this perspective, the work presented in this paper has begun to define two groups (or a range between two extremes) of class I molecules that are strategically evolved for different modes of resistance.

34One possible strategy is that fastidious class I molecules with pauci-clonal T cell responses might be better suited for rapidly evolving viruses with a limited scope for immune evasion, for which a particular peptide might be the most efficient way to achieve resistance. In contrast, promiscuous class I molecules might be better suited for pathogens with coding potential for many immune evasion genes and more stability over evolutionary time, such as large DNA viruses, bacteria, and even parasites. There are few reports about pathogen peptides that are presented in chickens, but the large literature on the MHC restriction of viruses, bacteria, and parasites in humans and mice (for example,;;;;; ) may be reinterpreted, as the peptide repertoire and cell surface expression levels of the class I molecules become known.

35Another potentially fruitful way of considering the strategies of class I molecules (not necessarily exclusive of the first view) might be as generalists and specialists. There is a large literature in biology that examines the generalist/specialist paradigm at the species level, typically testing the model that ‘the jack of all trades is the master of none’. Recent examples, among many others, include breadth of diet by insect herbivore species, niche breadth by bird species, competition on shared hosts by aphid parasitoids, and weasel predation of voles, as well as mathematical models to disentangle the contributions by various factors. Indeed, the terms generalist and specialist MHC alleles have already been used for describing correlations of class II alleles of the striped mouse in Africa with number of nematode species carried.

36In this view, the promiscuous class I molecules would be generalists, which one might expect to suffice for protection against a wide variety of the most common pathogens. However, such generalists might not suffice for protection from a new and/or an especially virulent pathogen that suddenly appears, at which point there would be a strong selection for a specialist class I molecule that was particularly suited to deal with the new threat. The properties of fastidious class I molecules are consistent with selection as specialists for particular pathogens, perhaps including those no longer a danger in the current population.

37The importance of these concepts for immunology and medicine may be clear from the discussion above, but there are also ramifications for evolutionary biology, ecology, and conservation. As one example, the number of MHC alleles is considered a key measure for population diversity in estimating the risk of extinction, both as a measure of overall genome diversity and in terms of fitness, including resilience to infection. However, a population with a few generalist MHC alleles might remain healthy compared to a population with many inappropriate specialist MHC alleles.

38Ex vivo chicken cells were from inbred chicken lines with known MHC haplotypes, kept at the University of Cambridge. All procedures were performed under appropriate Home Office Licenses and after review by the Ethics Committee at the University of Cambridge. Spleens were mashed through 100-μm nylon cell strainers (Falcon) in RPMI-1640 medium, supernatants taken after 5 min settling, and spleen cells recovered after centrifugation at 400×g for 5 min. All procedures were carried out under Home Office licenses and with ethical approval.

39Chicken cell lines were from the Pirbright Institute. AVOL-1 was derived from in vitro transformation of spleen cells from a line 0 (B21) chicken by the reticuloendotheliosis virus REV-T (;; W Mwangi and V Nair, unpublished data). The MDCC-265L cell line was established from a liver lymphoma of a line P2a (B19) chicken infected with the RB-1B virus derived from a BAC clone (as in; W Mwangi and V Nair, unpublished data). Both lines were maintained in RPMI 1640 medium containing 10% foetal bovine serum, 10% tryptose phosphate broth and 1% sodium pyruvate, and at 38.5°C in 5% CO2.

40Ex vivo human cells were from Anthony Nolan registrants typed as homozygous for particular HLA-A and HLA-B locus alleles, who signed written consent forms, and with all procedures carried out under Human Tissue Act licenses and with ethical approval. Blood samples were collected by general practice or Walk-in Clinic phlebotomists and were couriered to the Anthony Nolan Research Institute within 24 hr. Whole blood was diluted 1:1 upon arrival with transport media (RPMI 1640 [Lonza, Belgium] supplemented with 0.6% tri-sodium citrate and 50 nM 2-mercaptoethanol), and most samples were rocked at room temperature overnight. In a similar manner as originally described for cord blood, peripheral blood mononuclear cells were isolated (all steps at 20°C) using a density gradient centrifugation (Ficoll–Paque Plus 1077, GE Healthcare) at 840×g for 30 min with no brake, with the buffy coat washed twice with two volumes RPMI-1640 media, spun once at 680×g for 10 min and once at 540×g for 10 min. All samples were stained, fixed, and anonymized before transfer to Cambridge for analysis by flow cytometry.

41The human homozygous HLA-B*57 typing cell line WIN (alias IHW9095 from 10th International Workshop, gift of W Bultitude and S Marsh, Anthony Nolan Research Institute) was maintained in RPMI1640 with 10% foetal bovine serum and 1 mM glutamine in 5% CO2 at 37°C.

42As described by instructions for quantitative flow cytometry from manufacturer (QIFIKIT, Dako) and following previous work, 5 × 105 cells were incubated on ice in 96-well (U-bottom for chicken, V-bottom for human) microtiter plates (Nunc) with saturating primary antibody followed by washing and then by incubation with goat anti-mouse secondary antibody conjugated to fluorescein followed by washing, and data acquired using a FACscan (Becton-Dickenson). Set-up beads and calibration beads were stained separately with the secondary antibody for calibration curves to calculate the specific antigen binding capacity, which reflects the absolute numbers of epitopes on the cell surface. Primary mAb include 200 µl tissue culture supernatant of mouse mAb F21-2 for chicken class I molecules and three that react with certain HLA-B antigens but not certain HLA-A alleles: 200 µl tissue culture supernatant of Tu149 (; gift of J Trowsdale, University of Cambridge), 20 µl 1 mg/ml B1.2.23 (; bought from eBioscience) diluted in PBS and 20 µl 1 mg/ml 22E1 (; bought from Caltag Medsystems) diluted in PBS. Saturation was confirmed by staining with dilutions of antibodies on each set of cells, except for human ex vivo cells, for which saturation was confirmed by staining WIN cells.

43For the inhibition assay, Tu149 conjugated to APC (Invitrogen; kind gift of S Ashraf and J Trowsdale, University of Cambridge) and purified B1.23.2 conjugated to Alexa Fluor 647 (purified mAb from eBioscience conjugated using the Antibody Labeling kit, Molecular Probes/Life Technologies, according to manufacturer's instructions) were used. As above, 5 × 105 WIN cells were incubated with saturating amounts of unlabelled B1.23.2 or Tu149 (or with PBS) for 1 hr, washed and then incubated with a directly labelled antibody for 1 hr, before washing the cells again. Data were acquired using the red laser on a Cytek FACSanalyser (Becton Dickinson).

44Inbred chicken lines, with known MHC haplotypes, were kept at the Institute for Animal Health at Compton. All procedures were performed under appropriate Home Office Licenses and after review by the Ethics Committee at the Institute for Animal Health. As previously described, erythrocytes (or whole mashed up spleens) were solubilized in detergent, and class I molecules were isolated by affinity chromatography using mAb F21-2 against chicken class I molecules or F21-21 against chicken β2m. Peptides were eluted using trifluoroacetic acid and separated by reverse phase high-pressure liquid chromatography with single peaks of abundant peptides as well as pools of non-abundant peptides sequenced by Edman degradation.

45Immunoaffinity beads were produced, with all steps at room temperature. Protein G-Sepharose beads (Expedeon) were washed with 50 mM borate, 50 mM KCl, pH 8.0, the equivalent of 1-ml packed beads was incubated with 3 mg F21-2 (produced by the Microbiological Media Services of the Pirbright Institute) for 1 hr, treated with 40 mM dimethyl pimelimidate dihydrochloride (Sigma) in 0.1 M triethanolamine, pH 8.3 for 1 hr to cross-link the antibody to the protein G, washed with 100 mM citric acid pH 3.0, and equilibrated in 50 mM Tris, pH 8.0. The two cell lines AVOL1 and 265L were washed with PBS. Pellets of 109 cells were incubated with 10 ml 1% Igepal 630, 300 mM NaCl, 100 mM Tris pH 8.0 for 30 min at 4°C, subcellular debris was pelleted by centrifugation at 300×g for 10 min and 15,000×g for 30 min at 4°C, and the cleared lysates were incubated with 1 ml immunoaffinity beads for 1 hr at 4°C. The beads were washed with 50 mM TrisCl, pH 8.0 buffer, first with 150 mM NaCl, then with 400 mM NaCl and finally with no salt. Bound material was eluted with 10% acetic acid. The eluted material was dried, resuspended in 3% acetonitrile, 0.1% formic acid in water, loaded directly onto on a 4.6 × 50 mm ProSwiftTM RP-1S column (ThermoFisher) and eluted at 500 μl/min flow rate for 10 min with a linear gradient from 2 to 35% buffer B (0.1% formic acid in acetonitrile) in buffer A (0.1% formic acid in water) using an Ultimate 3000 HPLC system (ThermoFisher), with fractions collected from 2 to 15 min. Protein detection was performed at 280 nm absorbance, with fractions eluting before β2m pooled and dried.

46For liquid chromatography tandem mass spectrometry (LC-MS/MS), peptides were analysed using either a Q-Exactive (Thermo Scientific) or a TripleTOF 5600 (AB Sciex) system. For the Q-Exactive system, peptides were separated on a Ultimate 3000 RSLCnano System utilizing a PepMap C18 column, 2 μm particle size, 75 μm × 50 cm (Thermo Scientific) with a linear gradient from 3% to 35% buffer B in buffer A (as above) at a flow rate of 250 nl/min (∼65 MPa) for 60 min, and the 15 most intense precursors per full MS scan were selected for MS/MS analysis using HCD fragmentation. For the TripleTOF system, peptides were separated with a 15 cm × 75 µm ChromXP C18-CL (3 µm particle size) using an ekspert nanoLC 400 cHiPLC system (Eksigent) with a linear gradient from 8% to 35% buffer B in buffer A (as above) at a flow rate of 300 nl/min (∼1600 psi) for 60 min, and CID fragmentation was induced on the 30 most abundant ions per full MS scan. All fragmented precursor ions were actively excluded from repeated selection for 15 s. Data were analysed using Peaks 7 (Bioinformatics Solutions) using a database containing all 24,092 Uniprot entries for the organism Gallus gallus combined with protein translations (>8 amino acids) of either all six reading frames of gallid herpesvirus 2 (NCBI entry NC_002229.3; 10,026 entries) or reticuloendotheliosis virus (NC_006934.1; 412 entries). Results were filtered using a false discovery rate of 1% that was determined by parallel searching of a randomized decoy database.

47As previously described, the extracellular sequence of mature BF2*2101 heavy chain and chicken β2m cloned in pET22b(+) were expressed separately as inclusion bodies in BL21 (λDE3) pLysS Rosetta bacterial cells, solubilized in urea and assembled together with synthetic peptide (synthesized commercially by fluorenyl-methoxy-carbonyl [fMOC] chemistry) by dilution in a renaturation buffer. Assembled class I molecules were isolated after SEC using a HiLoad 26/60 Superdex 200 column (GE Healthcare, UK). Similar procedures were used for BF1*0201, BF2*0201 and BF2*1401, except that a codon-optimized gene was synthesized (GenScript) for BF2*0201, which was then cloned into a pET28a(+) vector followed by a factor X site for cleavage, a BirA site for biotinylation and a His tag for purification.

48As previously described, heavy chains and β2m were expressed in bacteria as inclusion bodies and denatured in urea; β2m was refolded and purified by SEC. For each renaturation sample, solutions containing 30 µg heavy chain plus or minus 42 µg β2m and/or 10 µg synthetic peptide were added to refold buffer and incubated at 4°C with stirring for roughly 40 hr. After centrifugation and passage through a 0.45-µm sterile filter, each sample was loaded on a Superdex 200 10/300 GL SEC column (GE Healthcare, UK) as part of an AKTA 920 with 100 mM NaCl, 25 mM TrisCl, pH 8.0 running at 1 ml/min at room temperature. Peak fractions were collected and concentrated first using a pre-rinsed Amicon Ultra-4 10 kDa column and then a Vivaspin 500 10 kDa column to roughly 100 µl, buffer-exchanged into 50 mM NaCl, 10 mM Tris, pH 8.0, concentrated to roughly 20 µl, and then transferred into a polypropylene microfuge tube. Acetic acid was added to 5% and the sample was concentrated to 2–4 µl using a SpeedVac at 40°C before analysis by MALDI-TOF (Protein and Nucleic Acid Chemistry services, Department of Biochemistry, University of Cambridge).

49Recombinant MHC class I complexes were crystallized using the sitting-drop method (see Table 3 for conditions). Crystals were flash frozen in liquid nitrogen and native data sets for each crystal were collected at 100K [Diamond Light Source, Harwell (beamlines I02, I03, I04 or I04-1), or the ESRF, Grenoble (beamline ID29)]. Data were processed using either AUTOPROC or XIA2 with XDS for integration and AIMLESS or SCALA for scaling. All structures were solved by molecular replacement, as implemented in Phaser, part of the CCP4 software package. Starting molecular replacement models were generated using CHAINSAW and the atomic co-ordinates of the chicken B21 MHC class I molecule (PDBID: 3BEV) with the peptide removed. Model building and refinement were carried out using COOT and AUTOBUSTER or REFMAC, with the heavy and light chains of the MHC molecule rebuilt first before the peptide was modelled into residual electron density (see Table 1 for refinement statistics, Table 3 for data collection and refinement information, Table 4 for Ramachandran statistics).