Author response:
eLife Assessment
This important study investigates the peptide-binding principles of promiscuous chicken MHC molecules. The data from crystallography, mass spectrometry, and modeling are convincing. However, the presentation would benefit from streamlining and clear links between data and conclusions. This paper will be of broad interest to immunologists and those interested in vaccine development.
Overall, we are delighted and grateful to the eLife editors and the two reviewers for the careful and thoughtful assessments and reviews of our paper. We are glad that the strengths of the paper were apparent and appreciated. And of course, every paper has weaknesses, especially for a story as complex as this one.
We are making only minor changes in our revision, so we would be happy if the editors decide to evaluate the revised manuscript without involving the reviewers further.
Before answering the comments and questions directly, perhaps a few points would help clarify why the paper is as it is.
First, the experiments cover over three decades of work, with the first gas phase sequencing results done in 1992. Unlike some of the chicken class I alleles which immediately gave completely clear stringent motifs (B4, B12 and B15 in Wallny et al 2006 PNAS, B19 in Han et al 2023 J Immunol), we harvested nothing but confusion from the B21 class I results (Fig. 1). Initially, we thought that the lack of a clear motif for B21 was due to multiple well-expressed class I molecules but only one dominantly-expressed class I molecule was found (Wallny et al 2006 PNAS, Shaw et al 2007 J Immunol) and, to our surprise, bacterially-expressed BF2*21:01 heavy chain and b2-microglobulin refolded with two synthetic peptides without sequence in common, and the crystal structures showed that this molecule remodeled the binding site to accommodate two such disparate peptides (Koch et al 2008 Immunity). This was the beginning of our understanding of the spectrum of class I alleles from promiscuous generalists to fastidious specialists, which we have explored in a series of further papers (in particular, Chappell et al 2015 eLife, Tresgaskes et al 2016 PNAS, Kaufman 2018 Trends Immunol, Tregaskes and Kaufman 2022 Mol Immunol).
Second, over these many years, we continued to explore the binding properties of BF2*21:01 in ever more detail, resulting in the current manuscript. We learned only slowly how to probe this unexpected promiscuity, unprecedented in the MHC literature, so that the experiments proceeded with our best understanding at the time, including taking advantage of new approaches as they become available. Each experiment built on the previous set of experiments and each brought us closer to an understanding.
Third, having amassed a collection of data, we chose eLIFE exactly because it allows us to present the entire story from beginning to end without compromise, not just the highlights with the major points illustrated by a few main figures and with the supporting data in many supplementary figures. We include all the data, because it is all part of the story, and so interested researchers to look at the data from their own perspective. Although mostly we provide bar graphs, we include the raw data (or close to it) for the final experiments (illustrated by Figs. 10 and 18) in the single supplementary data spreadsheet, so these can be assessed easily by others in the field, perhaps using approaches that we may not feel competent to perform.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Combining in vitro refolding, SEC-based assembly assays, peptide-library screening, MALDI-TOF, LC-MS/MS, structural analysis and immunopeptidomics, this manuscript investigates the peptide-binding principles of the promiscuous chicken MHC-I molecule BF2*21:01.
Strengths:
Although the peptide motif of BF2*21:01 is highly complex, this manuscript identified several principles, including a preference for 10-mer peptides, co-variation between P2 and Pc-2, effects of P3 and Pc-3, and a strong cellular preference for Leu at Pc. The results are important for avian MHC biology and poultry vaccine epitope prediction.
Weaknesses:
The manuscript is sometimes difficult to follow because the authors present a large amount of peptide-library, structural and immunopeptidomics data. without always clearly explaining how these datasets support the proposed simplifying principles.
We are delighted and grateful to the reviewer 1 for the careful and thoughtful comments and questions concerning our manuscript. We are glad that the strengths of the paper were apparent and appreciated, and acknowledge the weaknesses that come with such a complex story with experiments performed over decades.
Major Issues - Points Requiring Clarification or Additional Support:
(1) (Line 282-301, 537-545)
The immunopeptidomics conclusions are mainly based on one B21 cell line with one biological replicate and at least two technical replicates. Given the complexity of the BF2*21:01 peptide repertoire, this is a major limitation. The authors should either provide additional biological replicates or clearly state this limitation in the Abstract, Results and Discussion.
This limitation is clearly stated in lines 537-545, as part of a paragraph covering the various ways in which the data presented in this manuscript could be improved. In fact, we have performed immunopeptidomics of several different B21 cell types, with many replicates and found similar data as presented, giving us confidence in our interpretations. However, these other experiments belong in different stories, so it is not appropriate that the data be reported in this manuscript.
(2) (Lines 290-313)
The B21 cell preparations contain both BF2 and the lowly expressed BF1 molecule. Some peptides, especially 8-mers or peptides with atypical motifs, may derive from BF1*21:01. The authors should clarify how BF2*21:01-bound peptides were distinguished from possible BF1-derived peptides, or interpret the immunopeptidomics motif more cautiously. The authors should also provide or cite evidence confirming the B21 haplotype identity of the cell line and chicken materials used for immunopeptidomics.
The concern about the contribution of BF1*21:01 to the immunopeptidomics is clearly stated in the manuscript, both lines 290-313 and as part of the paragraph describing the limitations of the experiments (lines 542-543). In fact, the expression of BF1 molecules has long been known to be less than 10% of BF2 molecules at the RNA level, and much less at the protein level (Wallny et al 2006 PNAS, Shaw et al 2007 J Immunol). The proportion of 8mers identified by immunopeptidomics is also low (Fig. 14), and it is not impossible that most 8mers are due to BF1*21:01. We have used assembly assays with peptide libraries, immunopeptidomics and a crystal structure to determine the peptide motif for typical BF1 molecules, of which BF1*21:01 is one and found it may contribute to 8mer peptides but very seldom to longer peptides. This work is unpublished but gives us confidence that the characteristics of BF2*21:01 are not misrepresented by the data in this manuscript.
The sources of the chicken samples and the cell lines are described in detail under Materials and Methods (lines 577-590), citing relevant publications.
(3) (Lines 217-221, 243-253)
The authors acknowledge that MALDI-TOF cannot reliably distinguish peptide combinations with identical or similar masses, nor determine residue positions in some cases. Therefore, MALDI-TOF results should not be over-interpreted as precise evidence for residue preference. The authors should clearly indicate which conclusions are supported by LC-MS/MS.
As described, the experiments follow each other in temporal sequence, so that we started with single peptides, then peptide libraries that varied in one position, then peptide libraries that varied in two positions first analysed by MALDI-TOF and later by LC-MS/MS. The final experiment (Fig. 10, with the original data in the supplementary spreadsheet) directly compares MALDI-TOF and LC-MS/MS results for six peptide libraries, so that the strength of the evidence for residue preference is clear. Throughout the manuscript, we do our best to not to overstate conclusions based on the data of any particular experiment.
(4) (Lines 297-301, 316-330)
The authors suggest that longer peptides may bulge in the middle or extend out of the groove at the C-terminal end. The rationale for the C-terminal extension is not clearly explained. Why is the C-terminal extension considered rather than the N-terminal extension? If the binding register is uncertain, long peptides should be analyzed separately from canonical-length peptides.
When the first sequence of a chicken class I cDNA was determined, an immediate mystery was why one of the so-called invariant residues that coordinate the N- and C-termini of the bound peptide is not conserved (Kaufman et al 1992 J Immunol). In fact, this residue Tyr at position 86 in HLA-A2 and the equivalent position in all mammalian classical class I molecules is an Arg in the classical class I molecules of all non-mammalian vertebrates and is common with class II molecules (Kaufman et al 1995 Semin Immunol). Similar to class II molecules, this Arg in chicken class I molecules allows the peptide to extend out of the C-terminus, as shown by a crystal structure (Xiao et al 2018 J Immunol). The concern that we might be misidentifying the C-terminal amino acid was the basis for the analysis in Figs. 23 and 24, but in the absence of crystal structures, we are not able to provide a final answer this question. Perhaps relevant is the fact that a chicken class II molecule can bind exactly the same peptide in two conformations, one with a canonical 9mer core and the other with an unexpected 10mer core (Goryanin et al 2026 J Virol).
By contrast, N-terminal extensions are only found for some class I alleles and thus far depend on the substitution of small amino acid sidechains for W166 (Li et al 2011 J Virol for bovine, Ma et al 2020 J Immunol for Xenopus, Wei et al 2022 J Immunol for ovine). Thus far, no chicken BF2 sequences have this substitution, consonant with the many crystal structures, including those for BF2*21:01 (Koch et al 2008 Immunity, Chappell et al 2015 eLlife, this manuscript). However, in unpublished data, we find that most BF1 sequences have sequence differences that could allow N-terminal extensions, although we have no crystal structures to support this possibility.
(5) (Lines 406-439)
In vitro assembly assays show that several hydrophobic residues can be tolerated at Pc, whereas immunopeptidomics shows a strong Leu preference at this position. The authors should clarify whether this Leu preference reflects intrinsic BF2*21:01 binding specificity, TAP-mediated peptide transport, antigen processing, peptide loading, or a cell-line-specific effect. Additional experimental support, such as TAP transport analysis, would strengthen this conclusion.
The preference for Leu at the final position of the peptide by immunopeptidomics of the B21 cell line is strong but not absolute and is certainly affected at the least by the length of the peptide (Figs. 23 and 24). Unpublished immunopeptidomics results (mentioned above) show that this is not a cell line-specific result. The evidence from assembly assays of various peptides is that several hydrophobic amino acids are tolerated with sufficient stability of BF2*21:01 that they are detected in the assay (Figs. 3, 5, 9 and 10). Thermostability assays (Fig. 6) show that peptides with these same hydrophobic amino acids are stable to at least body temperature of chickens. These experiments show that such stability is peptide-dependent (that is, whether a particular amino acid is tolerated depends on the stability conferred by the rest of the peptide). Finally, peptide translocation assays using B21 cells have been done (Tregaskes et al 2016 PNAS) and show that peptides with several hydrophobic amino acids can be pumped into the lumen of the endoplasmic reticulum. However, the assays are with single synthetic peptides, so the data are not extensive enough to separate the effects of the final amino acid from the rest of the peptide. Certainly, peptides with amino acids other than Leu at the C-terminus can be translocated. So, it is not yet clear at which point the preference for Leu at the C-terminus of the peptide arises.
(6) (Lines 172-178, 243-279, 442-457)
The structural analysis explains some residue combinations, such as Arg at P2 with Glu at Pc-2 or Trp at Pc. However, the structural interpretation is not fully integrated with the large-scale peptide library and immunopeptidomics results. Representative high- and low-frequency combinations should be discussed structurally.
Six crystal structures show that BF2*21:02 remodels the binding to accommodate a variety of anchor residues (Koch et al 2008 Immunity, Chappel et al 2015 eLife). These crystal structures are representative of sequences found by the immunopeptidomics from very frequent (H-E at roughly 15% 8-12mers) to moderately frequent (E-L at roughly 6% 8-12mers) to infrequent (N-F, A-D and E-D at roughly 1.5%, 1.6% and 0.7% 8-12mers) based on Fig. 18. All but one of the structures has Leu at the C-terminus, with the last one having Val which is found but not frequently by immunopeptidomics.
Similar numbers are found by LC-MS/MS of double-substitution libraries of the two original peptide sequences in Fig. 10 with H-E found frequently (8.1% in P390, 3.8% in P498) and the others infrequently (0.1, 0.9, 1.0, 0.3% in P390, 0, 1.4, 1.0, 0.3% in P498), as calculated from the numbers in the Supplementary data spreadsheet. As discussed in the manuscript, for single-substitution peptide libraries of the two original peptides, Ile/Leu at the C-terminus was very frequent but at the same or slightly less level as Phe, with Met less frequent and Val even less so (Fig. 7).
In addition, there are two more structures along with models explicitly testing some substitutions (Fig. 5). Attempting more current modelling approaches, we found AlphaFold 3 was unable to correctly predict most of the conformations that are found in the crystal structures of BF2*21:01, so we don’t feel confident in using them to predict unknown structures of this kind.
(7) The inference of co-variation between P2 and Pc-2, as well as the modulatory effects of P3 and Pc-3, should be better explained. At present, some conclusions appear to be based mainly on residue-frequency patterns, and the logical connection between these observations and the proposed binding principles is not always clear. Statistical analyses, such as mutual information, chi-square tests or permutation tests, and representative structural explanations would strengthen this conclusion.
We endeavored to do our best to explain the data, our interpretations and our reasoning, so we apologise if we have not managed to be as clear as might be desired. We have included as close to raw data as possible for the LC-MS/MS and MALDI-TOF (Fig. 10) and for the immunopeptidomics (Fig. 14 and 18) in the Supplementary Data spreadsheet, exactly so that competent practitioners can carry out further analyses (including the sophisticated statistical tests mentioned).
Reviewer #2 (Public review):
Summary:
The study presents an in-depth analysis of the peptide repertoire bound by a promiscuous chicken MHC molecule using mass spectrometry, x-ray crystallography and modelling. While the MHC can bind a very diverse set of peptides, the authors have found some new rules that govern peptide binding to this MHC that could help to build a predictive model to study the repertoire of pathogen-derived peptides.
Strengths:
The study uses a range of well performed experiment across multiple techniques and provides an in-depth analysis of the peptide repertoire, including peptide sequences, length, preferred residues, stability and MHC presentation.
Weaknesses:
The data overall support the analysis and conclusion well. The only caveat is linked to Figure 4, which does not describe the stability of the peptide-MHC complex, but instead shows refold yield, and the two are not always linked.
We are grateful for the clear understanding of the strengths of the work. With regards to Fig. 4, we agree with the reviewer that there are differences in refold yield but that measure may not be correlated with stability of the peptide-MHC complex. However, we were basing our interpretation of stability on the position and quality of the monomer peak, as illustrated by the trace in Fig. 2, in which a sharp peak at the monomer position represents a stable complex (as seen for the 10 and 11mer peptides) and later peaks represent unstable complexes falling apart during the chromatography (as seen for the 7, 8 and 9mer peptides).
