Simplifying principles that underlie the highly complex peptide motif of the promiscuous chicken class I molecule, BF2*21:01

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.

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.

(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.

(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 overinterpreted as precise evidence for residue preference. The authors should clearly indicate which conclusions are supported by LC-MS/MS.

(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.

(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.

(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.

(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.

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.