Temperature-based MHC class-I multimer peptide exchange for human HLA-A, B and C

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

A fundamental technique for the identification of peptide-specific CD8 T cells is the use of fluorophore-conjugated and peptide loaded MHC tetramers. Classically, refolding of specific peptides with MHC monomers can be labour intensive, and not optimal for screening large numbers of different peptides. Hence, UV-exchanged tetramers have been developed to upscale this, however, still has some associated challenges such as UV-mediated damage to peptide complexes. Here, Pothast, C.R. et al demonstrate the efficacy of using temperature exchanged tetramers for the prevalent alleles HLA-A*03:01, A*11:01, B*07:02, and C*07:02. Building upon their previous work with HLA-A*02:01, H-2Kb, and HLA-E. They first demonstrate the complex stability of tetramers with different affinity peptides at high temperature, showing complex destabilisation can be rescued with higher affinity peptides. This is followed by an optimisation of peptide exchange temperatures, tailored for each allele. The authors then demonstrate successful binding to clonal T cell lines, and then a step further with viral peptides against PBMCs from individuals with confirmed infection history. For the latter they compare to conventional tetramers and demonstrate comparable signal.

Due to the prevalence of these 4 alleles, the ease-of-handling, and short time requirements, these tetramers are likely to show high utility.

Strengths:

The manuscript is well-written and results are solid, although more detail may add clarity to some of the results, in particular figure 1 and 2. Other than the points reported below, the study uses accurate controls to demonstrate the specificity of the tetramers, and the data is convincing.

Overall, their interpretation of the results is accurate and the discussion thorough, additional comments may be included to cover potential tetramer batch variability, and differences in the stability of different alleles. Specifically, whether certain alleles require higher affinity peptides to be stable, compared to others.

Comments on revised version.

The authors addressed the weaknesses highlighted in the previous version, specifically addressing that they do not conclude that thermal peptide exchange is not superior to existing technologies, but may be more feasible, as well as including a section on the stability of the different alleles and template peptides. The authors also updated the labelling and description of figures, making them much more interpretable. Addition of the LILRB1 reporter cell line to demonstrate successful peptide exchange and stability adds extra validity to the results.

Reviewer #2 (Public review):

Summary:

This work highlights a new approach to build an array of highly stable peptide MHC-I using temperature exchange, which can be used to identify antigen specific CD8+ T cells.

Strengths:

In this work, the authors have proposed an alternative method to reload the peptide MHC-I molecule. Their temperature-exchange approach is distinct from current reloadable peptide MHC technologies involving photolabile peptide, empty MHC-I (Nat Commun 11, 1314 (2020). https://doi.org/10.1038/s41467-020-14862-4), tapasin/TAPBPR chaperone-assisted (eLife 7:e40126.), enzyme exchangeable (https://pubmed.ncbi.nlm.nih.gov/31201791/) and small alcohol (Curr Res Immunol. 2022 Aug 18;3:167-174. doi: 10.1016/j.crimmu.2022.08.002) approaches

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

A fundamental technique for the identification of peptide-specific CD8 T cells is the use of fluorophore-conjugated and peptide-loaded MHC tetramers. Classically, refolding of specific peptides with MHC monomers can be labour-intensive and not optimal for screening large numbers of different peptides. Hence, UV-exchanged tetramers have been developed to upscale this, however, still has some associated challenges such as UV-mediated damage to peptide complexes. Here, Pothast, C.R. et al demonstrate the efficacy of using temperature-exchanged tetramers for the prevalent alleles HLA-A*03:01, A*11:01, B*07:02, and C*07:02. Building upon their previous work with HLA-A*02:01, H-2Kb, and HLA-E. They first demonstrate the complex stability of tetramers with different affinity peptides at high temperature, showing complex destabilisation can be rescued with higher affinity peptides. This is followed by an optimisation of peptide exchange temperatures, tailored for each allele. The authors then demonstrate successful binding to clonal T cell lines, and then a step further with viral peptides against PBMCs from individuals with confirmed infection history. For the later they compare to conventional tetramers and demonstrate comparable signal.

Due to the prevalence of these 4 alleles, the ease of handling, and short me requirements, these tetramers are likely to show high utility.

Strengths:

The manuscript is well-written and results are solid, although more detail may add clarity to some of the results, in particular figure 1 and 2. Other than the points reported below, the study uses accurate controls to demonstrate the specificity of the tetramers, and the data is convincing.

Overall, their interpretation of the results is accurate and the discussion thorough, additional comments may be included to cover potential tetramer batch variability, and differences in the stability of different alleles. Specifically, whether certain alleles require higher affinity peptides to be stable, compared to others.

Comments on revised version.

The authors addressed the weaknesses highlighted in the previous version, specifically addressing that they do not conclude that thermal peptide exchange is not superior to existing technologies, but may be more feasible, as well as including a section on the stability of the different alleles and template peptides. The authors also updated the labelling and description of figures, making them much more interpretable. Addition of the LILRB1 reporter cell line to demonstrate successful peptide exchange and stability adds extra validity to the results.

We thank the reviewer for the careful evaluation of our work and for the positive assessment of the manuscript. We are pleased that the reviewer finds the study well written, the data convincing, and the approach likely to be of practical utility for the analysis of antigen-specific T-cell responses.

Reviewer #2 (Public review):

Summary:

This work highlights a new approach to build an array of highly stable peptide MHC-I using temperature exchange, which can be used to identify antigen specific CD8+ T cells.

Strengths:

In this work, the authors have proposed an alternative method to reload the peptide MHC-I molecule. Their temperature-exchange approach is distinct from current reloadable peptide MHC technologies involving photo labile peptide, empty MHC-I (Nat Community 11, 1314 (2020). https://doi.org/10.1038/s41467-020-14862-4), tapasin/TAPBPR chaperone-assisted (eLife 7:e40126.), enzyme exchangeable (https://pubmed.ncbi.nlm.nih.gov/31201791/) and small alcohol (Curr Res Immunol. 2022 Aug 18;3:167-174. doi: 10.1016/j.crimmu.2022.08.002) approaches

Weaknesses:

The temperature-exchange approach identifies antigen-specific T cells at the same frequency of the photolabile peptide MHC-I molecules. The time saved using temperature-exchange protocol may not be a pull factor as the photolabile peptide MHC-I approach is not unreasonably laborious.

As with other exchange systems, performance can vary by HLA allele and complex stability, so each new allele needs empirical optimization.

In short, temperature-based p-MHC exchange is a practical and fast loading strategy with good utility for multimer generation, but its main limitation is dependence on thermal stability and careful template selection.

We thank the reviewer for the positive evaluation of our work and for recognizing temperature-mediated peptide exchange as a practical approach for the generation of peptide–MHCI multimers.

We appreciate the reviewer placing our methodology within the broader context of existing peptide loading technologies, including photolabile peptide, empty MHC-I, chaperone-assisted, enzyme-mediated, and small alcohol-based exchange systems. In response to comments raised during review, we have expanded the Introduction to include a more comprehensive discussion of these alternative approaches and their respective strengths and limitations.

We agree that the primary advantage of thermal peptide exchange is not necessarily improved sensitivity or detection of antigen-specific T cells compared with established methodologies. Rather, its value lies in providing an additional peptide-exchange platform that is simple to implement, requires only standard laboratory equipment, and enables rapid generation of peptide–MHC multimers. We also agree that successful implementation requires empirical optimization for each HLA allele and relies on the identification of suitable template peptides.

We are grateful for the reviewer's constructive comments, which have helped us improve the clarity and balance of the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

No additional experiments required.

Resolution of figures could be improved unless it is a caveat of the software used.

We thank the reviewer for this positive assessment and constructive suggestion. High-resolution versions of all figures have been included in the revised manuscript

Reviewer #2 (Recommendations for the authors):

The following sentences are incorrect.

Line 101: "The MHC-multimers are multimerized and fluorescently labeled prior to peptide exchange" is incorrect. Instead, combinatorial peptide MHC multimers involves peptide exchange prior to multimerization during barcoding and not vice versa (https://pubmed.ncbi.nlm.nih.gov/23748502/).

We thank the reviewer for carefully noting this inaccuracy. As suggested, we have revised the text to better reflect the different workflows used across MHC multimer platforms. The sentence now reads: “Depending on the platform, peptide exchange may be performed either before or after multimerization and labeling.”

Line 296: "Quantifying the efficiency of UV-induced peptide destruction and subsequent peptide exchange remains challenging" is incorrect. The thermal stability of empty MHC-I vs loaded peptide MHC-I after UV irradiation or thermal exchange can be measured using either gel filtration chromatography or SyPro orange thermal shift.

The sentence now reads: “While the stability of UV-exchanged p-MHC complexes can be assessed using methods such as thermal shift assays and size-exclusion chromatography, the efficiency of peptide exchange is more difficult to quantify directly.”