Temperature-based MHC class-I multimer peptide exchange for human HLA-A, B and C
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eLife Assessment
This study presents an alternative to conventional and UV-based tetramers, which are easy to use and reliable for the identification of antigen-specific CD8 T cells. The authors demonstrate that tetramers for HLA alleles A0301, A1101, B0702, and C0702 can be subjected to specific temperatures that facilitate peptide exchange, whilst maintaining structural integrity. Whilst the strength of the evidence is currently incomplete, further development and validation of this approach is likely to provide a useful alternative to generating reagents for examining T cell specificities.
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Abstract
Abstract
T cell recognition of specific antigens presented by major histocompatibility complexes class-I (MHC-I) can play an important role during immune responses against pathogens and cancer cells. Detection of T cell immunity is based on assessing the presence of antigen-specific cytotoxic CD8+ T cells using MHC class-I (MHC-I) multimer technology. Previously we have designed conditional peptides for HLA-A*02:01, H-2Kb and HLA-E that form stable peptide-MHC-I-complexes at low temperatures and dissociate when exposed to a defined elevated temperature. The resulting conditional MHC-I complex can easily and without additional handling be exchanged with a peptide of interest, allowing to exchange peptides in a ready-to-use multimer and a high-throughput manner. Here we present data that this peptide-exchange technology is a general applicable, ready-to-use and fast approach to load many different peptides in MHC-I multimers for alleles of the HLA-A, HLA-B and HLA-C loci. We describe the development of conditional peptides for HLA-A*03:01, HLA-A*11:01, HLA-B*07:02 and HLA-C*07:02 that only form stable peptide-MHC-I complexes at low temperatures, allowing peptide exchange at higher defined temperature. We document the ease and flexibility of this technology by monitoring CD8+ T cell responses to virus-specific peptide-MHC complexes in patients.
Article activity feed
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eLife Assessment
This study presents an alternative to conventional and UV-based tetramers, which are easy to use and reliable for the identification of antigen-specific CD8 T cells. The authors demonstrate that tetramers for HLA alleles A0301, A1101, B0702, and C0702 can be subjected to specific temperatures that facilitate peptide exchange, whilst maintaining structural integrity. Whilst the strength of the evidence is currently incomplete, further development and validation of this approach is likely to provide a useful alternative to generating reagents for examining T cell specificities.
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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, …
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 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 the results are solid, although more detail may add clarity to some of the results, in particular Figures 1 and 2. Other than the points reported below, the study uses accurate controls to demonstrate the specificity of the tetramers, and the data are convincing.
Overall, the interpretation of the results is accurate, and the discussion is 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.
Weaknesses:
The authors demonstrate the equivalence of temperature-exchanged tetramers to conventional ones, however, as they are an advancement on UV-exchange, it would be useful to show data on how their stability, exchange efficacy, and binding to T cell lines compare to UV-based tetramers. It would be supportive to show that temperature does not impact fluorophore intensity as well.
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Reviewer #2 (Public review):
Summary:
The majority of CD8+ T cell responses rely on the proper presentation of antigens through stable MHC-I (but not requiring a stable immunological synapse). This work highlights a new approach to build an array of 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 (WO2020226570) and small alcohol (Curr Res Immunol. 2022 Aug 18;3:167-174. doi: …
Reviewer #2 (Public review):
Summary:
The majority of CD8+ T cell responses rely on the proper presentation of antigens through stable MHC-I (but not requiring a stable immunological synapse). This work highlights a new approach to build an array of 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 (WO2020226570) and small alcohol (Curr Res Immunol. 2022 Aug 18;3:167-174. doi: 10.1016/j.crimmu.2022.08.002) approaches.
Weaknesses:
However, the proposed temperature-exchange approach does not substantially improve the quality of antigen-specific T cells that can be identified using the photolabile peptide MHC-I molecules.
The time saved using the temperature-exchange protocol may not be a pull factor as the photolabile peptide MHC-I approach is not unreasonably laborious.
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Reviewer #3 (Public review):
Summary:
The study by Pothast and colleagues outlines an extension of their previously described temperature-based MHC-I peptide exchange method on 4 common HLA alleles, to enable the generation of peptide/MCH-I tetramers for characterization of antigen-specific T cells by flow cytometry.
Strengths:
This work outlines a protocol for generating MHC-I tetramers on 4 common HLA allotypes, which can then be applied to monitor T cell responses by flow cytometry studies. The work provides conditional ligands for exchange on each HLA and demonstrates proof of concept studies using clonotypic T cells and CD8+ PBMCs.
The results support that the temperature-exchanged tetramers can perform similarly to conventional tetramers in some settings.
Weaknesses:
Given that there are several proposed methodologies addressing …
Reviewer #3 (Public review):
Summary:
The study by Pothast and colleagues outlines an extension of their previously described temperature-based MHC-I peptide exchange method on 4 common HLA alleles, to enable the generation of peptide/MCH-I tetramers for characterization of antigen-specific T cells by flow cytometry.
Strengths:
This work outlines a protocol for generating MHC-I tetramers on 4 common HLA allotypes, which can then be applied to monitor T cell responses by flow cytometry studies. The work provides conditional ligands for exchange on each HLA and demonstrates proof of concept studies using clonotypic T cells and CD8+ PBMCs.
The results support that the temperature-exchanged tetramers can perform similarly to conventional tetramers in some settings.
Weaknesses:
Given that there are several proposed methodologies addressing the same task (including UV-mediated, disulfide-bond based stabilization of empty MHC-I conformers, and chaperone-based methods), the relevance of the proposed temperature-mediated technology is questionable.
More specifically, important limitations of the study include:
(1) A lack of quantification of exchanged molecules relative to molecules that retain the original placeholder peptides, or completely empty molecules present in the same sample.
(2) A lack of validation that peptide exchange has occurred in the absence of a reporter T cell line appears to be a significant limitation of the methodology for antigen / T cell discovery.
(3) The sub-optimal exchange efficiency relative to conventional prepared pMHC-I molecules, shown in Figure 4, is a significant limitation of the approach.
(4) There are no data to support that exchange proceeds through the generation of empty molecules during the temperature cycle, or by peptide binding on empty molecules that are already present in the sample. Understanding the mechanism of exchange is important for the necessary improvements to the methodology.
(5) It is possible that the temperature cycle causes protein aggregation or other irreversible changes to the sample - this should be explicitly quantified and addressed in the paper, since misfolded MHC-I molecules can lead to high levels of background staining.
(6) These potential limitations should limit detection of low-affinity/low-avidity interactions between TCRs and their cognate pMHC antigens - this should be addressed explicitly in a model antigen setting.
(7) The approach appears to be limited to the HLAs showing high thermal stability, which have been explored in this study. However, a large fraction of HLAs show sub-optimal thermal stabilities. It seems that explicit validation of peptide exchange would be required for any new HLA allele introduced into this process.
(8) Whether the approach can be used to load suboptimal peptides with lower thermal stabilities that are emerging immunotherapy targets is not addressed in the present study.
Because of these limitations, the present manuscript does not conclusively support the claim that temperature-based exchange can be used as a robust methodology to generate pMHC-I tetramers with desired peptide specificities.
As a result, the scope of applications using these suboptimal exchanged pHLA tetramers is limited, and should be addressed with further improvements of the methodology, including better characterization of exchange efficiency, demonstration of functionality across a broader range of HLA allotypes with varying thermal stability profiles, and validation with clinically relevant low-affinity peptides that would strengthen the potential utility of this approach in immunotherapy development and basic T cell biology research.
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