Author response:
The following is the authors’ response to the original 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 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.
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.
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.
We are submittng a revised version that addresses the major points raised. We believe that the manuscript in its present form clearly communicates the development of a robust and versatile temperature-based peptide exchange platform applicable across multiple HLA class I alleles, with relevance for both fundamental and clinical immunology. While we appreciate the constructive feedback provided, we feel that the core findings and conclusions are already well supported and suitable for publication.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The main recommendation here is to include a stability and staining comparison to UV-exchanged tetramers, as this will directly address why temperature-exchanged tetramers are superior. It could also be discussed that research groups may have better access to temperature-regulating equipment than UV. Other minor corrections may include:
We thank the reviewer for this suggestion. We have previously demonstrated that HLA‑A*02:01 and H‑2Kb remain stable as frozen monomers, and similarly showed the stability of thermal peptide exchange for HLA‑E, both at the monomer stage and after multimerization into thermal‑exchange HLA‑E tetramers. In addition, we have directly compared these reagents with conventional tetramers (see Figure 5). Conventional monomers and tetramers are routinely stored at −80 °C for several months without loss of staining performance, and we applied the same storage conditions to thermal‑exchange HLA‑E tetramers. Consistent with conventional tetramers, thermal‑exchange tetramers maintained their ability to stain TCR‑expressing cells for up to six months, with optimal staining preserved at both −20 °C and −80 °C. These findings have been published previously and are discussed in the current manuscript.
We would like to emphasize that we do not conclude that thermal peptide exchange is superior to existing technologies. Our intention is to demonstrate that this approach is feasible and to present its advantages and limitations, which are discussed in detail in the manuscript.
In the discussion we have included a sentence about the accessibility of heat exchange in relation to the equipment needed for UV exchange.
“Moreover, the broader availability and simplicity of equipment needed for thermal exchange compared to UV sources enables wider adoption of this approach.”
(1) Greater explanation of the different coloured curves in Figures 1 and 2, and whether all curves represent samples that are incubated at 50 degrees Centigrade.
We have now clarified in the figure legends that the different coloured curves represent the distinct peptide–HLA complexes analysed, and we explicitly state which curves correspond to samples incubated at 50 °C.
(2) Addition of X-axis title for Figures 1 and 2.
An X‑axis title was already present in Figures 1 and 2, but it was minimal; we have now revised it to make the labeling clearer and more informative.
(3) Clarify whether the background signal (negative) increases with the addition of more tetramer in Supplementary Figure 3 (non-specific binding).
The background (negative) signal did not appreciably increase with the addition of higher tetramer concentrations, indicating minimal non-specific binding under these conditions. This is consistent with observations in the MHC class I multimer field, where non-specific binding is generally limited under optimized staining conditions, and MHC class I multimers can be reliably used to detect antigen-specific T cells.
(4) Address stability of different alleles, or whether they are similar.
Peptide stability varies for each peptide–HLA combination rather than being strictly allele‑specific. We have therefore clarified this point in the manuscript. In addition, as requested by the reviewer, we have included a dedicated section describing the stability of the thermal‑exchange template peptides.
“The peptides that formed stable complexes at 4°C but unstable complexes at 20–32°C and 50°C showed affinities ranging from 4 to 11 µM. Such low affinities are uncommon for endogenous peptides and are unlikely to have physiological relevance, given that the complexes are highly unstable at 37°C.”.
(5) Measure fluorescence signal of tetramer before and after temperature exchange, compared to UV, to show fluorophore stability.
Fluorescent streptavidin forms a highly stable complex. During UV-mediated peptide exchange, reagents are exposed to heat generated by the UV lamp, which can partially denature MHC-I proteins. For this reason, it is necessary to centrifuge the complexes after UV exchange to remove denatured protein aggregates. Importantly, the fluorescence of streptavidin itself is not affected by this process. In contrast, thermal exchange is performed at substantially lower temperatures than UV-mediated exchange and does not compromise MHC-I protein integrity. Therefore, in our view, it does not pose a significant risk to fluorophore stability. In addition, UV may destroy fluorescence by simple bleaching, which cannot be an issue with temperature-dependent peptide exchange.
(6) Use HPLC to show stability of tetramer following temperature exchange versus UV.
HPLC analysis of tetramers is not possible and this is also not performed by others. The reason is that the tetramers are very heterogeneous (clustering of streptavidin together and the amount of MHCI monomers attached to streptavidin). As discussed above we do not claim to be better or worse compared to UV-exchange. This technology has a some additional benefits compared to UV-exchange, such as the tetramerization can be done before the peptide exchange. Additional it does not need a calibrated UV lamp but just an heatblock is needed for the peptide exchange making the exchange less complicated and in our opinion easier to implement. The UV exposure may oxidize amino acids, which is unlike to occur under temperature dependent conditions.
Reviewer #2 (Recommendations for the authors):
Minor in figure:
The authors should explain what "inputs" are in the figure legend.
We have now clarified in the figure legends that the different coloured curves represent the distinct peptide–HLA complexes analysed, and we explicitly state the nature of the “inputs”.
Minor in text:
The authors should also state two important limitations when designing temperature-sensitive peptide MHC-I, which are peptide binding affinity and subsequent T cell recognition. Poorly designed peptides can result in unexchanged peptide MHC-I complexes that may be recognized by the CD8 T cells, leading to false positives.
We have adjusted this in the text (discussion) although we did not make poorly designed peptides. The peptide presented in this manuscript are only stable at 4°C and unstable from 20°C and higher. As the T cell staining are performed at room temperature, there will be no peptide MHC-I complex of the original thermal-exchange peptide present as this is disintegrated. Only the stable, higher-affinity peptide-exchanged MHC-I is in complex. This does pose a problem for peptide exchange with low-affinity peptides, which is mentioned in the current version. However, this does not differ from the other loading technologies as in each case the mother peptide has to be exchanged for a proper peptide and a low-affinity peptide will be lost in time in each other case as well
The authors should include other recent reloadable peptide MHC technologies, e.g. chaperone-assisted, enzyme-exchangeble, and small alcohol treatment.
We apologize for not including these technologies and in the current manuscript they are included in the introduction. (see text below)
“Several technologies have since been developed to allow the parallel production of multiple MHC-I multimers, facilitating high-throughput analysis. For instance, dipeptides have been used as catalysts for peptide exchange[7-10][FS1] , UV-sensitive conditional MHC ligands have been a widely adopted approach [11, 12], in which a photocleavable peptide is cleaved upon UV exposure. Additional peptide exchange technologies include: chaperone-mediated peptide exchange[13, 14] enzymatic cleavage of low-affinity peptides that are covalently linked [15] and small alcohol peptide exchange [16].”
Major in data:
The authors should show if any T cell was detected using the original unexchanged peptide MHC-I in cytometry, as the interpreted use of an irrelevant peptide also depends on the existence of antigen-specific T cells in circulation.
The unexchanged peptide–MHC‑I complex cannot be used for T‑cell staining under our standard conditions because the template peptide dissociates at room temperature, leading to disintegration of the complex. Since our T‑cell staining protocol is performed at room temperature, unexchanged complexes would not remain intact long enough to allow reliable detection. Performing this experiment would require modifying the standard protocol and carrying out all staining steps strictly at 4°C. We choose not to change the staining protocol to perform this experiment.
In addition, the thermal‑exchanged peptide–MHC‑I complexes do not produce elevated background staining. As shown in Figure 5, background staining would appear as single‑positive events (PE or APC only), which we do not observe. This indicates that the thermal exchange approach does not introduce nonspecific tetramer binding.
Reviewer #3 (Recommendations for the authors):
(1) The proportion of exchanged molecules should be quantified relative to those retaining the original placeholder peptides and completely empty molecules present in the sample, using quantitative mass-spectrometry analysis. This would provide critical information about exchange efficiency and help assess the method's reliability.
We thank the reviewer for this point. We have performed this experiment and quantified the efficiencies. This is in the current version in figure 2b.
(2) Validation studies demonstrating peptide exchange in the absence of reporter T cell lines should be included. This would strengthen the methodology's applicability for antigen/T cell discovery applications where such reporter lines may not be available.
We thank the reviewer for raising this important point. Indeed, incomplete or unstable peptide exchange is a general concern inherent to all MHC-I peptide exchange protocols. To address this, we implemented a T cell–independent validation strategy based on LILRB1 binding. The innate immune receptor LILRB1 selectively recognizes properly folded peptide–MHC-I complexes by interacting with β2-microglobulin (β2M) and the α3 domain of the MHC heavy chain in a peptide-independent manner.
Critically, in our system the conditional template peptides are thermally unstable and dissociate during the exchange step. As a result, only successfully exchanged complexes containing a stabilizing peptide remain intact. Consequently, LILRB1 binding serves as a functional readout of successful peptide exchange and pMHC stability.
To assess this, we used K562 cells retrovirally transduced with LILRB1 and stained them with the corresponding fluorophore-conjugated pMHC multimers. Flow cytometric analysis enables clear discrimination between stable (successfully exchanged) and unstable complexes.
This assay is fully independent of TCR-based reporter systems and can therefore be broadly applied in antigen discovery settings where such reagents are not available. Importantly, it complements our HPLC-based quantification of peptide exchange efficiency (Figure 2B) by providing a scalable quality-control readout of structural integrity (Figure 2C).
We have now included this analysis in the Results section and updated the Discussion and Materials and Methods accordingly.
(3) Optimization strategies to improve exchange efficiency should be explored, along with direct comparisons with established methods.
In the revised manuscript, we have included the quantified exchange efficiencies of the thermal peptide exchange reaction, as determined by HPLC analysis on monomeric complexes. In addition, we now present a novel assay that enables routine quality control of each peptide exchange. This analysis is performed using LILRB1+ cells and can be readily applied to any peptide–MHC complex using standard flow cytometry.
Our data demonstrate that the exchange efficiencies obtained with our method are robust, and for the HLA alleles included in this study no further optimization was required. With respect to comparisons with established technologies, we chose to benchmark our approach against conventionally folded tetramers (Fig. 5). As stated above, we do not claim superiority over other peptide-exchange platforms; rather, we aim to present an alternative workflow with distinct practical advantages.
(4) Potential protein aggregation or other irreversible structural changes resulting from temperature cycling should be quantified and addressed. Since misfolded MHC-I molecules can contribute to high background staining, this characterization is essential for assessing the method's reliability. This can be done using DLS studies, which should report on the fraction of misfolded, high MW species present in the sample upon temperature treatment.
We thank the reviewer for raising this important point regarding potential aggregation or irreversible structural changes during temperature cycling. We agree that the presence of misfolded or aggregated MHC-I complexes could contribute to nonspecific staining and affect the reliability of the method.
Although we did not perform dynamic light scattering (DLS), we addressed this concern using complementary biochemical and functional approaches. First, monomeric peptide–MHC complexes were analyzed by size-exclusion HPLC throughout the study. These analyses consistently showed well-defined peaks corresponding to properly folded complexes, without detectable high–molecular weight species that would indicate aggregation (Figures 1 and 2).
Second, and importantly, we assessed the functional consequences of any potential misfolding or aggregation at the multimer level. If aggregated or misfolded MHC-I complexes were present, this would be expected to result in increased nonspecific binding and elevated background staining. However, across all experiments using both clonal T cell lines and primary PBMCs, we consistently observed low background staining and clear antigen-specific populations (Figures 3–5), arguing against the presence of aggregation-prone or sticky multimer species.
Third, we incorporated an additional quality control step based on LILRB1 binding (Figure 2C). Because LILRB1 selectively recognizes properly folded peptide–MHC-I complexes, this assay provides a sensitive readout for structural integrity. Unstable or improperly folded complexes do not bind LILRB1, allowing discrimination between functional and non-functional pMHC complexes.
Taken together, these complementary analyses indicate that temperature-based peptide exchange does not induce detectable aggregation or irreversible structural damage under our experimental conditions, and that the resulting multimer reagents are structurally intact and suitable for downstream applications. We have clarified this in the revised manuscript.
(5) The method's capacity to detect low-affinity/low-avidity TCR-pMHC interactions in a well-characterized model antigen system should be explicitly addressed. This would define the sensitivity limits of the approach compared to established methods.
The reviewer raises a valid and broadly relevant point that applies to all MHC-based technologies. However, we consider a full discussion of this topic to be outside the scope of the current manuscript. We do address this limitation for low-affinity peptides in the Discussion, where we write: “This technology is most likely not optimal for low-affinity peptides, as the template peptide itself must also have low affinity.” However, this problem may be universal since low-affinity peptides will simply release all MHC class I-peptide exchanged complexes during the staining process.
(6) Since the current study focuses on HLAs with high thermal stability, the approach should be tested with HLA alleles exhibiting lower thermal stability profiles. A validation framework could be developed that could be applied when introducing new HLA alleles to this process.
We thank the reviewer for this insightful comment. To our knowledge, there is no established classification of HLA alleles strictly based on “high” versus “low” intrinsic thermal stability; rather, stability is largely determined by the peptide–HLA combination.
Regarding the development of a validation framework for additional HLA alleles, our data indicate that suitable template peptide affinities generally fall within a range of approximately 4–11 µM. We anticipate that this range may vary between alleles, and therefore each HLA allele will require empirical identification of an optimal template peptide to achieve appropriate thermal stability and efficient peptide exchange.
This can be accomplished using the workflow presented in our study: first, assessing complex stability at 50 °C in the absence or presence of a high-affinity peptide (Fig. 1), followed by stepwise temperature reduction to determine optimal exchange conditions and incubation times (Fig. 2). In addition, LILRB1-based quality control can be incorporated to confirm the formation of properly folded and stable peptide–MHC complexes.
(7) It is encouraged that the approach be explored for its ability to effectively load suboptimal peptides with lower thermal stabilities. Demonstrating this capability would significantly enhance the method's clinical relevance.
We agree with the reviewer that evaluating the performance of the approach on suboptimal, low‑affinity peptides would be highly valuable and would further strengthen the clinical relevance of the method. In the current study, however, this falls outside our experimental scope. We are working on this in a follow-up project. We have now acknowledged the limitation of low-affinity peptides in the revised manuscript, noting that low‑affinity peptides are less suitable to this technology because the template peptide itself must also possess low affinity. Future work will be needed to systematically explore template design and exchange conditions optimized for such peptides.
