T cells discriminate between groups C1 and C2 HLA-C
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Evaluation Summary:
In this study, the authors use structural, functional and immunopeptidomics analysis to provide insights into how HLA-C C1/C2 dimorphism impacts T cell recognition. This knowledge is important in immunotherapies targeting HLA-C-specific T cells. Overall, the experiments are well-performed, and the conclusions are largely supported by the presented data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Dimorphic amino acids at positions 77 and 80 delineate HLA-C allotypes into two groups, C1 and C2, which associate with disease through interactions with C1 and C2-specific natural killer cell receptors. How the C1/C2 dimorphism affects T cell recognition is unknown. Using HLA-C allotypes that differ only by the C1/C2-defining residues, we found that KRAS-G12D neoantigen-specific T cell receptors (TCRs) discriminated between C1 and C2 presenting the same KRAS-G12D peptides. Structural and functional experiments, and immunopeptidomics analysis revealed that Ser77 in C1 and Asn77 in C2 influence amino acid preference near the peptide C-terminus (pΩ), including the pΩ-1 position, in which C1 favors small and C2 prefers large residues. This resulted in weaker TCR affinity for KRAS-G12D-bound C2-HLA-C despite conserved TCR contacts. Thus, the C1/C2 dimorphism on its own impacts peptide presentation and HLA-C-restricted T cell responses, with implications in disease, including adoptive T cell therapy targeting KRAS-G12D-induced cancers.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Sim and colleagues explores HLA C1 and C2 defining polymorphism and its impact on TCR recognition as opposed to the more documented impacts on KIR recognition. The manuscript is well written but requires some relatively minor changes prior to publication. The overall findings that subtle changes in peptide repertoire and peptide binding dictate TCR recognition is perhaps not surprising. The context of the study looking at KRAS G12D derived peptides provide additional interest to this manuscript. The work has been performed to a high level and includes reports of novel ternary complexes of HLA-C with a G12D heteroclitic peptide analogue along with associated biophysical characterization of the TCR interaction with these pHLA complexes.
We thank the reviewer for the kind …
Author Response
Reviewer #1 (Public Review):
The manuscript by Sim and colleagues explores HLA C1 and C2 defining polymorphism and its impact on TCR recognition as opposed to the more documented impacts on KIR recognition. The manuscript is well written but requires some relatively minor changes prior to publication. The overall findings that subtle changes in peptide repertoire and peptide binding dictate TCR recognition is perhaps not surprising. The context of the study looking at KRAS G12D derived peptides provide additional interest to this manuscript. The work has been performed to a high level and includes reports of novel ternary complexes of HLA-C with a G12D heteroclitic peptide analogue along with associated biophysical characterization of the TCR interaction with these pHLA complexes.
We thank the reviewer for the kind comments. We also agree that even subtle changes in peptide sequence can be expected to change recognition of HLA-I by TCR. What we did find somewhat surprising is that the HLA-C C1/C2 dimorphism alone, in HLA-C molecules that are otherwise identical, puts constraints on peptide sequences that can be accommodated in the peptide binding site. It is the difference in those amino acids that, unsurprisingly, affect recognition by TCR, independently of TCR contact with amino acids 77 and 80 in HLA-C.
Reviewer #2 (Public Review):
This manuscript by Sim et al. describes the impact of different HLA-C1 and -C2 allotypes on T cell receptor (TCR) recognition. The study demonstrates that dimorphic position 77 in the HLAC heavy chain affected amino acid preferences at the C-terminus of the bound peptide, resulting in a weaker TCR affinity for HLA-C2 allotypes. The manuscript is clearly written, the data is sound and the figures are of high quality. The study is interesting and original; however the overall biological relevance remains unclear. It is uncertain how generalizable the findings will be to TCR recognition of HLA-C1 vs -C2 alleles in general, or whether the findings are perhaps more limited to this particular system. Moreover, the link/relevance to KIR recognition (if any) was not explained.
We thank the reviewer for these comments. We agree that it is unclear how generalizable our finding that C2 allotypes are worse TCR ligands than C1 allotypes. Further study of HLA-C restricted TCRs would be required to establish whether the effect we observed is truly generalizable. Such studies could examine how substitutions at position 77 and 80 influence responses of HLA-C restricted TCRs. Although, our study indicates that the effect is generalizable as both TCRs use different V genes and recognise different epitopes, and in both cases the C2 allotype is a worse ligand. Further, some of our other findings appear generalizable, such as the impact of the C1/C2 dimorphism on HLA-C immunopeptidomes and HLA-C structure. Presently, the translational relevance of our findings appear limited to the context of patients with KRASG12D-induced cancers in individuals carrying C08:02 or C05:01. In the context of KIR, earlier work has shown that positions 7 and 8 of 9mer peptides bound to HLA-C can have a large impact on KIR binding. An ongoing study by us is exploring the relevance of peptide-specific recognition of HLA-C by different KIR family members.
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Evaluation Summary:
In this study, the authors use structural, functional and immunopeptidomics analysis to provide insights into how HLA-C C1/C2 dimorphism impacts T cell recognition. This knowledge is important in immunotherapies targeting HLA-C-specific T cells. Overall, the experiments are well-performed, and the conclusions are largely supported by the presented data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Sim and colleagues explores HLA C1 and C2 defining polymorphism and its impact on TCR recognition as opposed to the more documented impacts on KIR recognition. The manuscript is well written but requires some relatively minor changes. The overall findings that subtle changes in peptide repertoire and peptide binding dictate TCR recognition is perhaps not surprising. The context of the study looking at KRAS G12D derived peptides provide additional interest to this manuscript. The work has been performed to a high level and includes reports of novel ternary complexes of HLA-C with a G12D heteroclitic peptide analogue along with associated biophysical characterization of the TCR interaction with these pHLA complexes.
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Reviewer #2 (Public Review):
This manuscript by Sim et al. describes the impact of different HLA-C1 and -C2 allotypes on T cell receptor (TCR) recognition. The study demonstrates that dimorphic position 77 in the HLA-C heavy chain affected amino acid preferences at the C-terminus of the bound peptide, resulting in a weaker TCR affinity for HLA-C2 allotypes. The manuscript is clearly written, the data is sound and the figures are of high quality. The study is interesting and original; however the overall biological relevance remains unclear. It is uncertain how generalizable the findings will be to TCR recognition of HLA-C1 vs -C2 alleles in general, or whether the findings are perhaps more limited to this particular system. Moreover, the link/relevance to KIR recognition (if any) was not explained.
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Reviewer #3 (Public Review):
Sim et al. investigate the structural and functional differences between group C1 and group C2 HLA-C molecules, which differ only in two amino acids (position 77 and 80 of HLA-C) that line the peptide binding group. Nevertheless, the KRAS-D12D specific TCR can discriminate both HLA-C groups as demonstrated in this study using cellular immune assays, X-ray crystallography and immunopeptidomics. As a result, the manuscripts provides an important insight into the functional differences of the C1/C2 dimorphism, especially in the context of cancer immunotherapy using T cell based therapeutics.
The authors study two HLA-C*08:02 (group C1) and HLA-C*05:01 (group C2) restricted TCRs that recognize the KRAS mutant peptide G12D. Using cell based assays the authors establish that both TCRs (one specific for the KRAs …
Reviewer #3 (Public Review):
Sim et al. investigate the structural and functional differences between group C1 and group C2 HLA-C molecules, which differ only in two amino acids (position 77 and 80 of HLA-C) that line the peptide binding group. Nevertheless, the KRAS-D12D specific TCR can discriminate both HLA-C groups as demonstrated in this study using cellular immune assays, X-ray crystallography and immunopeptidomics. As a result, the manuscripts provides an important insight into the functional differences of the C1/C2 dimorphism, especially in the context of cancer immunotherapy using T cell based therapeutics.
The authors study two HLA-C*08:02 (group C1) and HLA-C*05:01 (group C2) restricted TCRs that recognize the KRAS mutant peptide G12D. Using cell based assays the authors establish that both TCRs (one specific for the KRAs 9mer peptide and the other for the KRAS decamer peptide) recognize HLA-C*08:02 presenting the mutant peptide but not the wildtype peptide, while HLA-C*05:01 expressing cells only stimulate the jurkat T cells very weakly and also only with the mutant peptide. Further, the authors identify using peptide elution studies that C*08:02 can also present peptides with terminal alanine anchor residues, albeit at a frequency of only 1.5%, while C*05:01 does not. They authors hypothesize that the amino acid differences at position 77 and 80 of HLA-C, which are close to the F pocket are influencing the amino acid preference at the C-terminus of the presented peptide. Using Surface Plasmon Resonance studies (SPR), the authors assessed the binding of both KRAS-specific TCRs to different synthetic KRAS peptides with different anchor residue at P(omega) and reveal that HLA-C*05:01 is bound with high affinity when the KRAS G12 peptide is modified with a preferred leucine anchor residue at P(omega), instead of the natural alanine. Therefore, the authors argue that the inability of T cell stimulation using C*05:01 is a result of the inability of this allele to stably present the KRAS G12D peptide. Finally, using Xray crystallography the authors further identified that the C1-C2 dimorphism has only a minimal impact on the overall TCR binding mode when C*05:01 is loaded with the well-binding KRAS G12D peptide that contains the terminal Leucine anchor residue. In addition, the dimorphic residues at HLA-C position 77 and 80 are not directly contacted by the TCR. By swapping out the dimorphic residues, the authors are further able to switch T cell responses from C*08:02 toward C*05:01 using the KRAS G12D peptide, suggesting that the dimorphic residues are directly involved in shaping the immunopeptidome for each allele and directly influencing the binding/presentation of the peptide that gives rise to the observed T cell response. Furthermore, the authors identify different amino acids usage in P(omega)-1 position, which is suggested to be a result of slight differences in the peptide binding orientation at the C-terminal end. Specifically, large residues at P(omega)-1 diminished T cell recognition for group C1 HLA-C.
The experiments are well planned and executed and support the conclusion. The proper controls are included. The findings are discussed appropriately and the references contain many of the original studies in the field.
This is an important study that investigates the amino acid differences of the HLA-C C1 and C2 groups and how they affect peptide binding, presentation, and recognition. This knowledge is fundamental in order to design HLA allele-specific TCR based therapeutics.
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