Canonical T cell receptor docking on peptide–MHC is essential for T cell signaling
Authors: Xavier Y X Sng
Pirooz Zareie
Nicholas R J Gascoigne
Christopher Szeto
Jan Petersen
Lukasz Wojciech
Sachith D Gunasinghe
Alex J Fulcher
Carine Farenc
Katharina Gaus
Jasmine Li
Nicole L La Gruta
Qianru Wei
Brian D Evavold
Jamie Rossjohn
Stephanie Gras
Chantelle Blyth
Angela Nguyen
Jesica R Jacobs
Elizabeth M Kolawole
Claerwen M Jones
Journal: Science (2021)
Discipline: Multidisciplinary
Keywords: T cell receptor; peptide-major histocompatibility complexes; docking polarity; signaling constraints; TRBV 17+ TCRs
Affiliations: Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia., European Molecular Biology Laboratory (EMBL) Australia Node in Single Molecule Science and the ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, University of New South Wales, New South Wales, Australia., Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA., Infection and Immunity Program and Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia., Monash Micro Imaging, Monash University, Clayton, Victoria, Australia., Immunology Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545.

Abstract:  T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRβ-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints. 

Retracted: No

PubPeer comments:
 Dear authors, We have with great interest and excitement discussed your paper u201cCanonical T cell receptor docking on peptide-MHC is essential for T cell signalingu201d during our weekly literature meeting. Your results on reversed TCR-pMHC docking are intriguing. It provides another important piece to the discussion on the initiation of TCR signaling and the role of CD4 8 co-receptors in this event. Additionally, it contains an important word of caution for people studying TCR repertoires using tetramers. However, after reading your paper, we were left wondering about a few things that we think would be interesting to see explored or commented on, either here, or in future publications: 1. Is it possible that reversed-oriented TCRs are not matched with their targets? Might it be they are actually recognizing antigens in the canonical manner, but only in the context of their u201crealu201d target presented by a different HLA molecule? That may explain their existence in the first place. How else would they have been selected for in the thymus if they do not provide Lck for the necessary signaling (PMID: 25284152)? 2. On that same note, could it be that particular HLA-allele both promotes the selection of TCRs on one HLA-molecule expressed by that haplotype and allows interaction in the reversed-orientation of the same TCR with another HLA-molecule, while other HLA-alleles do not? 3. You are showing that the prevalence of TCRs in the immune response is unrelated to TCR-pMHCI affinity. What if the expansion of these TCRs depends on other antigens? Can the prevalence of TCRs depend on antigen abundance? 4. The KD values of your TCRs seem to be quite low in comparison to the maximum capacity of strong TCR-pMHC pairs. For example, 1G4 TCR and HLA A2 loaded with 9V peptide KD is 7.07 x 10^-5 u03bcM (PMID: 32817332), while your high affinity reversed orientation-TCR is 6,34 u03bcM (10000x lower affinity). What would happen if your reversed TCR had 10000 higher affinity? Would it rescue its function? 5. What is your hypothesis on why you were unsuccessful in expressing B17.C1 TCR in vivo? 6. Have you tested immune synapse formation on lipid bilayers? It would be interesting to see the synapse dynamics in a kinetic manner. Would the dynamics of CD8 recruitment to the synapse be affected in reversed-TCR? 7. Fig. 6F-H u2013 there is much higher background signal in CD8Null and CD8CxC than in CD8WT. Might it be an evidence for your Lck-CD8 sequestration theory (in cells lacking CD4 CD8, Lck would have higher background activity)? 8. Is it possible that the reversed TCR-pMHC interaction does not produce signaling because the TCR is naturally asymmetrical? Could CD3gamma and CD3delta define TCR asymmetricity? Might it be a mechanism for preventing unspecific interactions of expanded populations? By constraining TCR-pMHC interaction only to the canonical docking, the possibility of recognizing other unspecific antigens is reduced by 2-fold (which, in the case of all the possible TCR-pMHC pairing, is a lot). We would greatly appreciate it if you could share your thoughts on our comments above, as this may aid us (and other readers) in better understanding your work and thus help us all contribute to a fruitful scientific discussion. , 
 Thank you for your interest in our paper. Please find below some replies to your queries:- Is it possible that reversed-oriented TCRs are not matched with their targets? Might it be they are actually recognizing antigens in the canonical manner, but only in the context of their u201crealu201d target presented by a different HLA molecule? That may explain their existence in the first place. How else would they have been selected for in the thymus if they do not provide Lck for the necessary signaling (PMID: 25284152)? Yes, it is possible that the reversed TCRs were selected in a canonical orientation on a structurally distinct self-ligand and were thus destined to recognize a different cognate antigen in the periphery. Following on from that, it is possible that the recognition of the H-2DbNP366 epitope is a case of stochastic cross reactivity. However, it is important to remember that this reactivity is observed for a significant proportion of the TRBV17+ TCRs expressing a wide variety of TCR alpha chains, and thus if it is cross-reactivity, it is not restricted to a few TCRs. On that same note, could it be that particular HLA-allele both promotes the selection of TCRs on one HLA-molecule expressed by that haplotype and allows interaction in the reversed-orientation of the same TCR with another HLA-molecule, while other HLA-alleles do not? This is formally possible, however in our earlier paper that originally described the reversed docking TCRs (Gras et al, Immunity 2016) we showed that there was no impact on H-2DbNP366 tetramer binding cells, and specifically on TRBV17+ cells, in H-2Kb- - mice, suggesting that the reversed TCRs are not selected on H-2Kb. Given that they are CD8+ and WT B6 mice only express H-2Db and H-2Kb, this would indicate that the reversed TCRs are selected on H-2Db. You are showing that the prevalence of TCRs in the immune response is unrelated to TCR-pMHCI affinity. What if the expansion of these TCRs depends on other antigens? Can the prevalence of TCRs depend on antigen abundance? I am not entirely sure what you mean by this. Our interpretation is that, although they can bind the DbNP366 antigen, T cells expressing reversed TCRs cannot be expanded by its presentation during an IAV infection. And we know from the robust DbNP366-specific T cell response, as well as other direct and indirect analyses of DbNP366 presentation (PMID: 31253788, 16418289) that DbNP366 is well presented. The KD values of your TCRs seem to be quite low in comparison to the maximum capacity of strong TCR-pMHC pairs. For example, 1G4 TCR and HLA A2 loaded with 9V peptide KD is 7.07 x 10^-5 u03bcM (PMID: 32817332), while your high affinity reversed orientation-TCR is 6,34 u03bcM (10000x lower affinity). What would happen if your reversed TCR had 10000 higher affinity? Would it rescue its function? The Kd value of the u2018high affinityu2019 reversed TCR (B17.R2) is 6.34 u03bcM. Antiviral TCR-pMHC affinity is typically between 1 and 10 u03bcM, so this is considered a relatively high affinity interaction within a physiological range. The affinities you mention are clearly supraphysiological. It is unclear what would happen if the reversed TCR had 10000 higher affinity. Have you tested immune synapse formation on lipid bilayers? It would be interesting to see the synapse dynamics in a kinetic manner. Would the dynamics of CD8 recruitment to the synapse be affected in reversed-TCR? We have investigated receptor clustering on lipid bilayers as shown in Fig 5A-C of the paper. Further analysis of synapse formation is underway. Fig. 6F-H u2013 there is much higher background signal in CD8Null and CD8CxC than in CD8WT. Might it be an evidence for your Lck-CD8 sequestration theory (in cells lacking CD4 CD8, Lck would have higher background activity)? There is no higher background signal in CD8Null lines. Please see Figure S4 for background measurements. ,