Despite mounting evidence that MHCI-restricted CD8+
T cells play a pivotal role in the development of T1D, the structural and biophysical basis for such an interaction with target β-cells has not been studied. To investigate the molecular basis for the recognition of an HLA A*0201-restricted preproinsulin epitope (A2-ALW) by a CD8+
T cell clone, we solved the structures of the autoreactive 1E6 TCR and A2-ALW in isolation and in complex. Overall, the 1E6 TCR bound to A2-ALW in a canonical diagonal fashion, with the CDR3 loops positioned over the central bulge of the peptide. Nevertheless, despite this standard docking orientation, the 1E6 TCR engaged A2-ALW with an extremely low affinity, attributable to a lower than average BSA and limited MHC contact footprint. Further, several other features of the complex are noteworthy. In contrast to most TCR-pMHC structures determined to date, this autoreactive TCR-pMHCI interaction was characterized by a rigid ‘lock and key’ conformation that is more typical of innate receptors or natural killer T cell TCR-CD1d-Antigen interactions32, 33
. Moreover, the interaction was dominated by the CDR3 loops, consistent with a previously proposed system in which non-germline-encoded CDR3 loops drive the energetic landscape of recognition34
, and yet conflicting with current theories regarding the role of the CDR1 and CDR2 loops as determinants of MHC-restriction28, 29, 35, 36
. Our findings, that T cells specific for this antigen are highly enriched in T1D patients20, 22
, and that 1E6 T cells kill resting β-cells in vitro
, suggest that ultra-low affinity binding and highly limited MHC footprint are still sufficient to have an important impact on the destructive processes that lead to insulin deficiency and T1D.
Unusually, the 1E6-A2-ALW interaction was predominantly focused through just two aromatic TCR residues, with Tyr97α and Trp97β accounting for 64% of the contacts. Residue-focused binding was mirrored by the peptide, in which just 2/5 TCR contact residues (Pro5 and Asp6) comprised 63% of the contacts. Thus, the interaction between the 1E6 TCR and the ALW peptide was exquisitely focused and governed by a binding mechanism dominated by just two TCR and two peptide residues. Aromatic residues, and surface exposed Trp residues in particular, commonly participate in protein-protein interactions31
, yet are relatively rare in TCR CDR3 loops. Nonetheless, Tyr97α and Trp97β appeared to be the driving force in this otherwise weak TCR-pMHCI interaction, acting as an aromatic cap that perches on a central region of the peptide.
There is considerable interest as to how T cells bearing TCRs that interact with autoantigens escape negative selection in the thymus and populate the periphery. Comparison of 1E6-A2-ALW and four other self-reactive human TCR-pMHC complexes with a typical HLA A*0201 pathogen-reactive complex highlights some of the differences in TCR binding to autoimmune and microbial antigens observed to date. These differences suggest several possible mechanisms to explain how self-reactive T cells might escape negative selection. First, a DR4-MBP-specific TCR was shown to bind cognate antigen with high affinity and normal topology14
. This is the strongest TCR affinity reported to date for any MHCII-restricted antigen, and lies in the range reported for pathogen-specific MHCI-restricted TCRs24
. However, the DR4-MBP epitope was highly unstable, leading to the hypothesis that T cells responding to low antigen densities in the thymus could receive a weak signal for positive selection, but then activate in response to higher antigen densities in the periphery. Second, self-reactive TCR-pMHC complexes, such as Ob.1A12-DR2a-MBP, 3A6-DR2-MBP and hy.1B11-DQ-MBP, were found to exhibit unusual conformations compared to more conventional pathogen-specific TCR-pMHC interactions13, 16, 17
. Such unusual TCR-pMHC binding orientations may allow T cells bearing these TCRs to bypass negative selection signals. Our own findings suggest a third route of thymic escape. The 1E6 TCR bound A2-ALW conventionally, but with a very low affinity that was highly peptide-centric. The low affinity of 1E6 TCR may be below the threshold required to induce negative selection but is sufficient to induce killing of β-cells20
. It is tempting to speculate that, for this TCR at least, the presence of cognate pMHCI in the thymus is highly determining with regard to selection. It is noteworthy that the INS
gene encoding PPI is transcribed and translated in the thymus and that possession of variable numbers of tandem repeats of a consensus sequence located 5′ of the coding region is the strongest genetic risk for T1D outside the MHC37
. Homozygosity for alleles containing few repeats (AA at rs689) confers a >2-fold relative risk of disease and is associated with 2-3 fold-reduced thymic transcription level compared with homozygous protection (TT at rs689)38
. It is proposed that reduced thymic INS
expression impairs central tolerance to PPI and this paradigm is supported by the demonstration that non-obese diabetic mice with targeted deletion of INS2
(syntenic to human INS
) display enhanced T cell reactivity to insulin and more rapid and penetrant disease39
. The T1D patient from whom 1E6 was obtained has AA at rs689. Notably, all three of the above mechanisms represent atypical recognition parameters that may help self-reactive T cells to bypass thymic deletion.
The observation that clone 1E6 has the lowest TCR affinity for pMHC described to date, yet remains able to kill resting human HLA-A*0201 β-cells expressing low levels of HLA class I, is a further potentially important observation in relation to disease pathogenesis, since it suggests that such clones have the potential to engage non-conditioned targets. Most disease models of T1D pathogenesis and initiation elaborated to date argue that islet inflammation comes first, and that the resulting cytokine-induced islet hyper-expression of HLA class I facilitates CD8+
T cell killing. Our data raise the possibility that CD8+
T cell killing could be an early event. Pre-existing low affinity CD8+
T cells could also be relevant in the setting of islet transplantation, in which donor β-cells express resting levels of HLA class I and therefore constitute targets for 1E6-like CD8+
T cells. Similarly, the future use of gene-modified β-cell replacement therapy may need to include designs that limit HLA class I expression to avoid disease recurrence. Finally, our observations raise the important consideration as to whether 1E6-like autoreactive T cells with ultra-low affinity TCRs are originally primed by self- or non-self ligands. We consider that our findings make the latter more likely, especially when viewed in the context of numerous studies implying a link between viruses, islet autoimmunity and T1D. Extensive searching of databases of viral proteins against ALWGPDPAAA, or modifications tolerated by 1E6 TCR, followed by examination of stimulation of 1E6 in vitro
have not to date revealed any potential virus-derived ligands (data not shown). However, the number of complete sequences of viruses that might be considered of highest relevance (eg the human enteroviruses), and are in the public domain, are currently limited and pathogen cross-reactivity will remain an important question to be that needs to be addressed. We have recently shown that the 1E6 clone recognizes over a million distinct decamer peptides in the context HLA A*020140
. A large number of peptides acted as substantially better agonists than the preproinsulin-derived peptide ALWGPDPAAA with the most potent peptide identified, RQFGPDFPTI, differing from this ‘index’ sequence at 7 of 10 amino acids. These findings serve to highlight the enormous potential of TCR degeneracy to be a causative factor in autoimmune disease.
In summary, we present the first structure of a human TCR in complex with an autoreactive MHCI-restricted antigen. Collectively, our observations suggest that CD8+
T cells expressing the 1E6 TCR could evade negative selection due to a ‘light-touch’ MHC contact footprint and weak TCR binding. In the periphery, β-cells operating in the presence of elevated glucose levels, combined with the pro-inflammatory milieu that leads to hyper-expression of MHCI, might drive high levels of surface A2-ALW expression and render these cells susceptible to antigen-experienced, 1E6-like CD8+
T cells20, 41
. Such T cells with low avidity for self likely require priming through contact with an infectious agent. It is possible that the unusual features of 1E6 TCR binding, focusing on just two residues in the peptide, as seen here, might increase the chances of pathogen-derived peptide cross-recognition. Indeed, it has been speculated that such ‘hotspot mimicry’ limited to a small number of key features within the TCR-binding footprint could contribute to the initiation of multiple sclerosis42
. Collectively, these data suggest a mechanism by which 1E6-like CD8+
T cells avoid thymic culling to populate the periphery and cause disease.