King’s Research Portal Link to publication record in King's Research Portal Citation for published version (APA): Peakman, M., Harbige, J. E., & Eichmann, M. (2017). New insights into non-conventional epitopes as T cell targets: the missing link for 1 breaking immune tolerance in autoimmune disease? Journal of Autoimmunity, 12(20). Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. General rights Copyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights. •Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research. •You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 25. Aug. 2020
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King’s Research Portal
Link to publication record in King's Research Portal
Citation for published version (APA):Peakman, M., Harbige, J. E., & Eichmann, M. (2017). New insights into non-conventional epitopes as T celltargets: the missing link for 1 breaking immune tolerance in autoimmune disease? Journal of Autoimmunity,12(20).
Citing this paperPlease note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this maydiffer from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination,volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you areagain advised to check the publisher's website for any subsequent corrections.
General rightsCopyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyrightowners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights.
•Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research.•You may not further distribute the material or use it for any profit-making activity or commercial gain•You may freely distribute the URL identifying the publication in the Research Portal
Take down policyIf you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access tothe work immediately and investigate your claim.
New insights into non-conventional epitopes as T cell targets: the missing link for 1
breaking immune tolerance in autoimmune disease? 2
James Harbige 1, Martin Eichmann 1, Mark Peakman 1, 2, 3 3
1 Department of Immunobiology, Faculty of Life Sciences & Medicine, King's College 4
London, UK 5
2 Division of Diabetes and Nutritional Sciences, King's College London, UK 6
3 Institute of Diabetes, Endocrinology and Obesity, King’s Health Partners, London, UK 7
Abstract 8
The mechanism by which immune tolerance is breached in autoimmune disease is poorly 9
understood. One possibility is that post-translational modification of self-antigens leads to 10
peripheral recognition of neo-epitopes against which central and peripheral tolerance is 11
inadequate. Accumulating evidence points to multiple mechanisms through which non-12
germline encoded sequences can give rise to non-conventional epitopes which in turn engage 13
the immune system as T cell targets. In particular, where these modifications alter the rules of 14
epitope engagement with MHC molecules, such non-conventional epitopes offer a persuasive 15
explanation for associations between specific HLA alleles and autoimmune diseases. In this 16
review article, we discuss current understanding of mechanisms through which non-17
conventional epitopes may be generated, focusing on several recently described pathways 18
that can transpose germline-encoded sequences. We contextualise these discoveries around 19
type 1 diabetes, the prototypic organ-specific autoimmune disease in which specific HLA-DQ 20
molecules confer high risk. Non-conventional epitopes have the potential to act as tolerance 21
breakers or disease drivers in type 1 diabetes, prompting a timely re-evaluation of models of 22
aetiopathogenesis. Future studies are required to elucidate the disease-relevance of a range 23
of potential non-germline epitopes and their relationship to the natural peptide repertoire. 24
2
Table 1: Definition of terms 25
26
27
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32
33
34
35
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44
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Neo-epitope: an epitope that is modified from a germline sequence, either by processing of a neo-antigen or by modification of an existing germline epitope
Conventional epitope: target generated by processing of a germline-encoded sequence recognised by T cells or antibodies
Non-conventional epitope: target derived by processing of a non-germline encoded sequence recognised by T cells or antibodies
Heteroclitic peptide: A modified peptide in which HLA anchor residues in the native sequence are substituted for optimal anchor residues to induce stronger T cell responses than the corresponding native epitope
Super-agonist: a ligand capable of eliciting a response which is greater than the native endogenous ligand for the target receptor
Super-charging event: an event (e.g. inflammation) resulting in the generation of highly immunogenic non-conventional epitopes which supplement a pre-existing autoimmune response to conventional epitopes to drive pathology
3
Background and introduction 46
T cell mediated immune tolerance 47
The mechanisms through which T cells can avoid recognising and responding to self-antigens 48
have become well established in recent years and form an important base from which to 49
understand immune tolerance, autoimmunity and autoimmune disease. In brief, developing T 50
cells (thymocytes) undergo a selection process in the thymus based on the affinity of their T 51
cell receptor (TCR) for self-peptide–MHC complexes [1]. The focus on self is refined by the 52
transcription factor AIRE (autoimmune regulator) which enables enhanced expression of 53
tissue specific antigens (e.g. preproinsulin) by medullary thymic epithelial cells to promote 54
central tolerance against self-proteins that are highly represented in the periphery [2]. A TCR 55
with high affinity for self-peptide–MHC complexes results in deletion (negative selection) and 56
thymocytes with a TCR that has insufficient affinity undergo death by neglect. Only thymocytes 57
with a TCR that has a “low affinity” (inadequate affinity to lead to T cell activation) for self-58
peptide–MHC complexes receive a survival signal and exit into the periphery. TCR-self-59
peptide–MHC interactions of intermediate affinity drive regulatory T cell (Treg) differentiation 60
[3]. Central tolerance mechanisms are not 100% efficient, in part because not all self-antigens 61
are expressed in the thymus. As a result, autoreactive T cells can be released into the 62
periphery, where they are restrained by a series of peripheral tolerance mechanisms that 63
prevent activation of effector responses. These include immunological ignorance i.e. an 64
autoreactive T cell may never encounter the relevant self-antigen in vivo; conversely, 65
depending on context, encounter with the self-antigen might induce anergy (controlled 66
unresponsiveness) or activation-induced cell death [4]. In addition, evidence has accumulated 67
for the active suppression of autoreactivity in the periphery by Tregs [5]. When these tolerance 68
mechanisms fail, self-antigens can become the target of a sustained immune response leading 69
to chronic inflammation and autoimmunity. 70
71
4
HLA restriction in type 1 diabetes 72
Type 1 diabetes (T1D) is a chronic autoimmune disease associated with loss of insulin-73
producing β-cells, resulting from a complex interaction between genetic and environmental 74
factors. Of these genes, those in the HLA region confer the strongest disease risk [6]. 75
Specifically, the major T1D susceptibility loci map to the HLA class II region. Allelic class II 76
genes encoding either the HLA-DR3-DQ2 (DRB1*03:01-DQA1*05:01-DQB1*02:01) or HLA-77
DR4-DQ8 (DRB1*04-DQA1*03:01-DQB1*03:02) haplotypes carry the greatest risk (odds 78
ratios of ~5) whilst for individuals who are heterozygous for HLA-DR3-DQ2/DR4-DQ8 79
haplotypes the risk is ~5-fold higher again [7]. Conversely, possession of HLA-DQ6 (HLA-80
DQA1*01:02–DQB1*06:02) is associated with dominant protection from the disease. The 81
class I HLA genes are also implicated in T1D risk (albeit with a lower odds ratio [8]) with the 82
recognition of HLA-A2 (A*02:01) or HLA-A24 (A*24:02)-restricted epitopes by CD8+ T cells 83
shown to mediate β-cell killing [9]. 84
Consensus model of type 1 diabetes pathogenesis 85
To date, most evidence points to the adaptive immune system (T and B lymphocytes) as 86
dominant in the process of β-cell destruction in T1D [10]. However, there is limited 87
understanding of the mechanisms through which tolerance to β-cell autoantigens is 88
incomplete, insufficient or fails. Hitherto, a consensus disease model based on studies in 89
animals and humans, has suggested that an initiating event (as yet unknown) causes damage 90
to the islets of Langerhans and inflammation [11]. Dendritic cells (DCs) endocytose released 91
β-cell autoantigens, migrate to the local lymph nodes and present short peptides via HLA class 92
I and II molecules to T cells, leading to CD8+ and CD4+ T cell activation, respectively. 93
Activated T cells are poorly restrained by dysfunctioning Tregs [12] and traffic in the blood to 94
the pancreatic islets where CD8+ T cells can kill β-cells directly [13, 14] and where CD4+ T 95
cells produce pro-inflammatory cytokines resulting in further damage to β-cells via various 96
mechanisms [15]. The model proposes that the adaptive immune response focuses 97
predominantly onto a discrete group of molecular targets, most of which were discovered 98
5
through the study of disease-related autoantibodies and include (pro)insulin, glutamic acid 99
heteroclitic peptides of the immunodominant preproinsulin epitope encompassing residues 15-422
24 elicit a much stronger T cell clone response than the native peptide [75]. This finding 423
suggests that a high sensitivity of TCR to minor alterations in peptide conformation can exist. 424
An alternatively scenario is that Treg responses to non-conventional epitopes are 425
compromised. One can argue that low or absent expression of non-conventional epitopes 426
within the thymus will result in a lack of thymus-derived Tregs [76]. Although, DCs can acquire 427
18
peripheral antigens and traffic them to the thymus to induce T cell selection [77, 78]. Therefore, 428
thymic generation of Tregs against non-conventional epitopes is possible. 429
We propose a model whereby non-conventional epitopes represent tolerance breakers or 430
disease drivers. As tolerance breakers, hybrid peptides represent a key initiation point in 431
triggering loss of tolerance within islet tissues, and causing a degree of β-cell destruction. 432
Subsequent inflammatory events could be more, or as dependent on responses to 433
conventional “natural” epitopes. This triggering event could arise under different 434
circumstances, including weaning, during which there is considerable β-cell remodeling [79], 435
or a virus infection. In this case, studying Stage 1 of type 1 diabetes and following its 436
progression in parallel with measurement of immune responses to non-conventional epitopes 437
may prove fruitful in discerning pathological pathways. Alternatively, non-conventional 438
epitopes may be important in driving the disease once β-cell damage has been initiated 439
through a more conventional loss of self-tolerance, especially if the processes of splicing and 440
hybridicity are enhanced by inflammation. In either setting, immune monitoring strategies 441
focused on recognition of non-conventional epitopes could render these as useful biomarker 442
tools for patient stratification. 443
Future perspectives 444
The pace of these exciting new developments is remarkable but has left many questions 445
unanswered, including the cellular compartments from which HIPs derive and the intracellular 446
pathways through which they are presented by HLA class II molecules. As yet it is not clear 447
whether the granule extract contains these hybrid peptide species or whether they derive from 448
a longer polypeptide species that requires immunological processing. Equally important to 449
address is the initial cue responsible for the generation of hybrid and spliced peptides. 450
Evidence suggests ER stress induces translation of the INS-DRiP polypeptide [42] and may 451
therefore be a shared mechanism contributing to the generation of non-conventional T cell 452
epitopes. 453
19
Hybrid peptides are yet to be identified in human pancreatic β-cells and studies on their 454
molecular interaction with the TCR will yield important insight into the extent to which the 455
junction region of the HIP is critical for T cell activation. Whether there is a bias for high-risk, 456
disease-associated HLA-DQ molecules to bind hybrid peptides with particularly high affinity, 457
and how these epitopes interact with disease-protective molecules such as HLA-DQ6 will also 458
be important lines of study. An important avenue of research will be the capacity of CD8+ T 459
cells to target hybrid peptides in T1D. Conventionally, CD4+ T cells would be required for a 460
break of tolerance, although activation of CD8+ T cells by cross-presentation may represent 461
a way of subverting central CD4+ T cell tolerance by bypassing the requirement for CD4+ T 462
cell help [80]. Identification of novel non-conventional peptide epitopes will be challenging until 463
robust search algorithms are developed to find non-germline sequences in mass spectrometry 464
data. Similarly, translational errors are typically excluded from searches of transcriptomic 465
materials. Finally, whether the generation of non-conventional peptides is targetable at a 466
therapeutic level will require a better understanding of peptide generation, and may have 467
implications for antigen-specific therapies designed to induce immunological tolerance. 468
469
20
Acknowledgements 470
Related work in our laboratory receives funding from the Innovative Medicines Initiative 2 Joint 471
Undertaking under grant agreement No 115797 INNODIA. This Joint Undertaking receives 472
support from the European Union’s Horizon 2020 research and innovation programme and 473
“EFPIA”, ‘JDRF International” and “The Leona M. and Harry B. Helmsley Charitable Trust”. 474
The laboratory is also supported via the National Institute of Health Research Biomedical 475
Research Centre Award to Guy’s and St Thomas National Health Service Foundation Trust 476
and King’s College London. JH is in receipt of a Guy's & St Thomas' Charity Prize PhD 477
Studentship. 478
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21
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Figure legends 719
Figure 1: A schematic model of non-conventional epitopes as tolerance breakers (①) 720
or disease drivers (②). The initial autoreactive T cell response against islet autoantigens 721
(tolerance breaker) is primed to non-conventional epitopes. Alternatively, non-conventional 722
epitopes supplement autoimmune responses to conventional epitopes to drive disease. T cell 723
responses against non-conventional epitopes are likely characterised by enhanced TCR-724
pMHC affinities compared to conventional epitopes. TCR-pMHC, T cell receptor-peptide-725
major histocompatibility complex. 726
Figure 2: Generation of non-conventional epitopes within the pancreatic β-cell. The 727
figure depicts sites within the β-cell reported to be involved in the generation of non-728
conventional epitopes. These epitopes can be generated by proteasomal peptide splicing, 729
translational errors and during enzymatic cleavage of polypeptide cargoes within the β-cell 730