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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).

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Download date: 25. Aug. 2020

1

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

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

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

decarboxylase (GAD65), zinc transporter 8 (ZnT8), insulinoma-associated antigen-2 (IA-2) as 100

well as islet-specific glucose-6-phosphatase catalytic subunit−related protein (IGRP) which 101

was discovered via reverse translation [16]. The model needs to account for the role of HLA 102

in these processes: as mentioned above, alleles at the HLA-DRB1, DQA1 and DQB1 loci 103

confer the greatest genetic predisposition to T1D [17]. Previous studies suggest intrinsic 104

stability of HLA-DQ molecules as a factor [18] and/or preferential presentation of self-peptides 105

or infectious agents to the immune system [19], but details are scant and overall there is a 106

dearth of compelling explanations as to how HLA-DQ-mediated predisposition operates within 107

the consensus model. 108

Non-conventional epitope generation: multiple models and mechanisms 109

Given the fundamental limitation of this model – which cannot adequately account for the 110

major gene effect – it is not surprising that the field has sought new insights. In particular, the 111

possibility that post-translational modification of self-proteins could be a key component in β-112

cell autoimmunity has begun to garner increasing interest. 113

Post-translational modification of autoantigens 114

Recently, attention has focused on the possibility that breakdown in self-tolerance and HLA-115

dominated disease associations could be linked to post-translational modification (PTM). Self-116

antigens undergoing PTM generate neo-antigens for which central (thymic) deletion events 117

are insufficient, allowing them to become the target of an immune response. The role of PTM 118

in autoimmune disease is not the focus of this review article, as it has been extensively 119

reviewed elsewhere [20-22]; for reference, common PTMs are presented in Table 1. Of 120

particular relevance to the T1D discussion are the autoantigen modifications described in the 121

autoimmune/inflammatory diseases, rheumatoid arthritis (RA) and celiac disease (CD), which 122

are viewed as the exemplars of this phenomenon [23]. In RA citrullination of aggrecan and 123

vimentin converts positively-charged arginine into a neutral citrulline residue, thereby 124

6

enhancing binding of peptide epitopes to RA-associated HLA-DR4 (HLA-DRB1*04:01/04) 125

molecules [24]. Similarly, in CD deamidation of gluten-derived peptides by tissue 126

transglutaminase converts glutamine to negatively-charged glutamic acid [25]. The 127

consequence of this modification is that the deamidated peptide binds more strongly to 128

disease-associated HLA-DQ2 and HLA-DQ8 molecules. This leads to a prolonged dwell-time 129

rendering the peptide highly immunogenic and results in a strong T cell response as measured 130

by interferon-γ (IFN-γ) secretion and T cell proliferation [25, 26]. T1D shares similar HLA 131

associations to RA and CD and so it is not unreasonable to hypothesize that PTM of self-132

peptides, resulting in enhanced self-presentation and self-recognition, may also have a role in 133

the pathogenesis of T1D. 134

135

136

137

138

139

140

141

142

143

144

145

146

147

7

Table 2: Common post-translational modifications associated with autoimmune 148 disease. 149

Autoimmune disease

Modification Antigen Reference

Multiple sclerosis Citrullination

Acetylation

Palmitoylation

MBP, GFAP

MBP

PLP

[27]

[28]

[29]

Rheumatoid arthritis

Citrullination

Deimination

Vimentin, aggrecan

Fibrin

[24]

[30]

Systemic lupus erythematosus

Phosphorylation

Deimination

RNA splicing factors

Histones

[31]

[32]

Celiac disease Deamination Gluten (gliadin) [33] Psoriasis Endoprotease

cleavage Pso27 [34]

Type 1 diabetes Deamidation

Disulfide bond

Citrullination

Oxidation

Proinsulin Insulin GAD65 Insulin

[35]

[36] [37] [38]

150 MBP, myelin basic protein; GFAP, glial fibrillary acidic protein; PLP, myelin proteolipid protein; 151 GAD65, glutamic acid decarboxylase 65. 152

153

The first evidence of modified peptides in T1D was provided by Mannering et al [36] showing 154

T cell responses to a HLA-DR4-restricted disulphide-modified insulin A chain (A1-13) epitope. 155

The wider concept has been adopted and additional evidence derives from the discovery of a 156

transglutaminated chromogranin A (ChgA; a β-cell granule protein) epitope which displays 157

increased recognition by several diabetogenic CD4+ T cell clones in non-obese diabetic 158

(NOD) mice [39]. van Lummel et al [35] described that deamidation of islet autoantigen 159

epitopes by tissue transglutaminase increases their binding affinity to HLA-DQ8 molecules 160

and HLA-DQ8trans molecules (expressed in cells heterozygous for the HLA-DQ8/DQ2 161

8

haplotypes, generating four unique HLA-DQ molecules). CD4+ T cells reactive against a 162

deamidated proinsulin peptide presented by HLA-DQ8 were subsequently isolated from a 163

patient with new-onset T1D. More recently, citrullinated and transglutaminated GAD65 164

epitopes restricted to DRB1*04:01 have been reported and are recognised by T cell clones 165

from T1D patients [37]. T cells specific for these modified GAD65 epitopes were detected at 166

significantly higher frequencies in the peripheral blood of T1D subjects than in healthy controls. 167

Taken together, these findings imply that T cell recognition of enzymatically modified peptide 168

epitopes in T1D can disrupt normal immune regulatory networks. However, these mechanisms 169

of PTM generation derive directly from our knowledge of RA and CD pathogenesis; as yet 170

there is little or no a priori evidence to suggest that citrullination and transglutamination are 171

important in T1D. 172

In effect, whilst the concept of post-translational modification of autoantigens is an appealing 173

one for our understanding of T1D pathogenesis, the PTM model as elaborated for RA and CD 174

has failed to convince. It is timely, therefore, that new concepts as to how non-conventional 175

epitopes are generated and become T cell targets have begun to emerge and these will be 176

the focus of this article. Hybrid insulin peptides have been recently defined as novel T cell 177

targets in T1D and comprise non-germline encoded epitope sequences [40]. In this case, 178

hybrid insulin peptides represent alterations that are not achieved by modifying amino acids 179

per se, but by fusing multiple amino acid sequences together. Therefore, when compared with 180

the putative PTMs previously described in T1D (as discussed above), hybrid peptides are a 181

more complex type of modification through which novel, non-germline encoded sequences 182

are generated by peptide fusion. This type of modification has now been described to occur 183

for HLA class II in the context of T1D (hybrid insulin peptides) but also occurs frequently for 184

HLA class I in tumour cells (spliced peptides) [41] although their mechanisms of generation 185

may be different. To add to the mechanistic complexity, we will also highlight recent evidence 186

that a defective ribosomal insulin gene product is a novel T cell target in T1D [42]. In each 187

case, these mechanisms of non-conventional epitope generation can circumvent central 188

9

tolerance and in some cases, have the potential to solve the riddle of the powerful HLA-DQ 189

disease associations with T1D that were first identified 30 years ago [43]. 190

Hybrid insulin peptides 191

WE14 is a ChgA-derived neuropeptide identified as a naturally processed ChgA epitope in 192

both NOD mice [44] and T1D patients [45]. However, the prototypical NOD-derived 193

diabetogenic T cell clone, BDC-2.5 and other T cell clones previously described to be reactive 194

to ChgA, respond only weakly to the wild-type WE14 sequence [44]. Indeed, modelling of the 195

interaction with the NOD MHC class II molecule, IA-g7, suggests that the WE14 peptide 196

epitope fills only half of the peptide binding groove leaving several positions unoccupied. As a 197

result, it was hypothesized that the critical ligand recognised by these pathogenic CD4+ T 198

cells in vivo may be a modification of WE14 peptide. Indeed, addition of the amino acids RLGL 199

to the N-terminus of the WE14 peptide (RLGLWSRMDQLAKELTAE) improves binding to IA-200

g7 as demonstrated by the crystal structure of the RLGL–WE14–IA-g7 complex [46]. The 201

RLGL-modified WE14 peptide was shown to be more potent than wild-type WE14 in 202

stimulating interleukin-2 (IL-2) production by a diverse set of ChgA-reactive T cells. 203

Subsequently, a hybrid insulin peptide (HIP) epitope generated by covalently cross-linking a 204

fragment of proinsulin C-peptide to the N-terminal region of WE14 (see below) greatly 205

increases activation of the ChgA-reactive T cell clone BDC-2.5 compared with unmodified 206

WE14 [40]. This discovery sparked an interest in HIP formation as a potential pathway to 207

generate a novel class of non-conventional epitopes relevant to autoimmune diabetes. 208

HIPs described to-date derive from the fusion of proinsulin C-peptide to peptide(s) derived 209

from other pancreatic β-cell proteins. It is likely that hybrid formation is favoured in a chemically 210

hyper-dense environment e.g. within the β-cell secretory granule, where large quantities of 211

polypeptide cargoes are present. This is supported by the findings of Delong et al [40] where 212

hybrid peptides are identified from different peptide species which reside within the islet 213

granules. Using a panel of BDC T cell clones from NOD mice, two hybrid peptides formed by 214

the fusion of C-peptide with WE14 or IAPP2 (islet amyloid polypeptide propeptide 2) were 215

10

capable of stimulating IFN-γ secretion. Importantly, a stronger T cell response was observed 216

against the HIP compared with the native non-modified epitope. These data suggest that HIPs 217

represent the natural ligands for these pathogenic T cell clones, which have previously been 218

described to have defined reactivities. The HIP containing the DLQTLAL C-peptide sequence 219

and the WSRM WE14 sequence has been identified by mass spectrometry from the granule 220

fraction of an islet β-cell tumour (insulinoma). Intriguingly, T cell reactivity has been found 221

against these HIPs from T cells isolated from pancreatic islets. These T cells recognise HIPs 222

formed by the fusion of C-peptide to neuropeptide Y (NPY) and IAPP2 but respond weakly to 223

native C-peptide. Additionally, these T cells could only respond to HIPs presented by antigen-224

presenting cells (APCs) expressing HLA-DQ8, hinting that hybrid peptides may offer an 225

explanation for the high risk associated with HLA-DQ haplotypes in T1D. 226

These findings have been further supported by a recent paper by Babon et al [47]. In this study 227

CD4+ T cell lines grown directly from islets from cadaveric donors with T1D where shown to 228

secrete IFN-γ in response to some of the same hybrid peptide epitopes described by Delong 229

et al [40] and discussed in the previous section. Reactivity was found against HIPs comprising 230

C-peptide fused with insulin A-chain, IAPP1 (islet amyloid polypeptide propeptide 1) and 231

IAPP2. However, it is unknown whether these T cell lines also cross-react with native peptides 232

or whether the response is comparable between hybrid and wild-type sequences. Wiles et al 233

[48] have progressed the work by using truncated or sequence-modified insulin-IAPP hybrid 234

peptides to derive a minimal epitope for the IAPP-specific BDC-6.9 clone and investigate 235

residues critical for antigenicity. The ability of insulin-IAPP hybrid peptides to induce T cell 236

activation appears to arise from residues in both the insulin- and IAPP- components, as hybrid 237

peptides truncated at the N- or C-termini abrogate IFN-γ production by the T cell clone. 238

Furthermore, amino acid substitution revealed that an amino acid with a negatively charged 239

side chain at the C-terminus may be favourable for activation of the BDC-6.9 clone. Evidence 240

exists that IA-g7, similar to human HLA-DQ8 molecules, has a preference for binding peptides 241

that contain negatively charged amino acids towards their C-terminus [49]. Optimal binding of 242

11

HLA exists when these hybrid peptides contain negatively charged residues at key anchor 243

residues. In summary, since their discovery, HIPs and HIP-reactive T cells have opened a 244

new scope of research in T1D which challenges our current dogma of disease pathogenesis. 245

There are some caveats that should be considered before the disease model in humans can 246

be unequivocally revised. First, evidence for the existence of HIPs at a molecular level is 247

largely derived from studies in the NOD mouse and was derived using granules from an 248

insulinoma cell line. Whether this represents a physiological process and whether naturally 249

processed and presented epitopes (NPPEs) can be generated by MHC class II and class I 250

antigen processing pathways is unknown at this stage. Importantly, it is yet to be reported at 251

the molecular level that HIPs exist in vivo in humans; because of TCR promiscuity [50] the 252

responses of T cell clones to HIPs, whilst compelling, is not proof that these are the primary 253

immunogens. If we take the position that HIPs are generated in vivo in humans, then several 254

questions regarding their interaction with the adaptive immune system arise. It may be the 255

case that an autoreactive T cell response is initially made against conventional epitopes of 256

WE14 and/or C-peptide, which in and of itself has insufficient potency to drive pathological 257

autoimmunity. However, encounter with the relevant HIP (non-conventional epitope) results in 258

a super-agonist effect resulting in pathogenic, disease-driver clones (Figure 1). This would 259

imply a 2-stage process of failed tolerance followed by a “super-charging” event when non-260

conventional epitopes are formed, for example during local inflammation. Alternatively, the 261

initial autoreactive response is due to the priming of T cells to non-conventional epitopes de 262

novo (tolerance breaker); once activated via this route, these T cells may also cross-react with 263

natural germline epitope sequences, allowing them a more abundant source of signal 1 (TCR 264

engagement). In either event, it remains to be established whether HIP formation confers 265

enhanced HLA binding, TCR recognition, or both over the wild-type sequences. 266

267

268

12

Spliced peptides 269

An additional and intriguing new area of non-conventional epitope generation is related to 270

spliced peptides, generated via the fusion of two peptide fragments resulting in a novel non-271

germline encoded sequence. In contrast with HIPs which are constituted by two different 272

peptide species, spliced peptides that have been described to-date are characterized by the 273

joining of peptide fragments from a single protein substrate (cis-spliced peptides). The 274

existence of trans-spliced peptides (joining of peptide fragments from two distinct protein 275

species) within cells remains unclear – absence of evidence is not evidence of absence. 276

Peptides generated by cis-splicing have been reported as a cancer phenomenon to give rise 277

to novel class I restricted epitopes. Splicing has been shown to be a post-translational event 278

[51] which occurs by the joining of two peptides end-to-end [52] or in the reverse order to that 279

which occurs in the parental protein (reverse splicing) [53]. 280

Recent data suggest that cis-splicing of peptides is not a rare event, with proteasome-281

generated spliced peptides being highly abundant in the human peptidome. Liepe et al [41] 282

analysed the peptidome of HLA class I molecules (immunopeptidome) on cell lines (GR-LCL 283

and C1R) and human primary fibroblasts by mass spectrometry. Traditionally, mass 284

spectrometry data are queried against a human proteome database, which do not contain 285

spliced peptides. Using an algorithm to generate a database containing spliced peptides, the 286

authors show that up to one-third of unique identified epitopes are derived from spliced 287

species. These epitopes make up one-fourth of the total amount of peptides presented on HLA 288

class I molecules, suggesting conventional and spliced epitopes are generated with 289

comparable efficiency. Peptide splicing can also occur between two splice-reactants 290

originating from two distinct proteins (trans-spliced peptides) [54]; however, due to limitations 291

of computational power the authors focused on the identification of cis-spliced peptides, with 292

algorithm-based discovery of trans-splicing remaining a future target for discovery. 293

Peptide splicing does not appear to occur at random but follows distinct rules whereby distinct 294

peptide motifs either promote or abolish peptide joining. Splicing of longer peptides 295

13

predominantly occurs via cis-splicing and shorter peptides also allow for trans-splicing [55]. 296

This implies that the splicing process is regulated. It is therefore conceivable that spliced 297

epitopes are available for thymic education although these species may not be generated as 298

efficiently in the thymus. Peptide splicing can be considered beneficial for the immune system, 299

particularly during bacterial or viral infections when the generation of spliced epitopes would 300

considerably increase the number of antigen-derived epitopes that can be targeted by 301

adaptive responses [41]. Furthermore, spliced peptides have been reported to be important 302

for T cell responses in cancer with 50% of melanoma patients harbouring CTLs in peripheral 303

blood that secrete IFN-γ in response to two spliced epitopes of the melanocyte protein 304

gp100mel [56]. In summary, spliced peptides represent a new area of immunobiology, of as yet 305

unknown relevance to autoimmunity and T1D. Particularly under inflammatory conditions, e.g. 306

stressed β-cells might have an upregulated proteasomal degradation pathway which may, in 307

turn, enhance generation and presentation of spliced peptides. 308

Mechanisms for the generation of hybrid and spliced peptides 309

The mechanism(s) governing the generation of hybrid peptides in vivo are currently unknown 310

with some arguing that it represents an example of transpeptidation [40]. Transpeptidation is 311

the process of two peptides being fused together to form a hybrid peptide [57]. The current 312

hypothesis is that these hybrid peptides form in areas with high enzymatic activity and high 313

protein concentrations. A prime example would be the secretory granule of the β-cell where 314

high concentrations of insulin, ChgA, IAPP and other β-cell proteins (molecular crowding) exist 315

and favour this reaction [40]. To what extent the process is catalysed by enzymes or occurs 316

spontaneously within the granule is currently unknown. Given that the proteasome has been 317

described to generate epitopes by transpeptidation (see below), there may be an opportunity 318

for hybrid peptides to be generated within the proteasome under conditions of endoplasmic 319

reticulum (ER) stress or protein mis-folding. 320

321

14

Spliced peptides are generated via a proteasome-dependent mechanism. Previously, in a 322

tumour setting, only five spliced epitopes have been described [51-53, 58, 59] each of which 323

is produced by the proteasome by a transpeptidation reaction [52, 54, 58, 60]. In contrast, 324

Liepe et al [41] were able to identify thousands of proteasome-generated spliced peptides, 325

suggesting this pathway is a major source of antigenic epitopes. Although this phenomenon 326

has only been described for HLA class I, it may also occur for HLA class II but perhaps to a 327

lesser extent and via different mechanisms (e.g. endosome- or lysosome-based). For 328

example, if spliced peptides are available for loading into HLA class I molecules, a reservoir 329

may be present within the cell cytoplasm. In the event of cell death, this reservoir of spliced 330

peptides can be taken up by APCs for processing and presentation via HLA class II molecules. 331

Similar to observations in the cancer field, other modes for generation of non-germline 332

encoded peptide sequences might also exist. A predominant feature of cancer cells is the 333

existence of translocation, interstitial deletion, or chromosomal inversion of genes to generate 334

hybrid/fusion genes [61]. Research is ongoing to identify novel tumour epitopes generated by 335

point mutations, transcriptional variation and/or gene translocation and if these can be 336

selectively targeted by cancer immunotherapy [62]. CD8+ T cells associated with tumour 337

regression following checkpoint blockade cancer immunotherapy target mutation-produced 338

neo-epitopes [63] and such neo-epitopes have also proved to be effective therapeutic 339

vaccines for melanoma [64]. Cis and trans-splicing of RNA to generate chimeric RNA also 340

results in non-germline sequences in cancer cells (trans-splicing describes the joining of exons 341

from two different pre-mRNA transcripts, while cis-splicing corresponds to splicing of exons 342

within a single pre-mRNA transcript) [65]. There are no reports yet that similar mechanisms 343

operate in the setting of autoimmunity, but there is the theoretical possibility that stressed or 344

damaged endocrine cells could generate hybrid peptides via trans-splicing of RNA, and this 345

mechanism may yet need to be excluded. 346

347

348

15

The relevance of defective ribosomal products (DRiPs) 349

Under steady state, mis-folded/non-functional proteins are generated despite translation from 350

the correct start codon. Such proteins, termed defective ribosomal products (DRiPs) are 351

directed to the proteasome for degradation and generation of epitopes for HLA class I 352

presentation [66]. Alternatively, a form of DRiPs also occurs when translation is initiated from 353

out-of-frame start codons, translating an entirely new amino acid sequence [67]. Evidence 354

suggests that these pathways give rise to non-conventional class I epitopes in several human 355

diseases, notably cancer and viral infections such as influenza, optimizing opportunities for 356

immune surveillance of infected/tumour cells [68]. 357

Such an example of this different form of DRiP has been recently showed by Kracht et al [42] 358

to give rise to class I and class II epitopes relevant in T1D. Translation from an alternative start 359

site within the preproinsulin mRNA determines an alternative open reading frame which lacks 360

a stop codon. The result is translation of an amino acid sequence which is different from 361

conventional preproinsulin, generating a defective ribosomal insulin gene product (INS-DRiP) 362

(Figure 2). An epitope from this INS-DRiP is presented on HLA-A*02:01 and HLA-DQ8 363

molecules, with CTLs specific for this epitope shown to kill human β-cells in vitro. Additionally, 364

expression of this INS-DRiP polypeptide is increased under conditions of ER stress (IL-1β and 365

IFN-γ) and high levels of glucose, shown by enhanced β-cell killing. It will be important to 366

establish the timing of responses to this peptide in the natural history of T1D, its relationship 367

to responses to native sequences and whether central tolerance is established against these 368

DRiPs. 369

Implications of non-conventional epitope generation for type 1 diabetes 370

The studies discussed above invoke a timely reconsideration of the targeting of self in 371

autoimmune diseases such as T1D. The study of Delong et al [40] identifies hybrid peptides 372

using an insulinoma cell line which, given that this is neoplastic, may suggest hybrid peptide 373

generation is favoured by the abnormal metabolic behaviour of tumour cells. However, 374

16

chimeric RNAs have been found to be present in non-cancer tissues and cells and are not 375

unique to cancer [69]. A key question will be whether cis and trans-spliced RNA molecules 376

have a physiological and/or tumour-promoting function to provide a rationale for their 377

generation. Evidence exists that these fusion RNA species are functional and are required for 378

cellular activities [69]. Although currently unexplored, hybrid peptides generated via this route 379

could indeed have a physiological role in β-cells. 380

Beyond germline, we can further conclude that cells have evolved to generate non-germline 381

encoded peptides and epitope sequences, whether this be essentially random, whereby two 382

peptides fuse under mass and/or location bias or whether it is a proteasome-catalysed event. 383

At the level of the proteasome this process appears regulated with regard to the partners that 384

are fused, as has been demonstrated for HLA-A*02:01 [55]. At this stage, the HLA-A*02:01 385

restricted splicing pattern published by Berkers et al [55] differs from that of Liepe et al [41] 386

probably as a reflection of technical and methodological differences. Yet to be elucidated is 387

whether presentation of cis-spliced epitopes is restricted to specific HLA class I molecules. If 388

so, the large diversity and abundance of spliced peptides may increase the possibility of 389

antigenic peptides with sequences which extend the human proteome, with clear implications 390

for autoimmune diseases, especially those such as T1D with HLA class I allele risk 391

associations [8]. Whether this phenomenon has cell or tissue restriction, and especially 392

whether spliced epitopes with the correct binding register are generated in the thymus to 393

induce central tolerance will also need to be explored. 394

Consideration will also turn to the role of non-conventional epitopes in T cell selection in the 395

thymus. Animal studies utilising NOD mice with a genetic deletion in both insulin genes but 396

expressing a mutant proinsulin, highlight the requirement for central tolerance to non-germline 397

epitopes [70]. The mutant proinsulin contains a tyrosine to alanine amino acid substitution at 398

residue 16 of the insulin B-chain, termed B16:A-dKO. In the B16:A-dKO mice, T cells were 399

readily found following insulin immunisation and showed a higher response to the insulin 400

molecule compared to T cells from NOD wild-type mouse. This suggests in the B16:A-dKO 401

17

mice, T cells which recognise the normal amino acid at the position where the substitution is 402

located (residue 16), are not negatively selected and escape thymic deletion. Therefore, 403

following priming with insulin these T cells are activated. Indeed, evidence indicates the T cell 404

response in the B16:A-dKO mice is directed against the region of insulin where the substitution 405

lies. Overall, this finding supports the notion that there is a failure to eliminate the T cell 406

repertoire reactive against non-germline encoded epitopes. 407

Knowledge of how PTM of antigens/autoantigens enhance either peptide:MHC and/or 408

pMHC:TCR interactions, constitutes a base from which to reflect on how non-conventional 409

epitopes lead to T cell activation. Although PTM of peptide epitopes has been reported to have 410

various effects [70], these typically include enhanced binding of the modified peptide to MHC 411

molecules (see above) [24, 25]. In addition, a second effect of PTM of peptides is that it will 412

activate a more diverse and different T cell repertoire than the germline sequence alone [71]. 413

Furthermore, these activated T cells express TCRs with high avidity for peptide-MHC 414

complexes [72]. In the case of non-conventional epitopes, poor or absent thymic expression 415

results in a lack of thymic deletion, and a potentially richer, more diverse T cell repertoire in 416

the periphery [73]. It could be argued such a T cell repertoire is more likely to contain high 417

affinity TCRs to the non-conventional epitope, leading to enhanced potential for autoreactivity 418

(Figure 3). PTM could also give rise to a heteroclitic effect, whereby the modifications in non-419

conventional epitopes contain HLA anchor residues that have greater binding optimization and 420

enhance peptide affinity for HLA and/or improve TCR recognition [74, 75]. Indeed, numerous 421

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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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

granule. DRiP, defective ribosomal product; HIP, hybrid insulin peptide. 731

Figure 3: Activation of autoreactive T cells and the dysfunction of regulatory T cells by 732

non-conventional epitopes. Treg cells are able to effectively suppress T cell responses 733

against conventional (native) epitopes at steady state (left). In an autoimmune activation state 734

(right), a large T cell pool reactive against non-conventional epitopes is present in the 735

periphery due to a lack of thymic education. High affinity TCRs exist within the large TCR 736

repertoire, resulting in increased activation of autoreactive T cells. A diminished pool of thymic-737

derived Tregs is resident in the periphery due to low or absent expression of non-conventional 738

epitopes in the thymus. Teff, effector T cell; APC, antigen presenting cell; Treg, regulatory T 739

cell; TCRs, T cell receptors. 740

741

27

742

28

743

29

744