Translational Mini-Review Series on Type 1 Diabetes:
Systematic analysis of T cell epitopes in autoimmune diabetes

Authors


  • Editor: Mark Peakman

Teresa P. Di Lorenzo PhD, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, USA.
E-mail: dilorenz@aecom.yu.edu

Summary

T cell epitopes represent the molecular code words through which the adaptive immune system communicates. In the context of a T cell-mediated autoimmune disease such as type 1 diabetes, CD4 and CD8 T cell recognition of islet autoantigenic epitopes is a key step in the autoimmune cascade. Epitope recognition takes place during the generation of tolerance, during its loss as the disease process is initiated, and during epitope spreading as islet cell damage is perpetuated. Epitope recognition is also a potentially critical element in therapeutic interventions such as antigen-specific immunotherapy. T cell epitope discovery, therefore, is an important component of type 1 diabetes research, in both human and murine models. With this in mind, in this review we present a comprehensive guide to epitopes that have been identified as T cell targets in autoimmune diabetes. Targets of both CD4 and CD8 T cells are listed for human type 1 diabetes, for humanized [human leucocyte antigen (HLA)-transgenic] mouse models, and for the major spontaneous disease model, the non-obese diabetic (NOD) mouse. Importantly, for each epitope we provide an analysis of the relative stringency with which it has been identified, including whether recognition is spontaneous or induced and whether there is evidence that the epitope is generated from the native protein by natural antigen processing. This analysis provides an important resource for investigating diabetes pathogenesis, for developing antigen-specific therapies, and for developing strategies for T cell monitoring during disease development and therapeutic intervention.

T cell reactivity in type 1 diabetes

It is now widely accepted that type 1 diabetes is an autoimmune disease associated with the activation of CD4 and CD8 T cells recognizing islet autoantigens [1,2]. It is considered likely by many that these autoreactive T cells are the mediators of islet β cell damage, although direct evidence for this is compelling only in rodent models of the disease in which adoptive transfer experiments are feasible ethically and technically. Epitopes represent the molecular code words through which the adaptive immune system generates cellular immunity with specificity and memory; islet autoantigen epitopes represent the targets of regulatory and effector T cells that preside over the fate of the β cell. Because type 1 diabetes is a relatively common disorder with several representative animal models, and because some of the major autoantigens in humans and rodents have been well characterized, spontaneous diabetes has come to be considered as the prototypic cell-mediated autoimmune disease.

The considerable advances in autoimmune serology in type 1 diabetes during the period 1975–91 led to the unequivocal identification of three major islet autoantibody specificities, targeted against insulin, glutamic acid decarboxylase (GAD) and the islet tyrosine phosphatase, IA-2 [3–5]. Not surprisingly, most T cell epitope studies have focused on these three autoantigens; the existence of class-switched IgG autoantibodies against these particular islet proteins strongly implies the influence of T cell help. From the 1990s onwards, increasing numbers of reports noted the detection of T cells directed against these three proteins in the peripheral blood, leading to an increasing emphasis on epitope discovery. To date, even a relatively partial unravelling of the epitope code in type 1 diabetes has enabled the characterization of T cell responses and some key research advances. These include the demonstration that human type 1 diabetes may be Th1-dominated [6,7]; identification of a previously unrecognized population of naturally arising CD4 T cells producing interleukin (IL)-10 and associated with late disease onset [6]; evidence for T cell cross-reactivity between β cell antigens and common pathogens [8]; identification of CD8 T cells associated with recurrent autoimmunity after islet transplantation [9]; the first steps towards developing T cell assays for standardization and use in trial monitoring [10]; and the development of epitope-based intervention strategies [11].

In light of these advances, this review is designed to take stock of the large portfolio of epitopes identified to date, and examine their cellular source and major histocompatibility complex (MHC) restriction. Our review highlights the question of what truly constitutes a T cell epitope, and seeks to provide some indication as to the likelihood that particular epitopes may represent useful research and therapeutic tools, by categorizing the evidence that led to their identification.

Epitope discovery

The starting-points for Tables 1–4 were previous listings of diabetes-relevant T cell epitopes compiled by our groups [12,13]. These previous compilations have been supplemented here by the results of web-based searches of PubMed (the US National Library of Medicine's database of biomedical citations and abstracts) conducted in April 2006 using the search terms ‘diabetes, T cell, epitope, antigen’ or ‘diabetes, T cell, peptide, antigen’. Peptides listed as T cell epitopes for mouse β cell antigens in Tables 2 and 4 are those reported to be recognized by T cells from non-obese diabetic (NOD) mice either spontaneously or as a consequence of peptide or protein immunization. Peptides listed as T cell epitopes for human β cell antigens in Tables 1 and 3 are those shown to be recognized by T cells from type 1 diabetes patients and/or at-risk individuals, or those recognized by human leucocyte antigen (HLA)-restricted T cells in HLA-transgenic mice (not necessarily of the NOD background). For epitopes reported multiple times by the same group, only a single reference is provided in Tables 1–4. However, for epitopes reported by more than one group, all are credited. In this way, it can be seen readily which epitopes have been verified by multiple groups. While the epitope listings are intended to be comprehensive, the authors will welcome notification of inadvertent omissions.

Table 1.  CD4 T cell epitopes for human β cell antigens.
AgaPositionSequenceType of T cell responseLevel of evidencecCommentsRef.
MHCHumanHLA Tg miceb
Clone or linePBMCControlsd
  • a

    GAD, glutamic acid decarboxylase; HSP, heat shock protein; IA-2, insulinoma-associated protein 2; ICA, islet cell autoantigen; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein.

  • b

    b Pept-imm, peptide-immunized; Prot-imm, protein-immunized.

  • c

    A, T cell clone, line, or hybridoma [human or human leucocyte antigen (HLA) Tg mouse] exists that responds to peptide and whole protein; human peripheral blood mononuclear cells (PBMCs) respond. B, other evidence of epitope presentation from whole protein (e.g. elution); human PBMCs respond. C, T cell clone, line, or hybridoma (human or HLA Tg mouse) exists that responds to peptide and whole protein; no positive human PBMC data. D, T cell clone, line, or hybridoma (human or HLA Tg mouse) or human PBMCs or T cells from HLA Tg mice respond to peptide; no evidence of epitope presentation from whole protein.

  • d

    d 1: Responses from cases were increased compared to normal controls or differed qualitatively from control responses. 2: Cases and normal controls responded similarly. 3: No normal controls were examined, or differences between cases and controls were unclear. MHC: major histocompatibility complex.

GAD6588–99NYAFLHATDLLPDR1Yes 3 C [43]
101–115CDGERPTLAFLQDVMDQ8 Yes2Prot-immA [44]
115–127MNILLQYVVKSFDDR4   Pept-immC [45]
115–130MNILLQYVVKSFDRSTDR4   Prot-immC [46]
116–127NILLQYVVKSFDDR53Yes 1 C [47]
121–140YVVKSFDRSTKVIDFHYPNEDQ8   Prot-immD [48]
126–140FDRSTKVIDFHYPNEDQ8 Yes2Prot-immA [44]
146–165NWELADQPQNLEEILMHCQTDR2Yes 3 C [49]
173–187TGHPRYFNQLSTGLDDQ8 Yes3 D [50]
174–185GHPRYFNQLSTGDR2Yes 3 C [49]
201–220NTNMFTYEIAPVFVLLEYVTDQ8   Prot-immD [48]
202–216TNMFTYEIAPVFVLLDR8 or DR9Yes 1 D [47]
206–220TYEIAPVFVLLEYVTDQ8 Yes2Prot-immA [44]
206–225TYEIAPVFVLLFYVTLKKMRDR2Yes 3 C [49]
231–250PGGSGDGIFSPGGAISNMYADQ8   Prot-immD [48]
247–266NMYAMMIARFKMFPEVKEKGDQ and/or DR Yes1, 2 D [51,52]
247–266NMYAMMIARFKMFPEVKEKGDR3YesYes3 D [53]
248–257MYAMMIARFKDR51Yes 3 C [43]
251–270MMIARFKMFPEVKEKGMAALDR12YesYes3 D [53]
260–279PEVKEKGMAALPRLIAFTSEDQ and/or DR Yes1, 2 D [51,52]
261–280EVKEKGMAALPRLIAFTSEHDQ8YesYes3 D [53]
270–283LPRLIAFTSEHSHFDR4Yes 3 C [49]
271–285PRLIAFTSEHSHFSLDR4   Prot-immC [46]
274–286IAFTSEHSHFSLKDR4   Pept-immC [45]
339–352TVYGAFDPLLAVADDR3YesYes3 A [54]
356–370KYKIWMHVDAAWGGGDR4   Prot-immC [46]
370–386GLLMSRKHKWKLSGVERDP2Yes 1 D [47]
376–390KHKWKLSGVERANSVDR4   Prot-immC [46]
417–429NCNQMHASYLFQQDR1Yes 1 C [47]
431–445KHYDLSYDTGDKALQDQ8 Yes2Prot-immA [44]
461–475AKGTTGFEAHVDKCLDQ8 Yes2Prot-immA [44]
471–490VDKCLELAEYLYNIIKNREGDQ8   Prot-immD [48]
481–495LYNIIKNREGYEMVFDR4   Prot-immC [46]
491–510YEMVFDGKPQHTNVCFWYIPDR3 and DQ5YesYes3 D [53]
493–507MVFDGKPQHTNVCFWDQ8 Yes3 D [50]
501–520HTNVCFWYIPPSLRTLEDNEDR1 and DR4YesYes3 D [53]
505–519CFWYIPPSLRTLEDNDQ1 or DR1YesYes2 D [55]
505–519CFWYIPPSLRTLEDNDQ8YesYes3Pept-immD [50]
506–518FWYIPPSLRTLEDDQ and/or DR Yes1 D [56]
511–525PSLRTLEDNEERMSRDR4   Prot-immC [46]
511–530PSLRTLEDNEERMSRLSKVADR3YesYes3 D [53]
521–535ERMSRLSKVAPVIKADQ8YesYes3Pept-immDGreater response in diabetic compared to non-diabetic twins[50]
521–535ERMSRLSKVAPVIKADQ8 or DR4YesYes1 D [55]
533–547IKARMMEYGTTMVSYDQ8 Yes3 D [50]
536–550RMMEYGTTMVSYQPLDQ8 Yes2Prot-immA [44]
546–560SYQPLGDKVNFFRMVDR4   Prot-immC [46]
551–565GDKVNFFRMVISNPADR4   Prot-immC [46]
555–567NFFRMVISNPAATDR4YesYes1 BEluted from MHC[57,58]
556–570FFRMVISNPAATHQDDR4   Prot-immC [46]
563–575NPAATHQDIDFLIDR53Yes 3 C [59]
566–580ATHQDIDFLIEEIERDR4   Prot-immC [46]
HSP6031–50KFGADARALMLQGVDLLADA? Yes3 D [60]
136–155NPVEIRRGVMLAVDAVIAEL? Yes3 D [60]
255–275QSIVPALEIANAHRKPLVIIA? Yes3 D [60]
286–305LVLNRLKVGLQVVAVKAPGF? Yes3 D [60]
436–455IVLGGGCALLRCIPALDSLT? Yes3 D [60]
437–460VLGGGCALLRCIPALDSLTPANED? Yes1 D [60]
466–485EIIKRTLKIPAMTIAKNAGV? Yes1 D [60]
511–530VNMVEKGIIDPTKVVRTALL? Yes3 D [60]
HSP701–20MAKAAAVGIDLGTTYSCVGV? Yes3 D [61]
166–185GLNVLRIINEPTAAAIAYGL? Yes3 D [61]
210–229TIDDGIFEVKATAGDTHLGG? Yes3 D [61]
225–244THLGGEDFDNRLVNHFVEEF? Yes3 D [61]
271–290KRTLSSSTQASLEIDSLFEG? Yes3 D [61]
391–410LLLLDVAPLSLGLETAGGVM? Yes3 D [61]
421–440PTKQTQIFTTYSDNQPGVLI? Yes3 D [61]
496–515KANKITITNDKGRLSKEEIE? Yes3 D [61]
511–530KEEIERMVQEAEKYKAEDEV? Yes3 D [61]
IA-2502–514GSFINISVVGPAL? Yes2 D [62]
575–587RSVLLTLVALAGV? Yes2 D [62]
601–618RQHARQQDKERLAALGPEDQ8   Pept-immD [63]
608–620DKERLAALGPEGA? Yes3 D [64]
616–633GPEGAHGDTTFEYQDLCRDQ8   Pept-immD [63]
646–663EGPPEPSRVSSVSSQFSDDQ8   Pept-immD [63]
654–674VSSVSSQFSDAAQASPSSHSSDR4 Yes1 BEluted from MHC[7]
661–678FSDAAQASPSSHSSTPSWDQ8   Pept-immD [63]
685–700ANMDISTGHMILAYMEDR4–DQ8 Yes3 D [65]
709–732LAKEWQALCAYQAEPNTCATAQGEDR4 Yes1 BEluted from MHC[7]
713–728WQALCAYQAEPNTCATDR4–DQ8 Yes3 D [65]
721–738AEPNTCATAQGEGNIKKNDQ8   Pept-immD [63]
745–760PYDHARIKLKVESSPSDR4–DQ8 Yes3 D [65]
751–770IKLKVESSPSRSDYINASPIDR4Yes 3 C [66]
752–775KLKVESSPSRSDYINASPIIEHDPDR4 Yes1 BEluted from MHC[6]
766–783NASPIIEHDPRMPAYIATDQ8   Pept-immD [63]
787–802LSHTIADFWQMVWESGDR4–DQ8 Yes3 D [65]
793–808DFWQMVWESGCTVIVMDR4–DQ8 Yes3 D [65]
797–809MVWESGCTVIVML? Yes3 D [64]
797–817MVWESGCTVIVMLTPLVEDGVDR4 Yes1 BEluted from MHC[7]
799–814WESGCTVIVMLTPLVEDR3–DQ2; DR4–DQ8 Yes3 D [65]
804–816TVIVMLTPLVEDG? Yes3 D [64]
805–820VIVMLTPLVEDGVKQCDR3–DQ2; DR4–DQ8 Yes3 D [65]
826–843DEGASLYHVYEVNLVSEHDQ8   Pept-immD [63]
830–842SLYHVYEVNLVSE? Yes2 D [62]
831–850LYHVYEVNLVSEHIWCEDFLDPB4·1YesYes3 A [66]
841–856SEHIWCEDFLVRSFYLDR3–DQ2; DR4–DQ8 Yes3 D [65]
841–860SEHIWCEDFLVRSFYLKNVQDPB4·1YesYes3 A [66]
845–860WCEDFLVRSFYLKNVQDR4–DQ8 Yes3 D [65]
847–862EDFLVRSFYLKNVQTQDR3–DQ2; DR4–DQ8 Yes3 D [65]
854–866FYLKNVQTQETRT? Yes3 D [64]
854–872FYLKNVQTQETRTLTQFHFDR4 Yes1 BEluted from MHC[7]
889–904DFRRKVNKCYRGRSCPDR4–DQ8 Yes3 D [65]
918–930TYILIDMVLNRMA? Yes2 D [62]
919–934YILIDMVLNRMAKGVKDR3–DQ2; DR4–DQ8 Yes3 D [65]
931–948KGVKEIDIAATLEHVRDQDQ8   Pept-immD [63]
933–945VKEIDIAATLEHV? Yes3 D [64]
955–975SKDQFEFALTAVAEEVNAILKDR4 Yes1 BEluted from MHC[7]
959–974FEFALTAVAEEVNAILDR4–DQ8 Yes3 D [65]
961–979FALTAVAEEVNAILKALPQDQ8   Pept-immD [63]
ICA6936–47AFIKATGKKEDE? Yes1 D [67]
IGRP13–25QHLQKDYRAYYTFDR3Yes 2 D [68]
23–35YTFLNFMSNVGDPDR4Yes 2 D [68]
226–238RVLNIDLLWSVPIDR3Yes 2 D [68]
247–259DWIHIDTTPFAGLDR4Yes 2 D [68]
InsulinL1–16MALWMRLLPLLALLAL? Yes3 D [69]
L1–24MALWMRLLPLLALLALWGPDPAAADQ8   Pept-immD [70]
L5–20MRLLPLLALLALWGPD? Yes3 D [69]
L9–24PLLALLALWGPDPAAA? Yes3 D [69]
L11–B2LALLALWGPDPAAAFVDR4   Prot-immC [71]
L13–B4LLALWGPDPAAAFVNQ? Yes3 D [69]
L17–B8WGPDPAAAFVNQHLCG? Yes3 D [69]
L21–B12PAAAFVNQHLCGSHLVDR4   Prot-immC [71]
L21–B12PAAAFVNQHLCGSHLV? Yes3 D [69]
B1–17FVNQHLCGSHLVEALYL? Yes1 D [72]
B6–22LCGSHLVEALYLVCGER? Yes3 D [69]
B9–23SHLVEALYLVCGERGDQ8 Yes1 D [73]
B10–25HLVEALYLVCGERGFF?Yes 3 D [74]
B11–27LVEALYLVCGERGFFYTDR16Yes 3 C [75]
B11–27LVEALYLVCGERGFFYT? Yes1 D [72]
B14–30ALYLVCGERGFFYTPKT? Yes3 D [76]
B16–32YLVCGERGFFYTPKTRR? Yes3 D [69]
B20–C4GERGFFYTPKTRREAED? Yes3, 1 D [53,72]
B20–C7GERGFFYTPKTRREAEDLQVDQ8   Pept-immD [70]
B21–C5ERGFFYTPKTRREAEDL? Yes3 D [76]
B24–C4FFYTPKTRREAEDDQ and/or DR Yes1 D [56]
B25–C8FYTPKTRREAEDLQVG?Yes 3 D [74]
B25–C9FYTPKTRREAEDLQVGQ? Yes3 D [69]
B30–C14TRREAEDLQVGQVELGG? Yes1 D [72]
C3–18EDLQVGQVELGGGPGADRYes 3 D [74]
C3–19EDLQVGQVELGGGPGAG? Yes3 D [69]
C8–24GQVELGGGPGAGSLQPL? Yes1 D [72]
C13–29GGGPGAGSLQPLALEGS? Yes3 D [69]
C13–32GGGPGAGSLQPLALEGSLQKDR4 Yes1 BEluted from MHC[6]
C17–A1GAGSLQPLALEGSLQKRGDR4 Yes3Prot-immA [71]
C18–A1AGSLQPLALEGSLQKRG? Yes1 D [72]
C19–A3GSLQPLALEGSLQKRGIVDR4 Yes1 BEluted from MHC[6]
C22–A5QPLALEGSLQKRGIVEQDR4 Yes1 BEluted from MHC[6]
C23–A6PLALEGSLQKRGIVEQC? Yes3 D [69]
C24–A7LALEGSLQKRGIVEQCC? Yes3 D [76]
C28–A11GSLQKRGIVEQCCTSIC? Yes1 D [72]
C29–A12SLQKRGIVEQCCTSICSDR4   Prot-immC [71]
A1–13GIVEQCCTSICSLDR4Yes 1 C [77]
A1–15GIVEQCCTSICSLYQDR4Yes 3 DT cells from pancreatic lymph nodes[35]
A1–15GIVEQCCTSICSLYQ?Yes 3 D [74]
A1–16GIVEQCCTSICSLYQL? Yes3 D [69]
A6–21CCTSICSLYQLENYCN? Yes1 D [72]
Table 2.  CD4 T cell epitopes for mouse β cell antigens.
AgaPositionSequenceMHCType of T cell responsebLevel of evidencecRef.
Clone, line or hybridomaIslet T cellsSpleen or lymph nodes
  • a

    GAD, glutamic acid decarboxylase; HSP, heat shock protein; IA-2, insulinoma-associated protein 2; ICA, islet cell autoantigen; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein.

  • b

    b Pept-imm, peptide-immunized; Prot-imm, protein-immunized; Spont, spontaneous.

  • c

    c A: Evidence of epitope presentation from whole protein (e.g. T cell clone, line, or hybridoma exists that responds to peptide and whole protein; elution); spontaneous T cell response. B: No evidence of epitope presentation from whole protein; spontaneous T cell response. C: Evidence of epitope presentation from whole protein; no positive spontaneous T cell response data. D: No evidence of epitope presentation from whole protein; no positive spontaneous T cell response data. MHC: major histocompatibility complex.

GAD65202–221TNMFTYEIAPVFVLLEYVTLAg7  Spont; prot-imm; pept-immB[78]
206–220TYEIAPVFVLLEYVTAg7Spont; prot-imm Pept-immA[79,80]
217–236EYVTLKKMREIIGWPGGSGDAg7  Spont; prot-imm; pept-immB[78]
221–235LKKMREIIGWPGGSGAg7Prot-imm  C[80]
247–266NMYAMLIARYKMSPEVKEKGAg7  SpontB[81]
286–300KKGAAALGIGTDSVIAg7Spont; prot-imm  A[80]
401–415PLQCSALLVREEGLMAg7Prot-imm  C[80]
509–528VPPSLRTLEDNEERMSRLSKAg7  SpontB[81]
524–543SRLSKVAPVIKARMMEYGTTAg7Prot-imm Spont; pept-immA[30,79,81]
561–575ISNPAATHQDIDFLIAg7Prot-imm  C[80]
571–585IDFLIEEIERLGQDLAg7Prot-imm  C[82]
GAD6729–48DTWCGVAHGCTRKLGLKICGAg7  Spont; pept-immB[78]
44–62LKICGFLQRTNSLEEKSRLAg7  Spont; pept-immB[78]
HSP6076–95DGVTVAKSIDLKDKYKNIGAAg7  Pept-immD[83]
166–185EEIAQVATISANGDKDIGNIAg7  Pept-immD[83]
195–214RKGVITVKDGKTLNDELEIIAg7  Pept-immD[83]
361–380KGDKAHIEKRIQEITEQLDIAg7  Pept-immD[83]
437–460VLGGGCALLRCIPALDSLKPANEDAg7Prot-imm SpontA[84]
526–545RTALLDAAGVASLLTTAEAVAg7  Pept-immD[83]
541–560TAEAVVTEIPKEEKDPGMGAAg7  Pept-immD[83]
IA-2676–693PSWCEEPAQANMDISTGHAg7  Pept-immD[63]
691–708TGHMILAYMEDHLRNRDRAg7  Pept-immD[63]
706–723RDRLAKEWQALCAYQAEPAg7  Pept-immD[63]
751–768IKLKVESSPSRSDYINASAg7  Pept-immD[63]
766–783NASPIIEHDPRMPAYIATAg7  Pept-immD[63]
781–798IATQGPLSHTIADFWQMVAg7  Pept-immD[63]
961–979FALTAVAEEVNAILKALPQAg7  Pept-immD[63]
IA-2β640–659KLSGLGADPSADATEAYQELAg7Prot-imm Pept-immC[85]
755–777QREENAPKNRSLAVLTYDHASRIAg7Prot-imm Spont; pept-immA[85]
ICA6936–47AFIKATGKKEDEAg7  Spont; prot-imm; pept-immB[86]
IGRP4–22LHRSGVLIIHHLQEDYRTYAg7  Spont; pept-immB[87]
123–145WYVMVTAALSYTISRMEESSVTLAg7  Spont; pept-immB[87]
128–145TAALSYTISRMEESSVTLAg7  Spont; pept-immB[87]
195–214HTPGVHMASLSVYLKTNVFLAg7  Spont; pept-immB[87]
Insulin 1/2A7–21CTSICSLYQLENYCNAg7SpontSpont A[88]
Insulin 1L7–23FLPLLALLALWEPKPTQAg7  Pept-immD[89]
L20–B11KPTQAFVKQHLCGPHLAg7  Pept-immD[89]
B9–23PHLVEALYLVCGERGAg7SpontSpontPept-immA[89,90]
C15–30SPGDLQTLALEVARQKAg7SpontSpontPept-immB[89]
C21–A5TLALEVARQKRGIVDQAg7  Pept-immD[89]
Insulin 2L14–B6LFLWESHPTQAFVKQHLAg7  Pept-immD[89]
L20–B11HPTQAFVKQHLCGSHLAg7  Pept-immD[91]
B2–17VKQHLCGSHLVEALYLAg7SpontSpont B[89]
B9–23SHLVEALYLVCGERGAg7SpontSpontPept-immA[89,90]
B24–C1FFYTPMSRREAg7  Spont; prot-immB[92]
C15–32GPGAGDLQTLALEVAQQKAg7  Pept-immD[89]
Table 3.  CD8 T cell epitopes for human β cell antigens.
AgaPositionSequenceMHCType of T cell responseLevel of evidencecCommentsRef.
HumanHLA Tg miceb
Clone or linePBMCControlsd
  • a

    GAD, glutamic acid decarboxylase; IA-2, insulinoma-associated protein 2; IAPP, islet amyloid polypeptide; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein.

  • b

    b Pept-imm, peptide-immunized; Spont, spontaneous.

  • c

    A: T cell clone, line, or hybridoma [human or human leucocyte antigen (HLA) Tg mouse] exists that responds to peptide and whole protein; human peripheral blood mononuclear cells (PBMCs) respond. B: Other evidence of epitope presentation from whole protein (e.g. elution); human PBMCs respond. C: T cell clone, line, or hybridoma (human or HLA Tg mouse) exists that responds to peptide and whole protein; no positive human PBMC data. D: T cell clone, line, or hybridoma (human or HLA Tg mouse) or human PBMCs or T cells from HLA Tg mice respond to peptide; no evidence of epitope presentation from whole protein.

  • d

    d 1: Responses from cases were increased compared to normal controls or differed qualitatively from control responses. 2: Cases and normal controls responded similarly. 3: No normal controls were examined, or differences between cases and controls were unclear. MHC: major histocompatibility complex.

GAD65114–123VMNILLQYVVA2Yes 1 C [93]
IA-2797–805MVWESGCTVA2Yes 2 D [94]
IAPP (prepro)5–13KLQVFLIVLA2 Yes1 D [95]
IGRP265–273VLFGLGFAIA2   SpontD [96]
InsulinB9–18SHLVEALYLVA2   Pept-immC [14]
B10–18HLVEALYLVA2   Pept-immC [14]
B10–18HLVEALYLVA2 Yes1 BGenerated by proteasome[15]
B10–18HLVEALYLVA2YesYes3 BGenerated by proteasome; translocated by TAP; response correlated with islet graft failure[9]
B14–22ALYLVCGERA3, A11 Yes3 BPrecursor generated by proteasome[15]
B15–23LYLVCGERGA24 Yes1 D [97]
B15–24LYLVCGERGFA24 Yes3 BPrecursor generated by proteasome[15]
B17–26LVCGERGFFYA1, A3, A11 Yes1 BPrecursor generated by proteasome[15]
B18–27VCGERGFFYTA1, A2, B8, B18 Yes1 BPrecursor generated by proteasome[15]
B20–27GERGFFYTA1, B8 Yes1 BGenerated by proteasome[15]
B21–29ERGFFYTPKA3 Yes3 BPrecursor generated by proteasome[15]
B25–C1FYTPKTRREB8 Yes1 BGenerated by proteasome[15]
B27–C5TPKTRREAEDLB8 Yes2 BPrecursor generated by proteasome[15]
C20–28SLQPLALEGA2   Pept-immC [14]
C25–33ALEGSLQKRA2   Pept-immD [14]
C29–A5SLQKRGIVEQA2   Pept-immC [14]
A1–10GIVEQCCTSIA2   Pept-immC [14]
A12–20SLYQLENYCA2   Pept-immC [14]
Table 4.  CD8 T cell epitopes for mouse β cell antigens.
AgaPositionSequenceMHCType of T cell responsebLevel of evidencecCommentsRef.
Clone, line or hybridomaIslet T cellsSpleen or lymph nodes
  • a

    DMK, dystrophia myotonica kinase; GAD, glutamic acid decarboxylase; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein.

  • b

    b Pept-imm, peptide-immunized; Spont, spontaneous.

  • c

    c A: Evidence of epitope presentation from whole protein (e.g. T cell clone, line, or hybridoma exists that responds to peptide and whole protein; elution); spontaneous T cell response. B: No evidence of epitope presentation from whole protein; spontaneous T cell response. C: Evidence of epitope presentation from whole protein; no positive spontaneous T cell response data. D: No evidence of epitope presentation from whole protein; no positive spontaneous T cell response data. MHC: major histocompatibility complex.

DMK138–146FQDENYLYLDbSpontSpont A [98]
GAD65206–214TYEIAPVFVKd  SpontA [99]
507–516WFVPPSLRTLKdPept-imm Pept-immC [100]
546–554SYQPLGDKVKd  SpontA [99]
GAD67515–524WYIPQSLRGVKdPept-imm Pept-immD [101]
IGRP21–29TYYGFLNFMKd Spont B [24]
206–214VYLKTNVFLKdSpontSpont AEluted from MHC[27]
225–233LRLFGIDLLDb Spont B [24]
241–249KWCANPDWIDb Spont B [24]
324–332SFCKSASIPKd Spont B [24]
Insulin 1/2B15–23LYLVCGERGKdSpontSpont AAg identified by expression cloning[102]
Insulin 2B25–C2FYTPMSRREVKd  Pept-immD [103]

Our registry also provides information that allows the epitopes to be evaluated according to criteria that indicate the probability that a given epitope is important in the pathogenesis of type 1 diabetes. In the case of epitopes identified using standard or HLA-transgenic NOD mice, those shown to be recognized by islet-infiltrating T cells during the development of spontaneous type 1 diabetes in the mice are most likely to be disease-relevant, while reactivities detected only in response to peptide immunization may be less so. For the epitopes of human β cell antigens, peptides recognized preferentially or differentially in type 1 diabetes patients and/or at-risk individuals compared to normal controls are the best candidates for disease-relevant epitopes that could ultimately be exploited to monitor autoimmune activity in at-risk individuals or patients undergoing intervention therapies. In addition, for both the murine and human systems, evidence that a given epitope is naturally processed and presented (e.g. elution from MHC, proteasome processing or the existence of a T cell clone that recognizes both the peptide and whole protein) further increases the probability that the epitope is a relevant one. This is the first time that islet cell epitopes have been documented with this level of stringency, and we believe that this approach also represents a first among the organ-specific autoimmune diseases.

CD4 T cell epitopes in mouse and man

Tables 1 and 2 show the CD4 T cell epitopes identified for human and mouse islet cell autoantigens. The relative contribution of the different islet cell autoantigens to the epitope lists is depicted graphically in Fig. 1a. It is noteworthy that for CD4 epitopes, the relative contributions of GAD65, proinsulin and IA-2 are similar in man and mouse, although over three times as many epitopes have been reported in man. It is possible that the greater number of epitopes identified in man represents a more diverse MHC class II background compared with the single MHC class II molecule present in the NOD mouse.

Figure 1.

Pie charts showing the antigenic distribution of (a) CD4 and (b) CD8 T cell epitopes in autoimmune diabetes in humans and mice. Data used are from Tables 1–4. For the sake of clarity, the single epitope of ICA69 is not shown in the pie charts in (a).

CD8 T cell epitopes in mouse and man

In contrast with CD4, for CD8 T cells the major contributor to epitopes recognized in the mouse is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), and in man is proinsulin/insulin (Tables 3 and 4; Fig. 1b). The latter phenomenon most probably reflects the influence of two recent publications in which large numbers of potential proinsulin/insulin epitopes were identified using approaches that lend themselves to large-scale epitope identification (proteasomal digestion of whole protein and scanning proteins in silico using binding algorithms), as well as the development and exploitation of HLA-A2 transgenic mice [14,15]. It is also noteworthy that there is a distinct lack of identification of CD8 T cell epitopes of IA-2, despite the fact that, in man at least, there is a high prevalence of CD4 T cell responses to this autoantigen (Figs 1a and 2), as well as the known importance of IA-2 autoantibodies in heralding the imminent development of the disease. It is likely that in the coming months and years these apparent species-based differences will balance out, and the pie charts for CD8 will come to resemble the harmonized appearance seen for CD4 epitopes. For example, there is now a considerable effort being expended on the search for IGRP epitopes in man, given the demonstration of their importance in NOD mice. This is also a good example of the use of animal models in instructing research into human disease.

Figure 2.

Representation of linear sequence and location of CD4 T cell epitopes of the major islet autoantigens preproinsulin, GAD65, and IA-2 in human type 1 diabetes. GAD65 and IA-2 are drawn to the same scale, whereas preproinsulin is enlarged three-fold for clarity. Epitopes shown are from Table 1. Where data exist that show an epitope to be unambiguously restricted by a particular HLA class II molecule, this is shown with shading, where black = HLA-DQ8; diagonal stripes = HLA-DR3 (DRB1*0301), and grey = HLA-DR4 (DRB1*0401). All other epitopes are shown as white boxes. TM = transmembrane.

Evaluating the importance of specific antigens in the pathogenesis of type 1 diabetes

An autoantigen could reasonably be termed important, and even essential, if it could be shown that genetic ablation of its expression led to protection from disease. Fulfilment of this very stringent criterion has been achieved for murine preproinsulin 1 [16]. More recently, the presence of the CD4 T cell epitope InsB9−23 (which also contains a CD8 T cell epitope) in particular has been reported to be required for the development of islet autoimmunity in NOD mice [17]. In contrast, NOD mice deficient in expression of IA-2 [18,19], IA-2β[19,20], ICA69 [21] or GAD65 [22,23] are not protected from disease, indicating that these antigens are not essential for disease development. We would argue, however, that such results do not necessarily imply that T cell responses to these antigens play no role in β cell elimination. Rather, in their absence, other specificities may more efficiently compete (e.g. at the level of the antigen-presenting cell) and fill the niche occupied normally by T cells specific for the now-ablated antigens. For example, when CD8 T cells specific for IGRP206−214/H-2Kd (which, as discussed below, is a disease-relevant epitope by several criteria) were completely depleted by high-dose treatment of NOD mice with an altered peptide ligand, disease occurred none the less, and the expansion of clonotypes specific for subdominant epitopes was observed [24].

A number of other strategies have been employed to address the issue of the importance of particular β cell autoantigens in the pathogenesis of type 1 diabetes in NOD mice. The ability of adoptively transferred T cell clones to accelerate diabetes in young NOD mice or to cause disease in NOD-scid mice provides evidence for the importance of the corresponding antigenic specificity. For example, adoptive transfer studies have demonstrated the pathogenicity of the CD8 T cell clone 8·3 [25,26], which is specific for IGRP206−214/H-2Kd[27]. Acceleration of disease in T cell receptor (TCR) transgenic mice is another indication of the pathogenicity of a particular specificity. Consistent with the adoptive transfer studies, NOD mice transgenic for the 8·3 TCR show accelerated disease [28]. Further evidence for the importance of IGRP in the development of diabetes in NOD mice comes from the finding that quantification of IGRP-reactive T cells in the peripheral blood can be used to predict which individual NOD mice will go on to develop disease [29].

For GAD65, adoptive transfer and TCR transgenic mouse studies are in apparent disagreement. A GAD65-reactive CD4 T cell line was capable of inducing diabetes in NOD-scid mice [30]. However, mice transgenic for a class II MHC-restricted TCR specific for either GAD65286−300 or GAD65206−220 were protected from disease [31,32], suggesting that at least certain GAD65-reactive CD4 T cell clonotypes may play a beneficial regulatory role. It should be noted here that two InsB9−23-reactive TCR transgenic mice have been described, one that develops disease [33] and one that does not [34]. These results support the notion that both pathogenic and non-pathogenic GAD65-reactive T cell clonotypes also exist.

For the most part, the types of experiments used to evaluate the importance of specific antigens in the development of diabetes in NOD mice cannot be applied to patients. In this case, quantitative or qualitative differences in T cell responses between patients or at-risk individuals and normal controls are the only indicator of disease-relevant antigens. This information is provided in Tables 1 and 3. However, it must be acknowledged that peripheral blood is the only T cell source that has been monitored routinely in humans, and the argument could always be made that T cells in the islets or the pancreatic lymph nodes might represent more accurately truly disease-relevant specificities. To this end, CD4 T cells from the pancreatic lymph nodes of type 1 diabetes patients have been examined recently [35]. Studies in NOD mice suggest the possibility of antigen-specific imaging of islet-infiltrating T cells [36], which may one day allow non-invasive examination of islet-infiltrating T cells in humans and a novel strategy to assess the disease relevance of particular β cell antigens.

Avoiding ‘tunnel vision’

It is, of course, entirely conceivable that β cell proteins other than those appearing in Tables 1–4 serve as targets in the autoimmune destruction in type 1 diabetes. Such candidates include imogen38, fetal antigen 1 (or Pref-1), and as yet unidentified components of β cells and their secretory granules [37–39]. By no means should the present compilation be regarded as comprehensive or fully representative. It merely reflects the current knowledge of proven and published epitopes as well as, most probably, a biased focusing on proinsulin, IA-2 and GAD65. It is also possible that the current selection is biased to those peptides displaying high binding affinity to HLA class I or II restriction elements. Although it remains to be demonstrated that binding affinity correlates with T cell recognition and possible autoimmune disease, it is remarkable that the majority of CD4 epitopes described to be recognized by T1D patients display high binding affinity to HLA [6,7]. In the case of HLA class I, epitope discovery has often been led by high binding affinity to the respective HLA class I molecules and as such could reflect a bias.

The exploitation of epitope discovery

The epitope lists contained in this review reflect a conjunction of human and animal model-based research, and as such have several potential utilities. In particular we anticipate that peptides based on these sequences could be used in the design of assays to detect T cell autoreactivity. Such assays are increasingly being deployed to provide secondary measures of clinical efficacy [40], as well as mechanistic insights, during intervention and prevention trials for type 1 diabetes. Some assays, of course, are entirely reliant upon the discovery of robust T cell epitopes, for example MHC-multimer-based technologies, as well as those that function optimally when peptides rather than whole proteins are used as stimuli. Recent mouse and human studies indicate that peptides targeted by CD8 T cells in new-onset and graft-recurrent type 1 diabetes overlap [15,41]. Thus, assays to monitor autoimmune activity in at-risk individuals and patients might also be applicable to the monitoring of islet graft rejection. A further use for epitope discovery may be in the design of novel intervention strategies. It has long been argued that antigen-specific immunotherapies for autoimmune diseases offer the best hope for the development or re-establishment of tolerance to islet autoantigens, and some of these may be peptide-based strategies [42]. Examples of the successful use of peptide immunotherapy in the prevention or treatment of type 1 diabetes in NOD mice are presented in Table 5.

Table 5.  Examples of the use of peptide immunotherapy in the prevention or treatment of type 1 diabetes in NOD mice.
AgaPositionSequenceMHCTreatmentAdjuvantOutcomeRef.
  • a

    GAD, glutamic acid decarboxylase; HSP, heat shock protein; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein.

  • b

    b Mimotope peptide; changes from natural peptide (VYLKTNVFL) are denoted by underlining. MHC: major histocompatibility complex.

GAD65247–266NMYAMLIARYKMSPEVKEKGAg750 µg of each peptide in a mixture administered intranasally at 2–3 weeks of age (single dose)NoneMice followed to 52 weeks of age; decreased incidence of disease with treatment[104]
509–528VPPSLRTLEDNEERMSRLSKAg7
524–543SRLSKVAPVIKARMMEYGTTAg7
539–558EYGTTMVSYQPLGDKVNFFRAg7
HSP60437–460VLGGGCALLRCIPALDSLKPANEDAg750 µg administered subcutaneously after the appearance of hyperglycaemia (single dose)Incomplete Freund'sMice followed to 40 weeks of age; increased survival with treatment[105]
IGRP206–214KYNKANAFLbKd100 µg administered intraperitoneally every 2–3 weeks beginning at 3 weeks of ageNoneMice followed to 32 weeks of age; decreased incidence of disease with treatment[106]
Insulin 2B9–23SHLVEALYLVCGERGAg740 µg administered intranasally for 3 consecutive days every 4–5 weeks beginning at 4 weeks of ageNoneMice followed to 32 weeks of age; decreased incidence of disease with treatment[88]

It is our hope that this annotated T cell epitope compilation will prevent duplication of effort, while at the same time encouraging verification, validation and wider use of the most promising epitopes identified to date. Antigen discovery efforts ongoing in our laboratories and others will probably reveal additional candidate peptides to be evaluated in the future.

Acknowledgements

Relevant research conducted in our laboratories was funded by the Juvenile Diabetes Research Foundation International, Diabetes UK, the National Institutes of Health and the Alexandrine and Alexander Sinsheimer Foundation. We also acknowledge the role of the Immunology of Diabetes Society in promoting this effort.

Note added in proof

After submission of our article, glial fibrillary acidic protein (GFAP) 143–151 (NLAQDLATV), GFAP 192–200 (SLEEEIRFL), GFAP 214–222 (QLARQQVHV), IAPP 5–13 (KLQVFLIVL), IAPP 9–17 (FLIVLSVAL), IGRP 152–160 (FLWSVFWLI), IGRP 215–223 (FLFAVGFYL), and IGRP 293–301 (RLLCALTSL) were reported to be recognized in the context of HLA-A2 by PBMC from T1D patients and/or at-risk individuals (Standifer NE, Ouyang Q, Panagiotopoulos C, Verchere CB, Tan R, Greenbaum CJ, Pihoker C, Nepom GT. Identification of novel HLA-A*0201-restricted epitopes in recent-onset type 1 diabetic subjects and antibody-positive relatives. Diabetes 2006; 55:3061–7), as were IA-2 172–180 (SLSPLQAEL), IA-2 482–490 (SLAAGVKLL), IAPP 5–13 (KLQVFLIVL), insulin L2–10 (ALWMRLLPL), and insulin B10–18 (HLVEALYLV) (Ouyang Q, Standifer NE, Qin H, Gottlieb P, Verchere CB, Nepom GT, Tan R, Panagiotopoulos C. Recognition of HLA class I-restricted β-cell epitopes in type 1 diabetes. Diabetes 2006; 55:3068–74).

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