PATHOGENESIS OF TYPE 1 DIABETES (A PUGLIESE, SECTION EDITOR)

Self-Antigen Expression in the Peripheral Immune System: Rolesin Self-Tolerance and Type 1 Diabetes Pathogenesis

Rebecca Fuhlbrigge & Linda Yip

Published online: 17 July 2014# Springer Science+Business Media New York 2014

Abstract Type 1 diabetes (T1D) may result from a breakdownin peripheral tolerance that is partially controlled by the ectopicexpression of peripheral tissue antigens (PTAs) in lymph nodes.Various subsets of lymph node stromal cells and certain hema-topoietic cells play a role in maintaining T cell tolerance. Thesespecialized cells have been shown to endogenously transcribe,process, and present a range of PTAs to naive Tcells andmediatethe clonal deletion or inactivation of autoreactive cells. Duringthe progression of T1D, inflammation leads to reduced PTAexpression in the pancreatic lymph nodes and the productionof novel islet antigens that Tcells are not tolerized against. Theseevents allow for the escape and activation of autoreactive Tcellsand may contribute to the pathogenesis of T1D. In this review,we discuss recent findings in this area and propose possibletherapies that may help reestablish self-tolerance during T1D.

Keywords Type 1 diabetes . Peripheral tolerance . Lymphnode stromal cells . Peripheral tissue antigens . Deformedautoregulatory factor 1 (Deaf1) . Alternative splicing .

Nonobese diabetic mice . Neoantigens . Self-antigenexpression . Peripheral immune system . Self-tolerance

Introduction

Type 1 diabetes (T1D) is a complex disorder that results froma combination of genetic, epigenetic, and environmental

factors. During disease progression, self-reactive T cells thatrecognize pancreatic antigens escape deletion or inactivationin the thymus and periphery and mediate the gradual destruc-tion of pancreatic beta cells. The initial beta cell damagehappens early in life and likely occurs as a consequence ofan environmental trigger, such as a viral infection or cytokinesreleased from nearby antigen-presenting cells [1]. Damagedcells are phagocytosed by dendritic cells (DCs) and macro-phages that migrate to the pancreatic lymph nodes (PLNs),where they present self-antigens to autoreactive T cells. Whilethe insulin B chain peptide (9-23) is the main diabetogenicepitope in the nonobese diabetic (NOD) mouse model and islikely to be important in many T1D patients, T cell reactivityto multiple antigens, including glutamic acid decarboxylase(GAD) 65 and 67, islet-specific glucose-6-phosphatase relatedprotein (IGRP), insulinoma-associated protein-2 (IA-2),chromogranin A, and zinc transporter 8, is detected in patientsand mouse models of disease [2–6]. This indicates that T cellsthat recognize multiple islet antigens are able to evade centraland peripheral tolerance mechanisms and participate in theautoimmune destruction of beta cells.

Negative selection of naive autoreactive T cells is mediatedin the thymus by medullary thymic epithelial cells (mTECs)that express a range of peripheral tissue antigens (PTAs) underthe transcriptional control of the autoimmune regulator, Aire[7]. Normally, maturing thymocytes that express T cell recep-tors specific for these PTAs are deleted, but some self-reactiveT cells can escape to the periphery. These self-reactive cellsmay be dealt with by additional mechanisms, such asactivation-induced cell death, the induction of anergy, orsuppression by regulatory T cells. Tolerance to certain anti-gens, such as the melanocyte antigen tyrosinase, may relysolely on peripheral mechanisms. Elimination of self-reactive CD8+ T cells specific to tyrosinase occurs not in thethymus but in the lymph nodes, where tolerance is mediatedby nonhematopoietic lymph node stromal cells (LNSCs) thatendogenously express and present tyrosinase on major

This article is part of the Topical Collection on Pathogenesis of Type 1Diabetes

R. Fuhlbrigge : L. Yip (*)Department of Medicine, Division of Immunology andRheumatology, Stanford University, 269 Campus Drive, CCSRRoom 2240, Stanford, CA 94305-5166, USAe-mail: lindayip@stanford.edu

R. Fuhlbriggee-mail: bfuhl@stanford.edu

Curr Diab Rep (2014) 14:525DOI 10.1007/s11892-014-0525-x

histocompatibility complex (MHC) class I molecules [8].Similarly, CD8+ T cell tolerance to intestinal self-antigenscan be established by LNSCs that present these endogenouslyexpressed antigens [9]. In this review, we will discuss how theT cell repertoire and self-tolerance are shaped by peripheralcells that endogenously transcribe, process, and present self-antigens and then examine how aberrations that occur duringthe inflammatory T1D disease process could block the induc-tion of self-tolerance.

Induction of Self-Tolerance in Peripheral Lymph Nodes

Ectopic self-antigen-expressing cells were first described inhuman peripheral lymph nodes by Pugliese and colleagues[10]. These hematopoietic cells expressed the mRNA andprotein of several common beta cell autoantigens, includinginsulin, GAD65, and IA-2, and were often surrounded byapoptotic lymphocytes, suggesting a role for them in theestablishment of peripheral self-tolerance [10, 11]. This issupported by early studies showing that bone-marrow-derived cells that ectopically express self-antigens canmediateself-tolerance to the antigens they express [12, 13]. It is nowrecognized that, in addition to hematopoietic cells residing inthe lymph nodes, LNSCs can ectopically express and presentPTAs to mediate the deletion or inactivation of autoreactive Tcells and, possibly, induce regulatory T cells.

The lymph nodes consist of T cells, B cells, and antigen-presenting cells that are organized into distinct regions byvarious LNSCs and extracellular matrix proteins (Fig. 1).The LNSC network provides an infrastructure that allowsfor efficient migration and interaction among lymphocytes,aids in efficient activation of immune responses, and facili-tates movement of cytokines, chemokines, and small mole-cules [14•]. LNSCs are heterogeneous and can be character-ized by their location, function, transcriptional profile, and cellsurface markers [14•, 15–17]. On the basis of the absence ofCD45 and the surface expression of the glycoproteins CD31and gp38 (podoplanin), LNSCs can be separated into thefollowing major subsets by flow cytometry: lymphatic endo-thelial cells (LECs; gp38+CD31+), fibroblastic reticular cells(FRCs; gp38+CD31−), blood endothelial cells (BECs; gp38−

CD31+), and double negative cells (DNs; gp38−CD31−). Inthe pooled lymph nodes of BALB/c mice, our lab showed thatthe FRCs are most abundant (~50 %), followed by the BECs(25 %), LECs (~15 %), and DNs (~5 %) [18••]. However,these proportions can differ between lymph nodes [19]. Eachsubset also represents a heterogeneous collection of cells. Forexample, the LEC subset can be further subdivided on thebasis of programmed death ligand-1 (PD-L1), intercellularadhesion molecule 1 (ICAM-1), mucosal vascular addressincell adhesion molecule 1 (MAdCAM-1), and lymphotoxin-βreceptor (LtβR) expression into medullary, cortical, and

subcapsular LECs that make up approximately 30 %, 50 %,and 20 % of the total LEC population, respectively [20•].Likewise, FRCs sorted on the gp38+CD31− phenotype con-tain marginal reticular cells and may contain a rare populationof follicular dendritic cells.

In addition to maintaining the lymph node environment,LNSCs have more recently been recognized for their ability toexpress and present self-antigens and to induce CD8+ T celltolerance to these antigens in a manner comparable to mTECsin the thymus [8, 9, 20•, 21–24, 25•]. While stromal cellsmake up a relatively small proportion (1–2 %) of the lymphnode, they maintain close contact with lymphocytes as theyenter the lymph nodes and, thereby, permit efficient monitor-ing of the naive T cell repertoire [19]. Among LNSCs, thetolerogenic properties of the FRCs and LECs have been themost extensively studied. The FRCs are localized mainly inthe T cell zone and are also present in the subcapsular andmedullary stroma [14•] (Fig. 1). These cells form a complexnetwork of collagen-rich fibers that guides the movement oflymphocytes. In mice, these cells endogenously express anarray of PTAs, including the pancreatic PTA genes proinsulin,Insulin2 (Ins2), and Gad67 [18••, 23, 24]. Using the transgen-ic intestinal fatty-acid-binding protein (iFABP)-OVA mousemodel, FRCs have been shown to act as antigen-presentingcells that induce proliferation and subsequent deletion ofautoreactive CD8+ T cell against intestinal self-antigen(OVA) [9, 24], likely through a mechanism that involves thePD-L1 inhibitory pathway [26].

The LECs line the afferent lymphatic vessels and enablethe migration of lymphocytes and movement of soluble mol-ecules into the lymph node. Inside the lymph node, LECsform dense networks in the cortex, subcapsular sinus, andmedullary sinus and facilitate the egress of lymphocytesthough the efferent lymphatics [16, 20•] (Fig. 1). These cellsexpress various PTA genes, including the gene for the mela-nocyte differentiation protein tyrosinase (Tyr) [18••, 20•, 23,24]. LECs have been shown to directly present tyrosinase tomediate the activation and deletion of tyrosinase-specificCD8+ Tcells through engagement of PD-L1 [23, 25•]. Recentstudies further demonstrated that this effect is mediated spe-cifically by LECs that reside in the medullary sinus [20•].Tolerance to tyrosinase does not occur in the thymus and is notmediated by conventional DC or Langerhans cells [8], thusemphasizing the critical role of LNSCs in peripheral toleranceto certain antigens.

The DN subset is poorly defined, with unknown functionand localization. It is the only subset that has consistently beenshown to expresses Aire [23]. As such, a distinct population ofextra-thymic Aire-expressing cells (eTACs) was identified inthis subset. The eTACs are localized in the subcapsular zoneand are relatively motile, as compared with FRCs, LECs, orBECs [27] (Fig. 1). The eTACs were initially thought torepresent a population of nonhematopoietic stromal cells

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[21]. However, recent findings show that eTACs are a radio-resistant, bone-marrow-derived population of cells that ex-press low levels of CD45 [27••]. Although eTACs are distinctfrom conventional DCs, they express many of the markers(CD11c, CD40, CD80, and CD8a) found on a previouslydescribed population of human self-antigen-presenting hema-topoietic cells that were thought to represent dendritic cells[10]. The eTACs may also overlap with a population ofperipheral blood mononuclear cells that have been shown toexpress the self-antigens proinsulin, GAD65, glucagon, andsomatostatin [11, 28]. The eTACs express an array of Aire-regulated PTAs that differ from those expressed in themTECs. In a transgenic setting, eTACs have been shown tomediate the deletion of CD8+ and CD4+ Tcells that react withthe pancreatic antigens IGRP and BDC2.5 (an antigen derivedfrom chromogranin A), respectively [21, 27••]. Interestingly,inactivation of antigen-specific CD4+ T cells is mediated bythe lack of CD28 co-stimulation, rather than by activation ofthe PD-L1 pathway, which is used by FRCs and LECs toinactivate CD8+ T cells. This finding is similar to those in

previous studies showing that stimulation of PTA-expressingbone-marrow-derived cells results in activation-induceddeath, rather than clonal deletion of self-reactive cells [12]. Itis possible that these bone-marrow-derived cells represent thenewly described eTACS.

The fourth major subset of LNSCs, the BECs, is function-ally well defined. These cells line the high endothelial venules(HEVs) and express gp38, which maintains the barrier func-tion of HEVs during lymphocyte trafficking, and variousadhesion molecules that control the entry of lymphocytes intothe lymph nodes [14•, 29] (Fig. 1). Aside from the ability toectopically express certain PTA genes, including Gad67, thetolerogenic properties of BECs have not been thoroughlyexamined [20•, 23, 24]. It is unknown whether these cellscan inactivate or delete autoreactive Tcells in a manner similarto FRCs, LECs, and eTACs. The BECs, LECs, and FRCshave also been shown to upregulate MHC class II in responseto inflammation, suggesting the possibility that tolerance maybe induced by the conversion of autoreactive CD4+ T cells toregulatory T cells [16]. Interaction of bone-marrow-derived

Fig. 1 Localization of PTA-expressing cells in the lymph node: A diagram showing the architecture of the lymph node and the localization of differentPTA-expressing cells within the various compartments

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eTACs with cognate CD4+ T cells has already been shown toincrease regulatory T cell numbers, although this plays aseemingly minor role in eTAC-mediated tolerance [27••].While it is clear that certain LNSCs and hematopoietic cellsplay an important role in maintaining peripheral T cell toler-ance by directly expressing and presenting self-antigens, fu-ture studies are required to fully identify the specific cellpopulations and thoroughly understand the complex mecha-nisms that are involved in this process.

Regulation of PTA Expression by Deaf1 in LNSCs

Studies using Aire reporter mice clearly show that Aire is notexpressed in LNSCs [27••] and, thus, cannot drive PTA ex-pression in these cells. We previously showed that the tran-scriptional regulator, deformed autoregulatory factor 1(Deaf1), may instead be responsible for the transcription ofPTA genes in LNSCs [30]. Deaf1 is expressed in all fourmajor LNSC subsets and is highly enriched in LNSCs, ascompared with T and B cells of the lymph node [18••, 24, 30].Similar to Aire, Deaf1 contains a SAND (Sp100, Aire-1,NucP41/75, and Deaf1) domain that binds to DNA, mediateschromatin-dependent transcription, and acts as a site for pro-tein–protein interaction [31, 32]. Deaf1 also contains a car-boxyl terminal ZF-MYND domain (zinc finger, myeloid,Nervy, and Deaf1) that is structurally similar to the planthomeodomain 1 region of Aire that interacts with modifiedhistone H3 [33]. By comparing gene expression in the pan-creatic lymph nodes of Deaf1-KO mice with that of BALB/ccontrol mice, we showed that Deaf1 regulates the expressionof approximately 600 genes, with approximately 300 genesupregulated and 300 genes downregulated by knockout ofDeaf1 [30]. Interestingly, the most downregulated genes

encoded tissue-restricted antigens. We showed that someDeaf1-regulated genes are regulated by Aire in mTECs [7],but few are regulated by Aire in eTACs [21], suggesting thatDeaf1 and Aire may control the expression of distinct sets ofPTA genes in peripheral lymph nodes.

Since each LNSC subset expresses a distinct array ofPTA genes, we compared the expression of several PTAgenes in different LNSC subsets isolated from Deaf1-KOand BALB/c control mice. The gene alpha 1 microglobulin/bikunin (Ambp) is reduced by 6.5-fold in the PLN of Deaf1-KO mice and is expressed in the FRCs and DNs of pooledlymph nodes isolated from control mice [18••, 30]. Knock-out of Deaf1 completely eliminated Ambp expression in theDNs and reduced expression in the FRCs by more than 2-fold (Fig. 2). The chymotrypsin-like elastase family member1 gene (Cela1) is reduced 4-fold in the PLN of Deaf1-KOmice and is expressed in the DNs of pooled lymph nodesisolated from control mice [18••, 30]. Similar to Ambp,Cela1 expression was absent in the DNs of Deaf1-KO mice(Fig. 2). Comparable expression of Ins2 was also detected inthe FRCs and DNs of pooled control lymph nodes [18••].Loss of Deaf1 abolished Ins2 expression in the DNs but didnot reduce its expression in the FRCs (Fig. 2). We alsoexamined the expression of Tyr in LECs and found that itwas not different between Deaf1-KO and control mice(Fig. 2). On the basis of these findings, Deaf1 appears tohave more control over PTA expression in the DN subset.Since this subset is poorly defined and contains a populationof CD45logp38−CD31− eTACs [27••], we examined theexpression of Aire in DNs and found that loss of Deaf1did not significantly alter the expression of Aire (Fig. 2). It isunclear whether Deaf1 regulates PTA expression specificallyin eTACs, since these cells are not easily isolated fromDeaf1-KO and control mice.

Fig. 2 Deaf1 controls the expression of various PTAs in different subsetsof LNSCs. QPCR data showing the expression of PTA genes (Ambp,Cela1, Ins2 and Tyr) and Aire in different subsets of LNSCs (fibroblasticreticular cells, FRCs; double negatives, DNs; and lymphatic endothelialcells, LECs) extracted from the pooled lymph nodes (cervical, inguinal,

mesenteric, axillary, brachial, and pancreatic lymph nodes) of 3 Deaf1-KO mice, as compared with 3 wild-type control mice. Data represent themean±SD of two independent studies (unpublished data; detailedmethods are found in [18••])

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In addition to controlling PTA transcription, Deaf1 couldalso regulate translation of specific mRNA transcripts inLNSCs by controlling the expression of the eukaryotic trans-lation initiation factor 4 gamma 3 (Eif4g3) gene that encodeseIF4GII [18••]. In Deaf1-KO mice, Eif4g3 gene expression isdrastically reduced in all four major subsets of LNSCs. Thisreduced expression resulted in the diminished translation ofvarious genes, including the gene for Aminopeptidase N(Anpep), which encodes an enzyme involved in fine-tuningantigen presentation on MHC class II [18••]. Together, thesedata clearly demonstrate a role for Deaf1 in regulating ectopicPTA expression in peripheral lymph nodes.

Splicing of Deaf1 Results in Reduced PTA Expressionin the PLN During the Onset of Destructive Insulitis

Since it is not possible to obtain tissues from prediabeticsubjects at various stages of disease, our laboratory used thewell-established NOD mouse model to study disease progres-sion. These mice express the I-Ag7 MHC that is structurallyand functionally similar to HLA-DQ8, the major T1D-associated MHC allele [34]. T cells of NOD mice react totargets that have been identified as autoantigens in humanT1D, including insulin, GAD, and IA-2 [35, 36]. FemaleNODmice develop disease in a spontaneous, highly penetrantmanner with a predictable course. Peri-insulitis develops be-tween 4 and 8 weeks of age, and the onset of infiltrative/destructive insulitis is observed between 12 and 16 weeks ofage. This is followed by substantial beta cell loss and hyper-glycemia. Control NOD.B10 mice are isogenic to the NODmice outside of the MHC complex and do not develop anysigns of disease.

By performing a series of microarray experiments in thePLN of NOD and NOD.B10 mice of different ages, we foundthat the expression of various islet-specific/enriched genes,including Insulin 1 and 2 (Ins1 and Ins2), Glucagon (Gcg),Pancreatic polypeptide (Ppy), Chromogranin A (Chga),Regenerating islet derived protein 2, 3a, and 3g (Reg2, Reg3a,and Reg3g), were significantly reduced in the PLN of NOD,as compared with NOD.B10, mice at 12–16 weeks of age,coincident with the onset of destructive insulitis. In NODmice, Ins2 is expressed in the thymus and promotes toleranceto insulin: Knock-out of Ins2 has been shown to increase theincidence and rate of progression of T1D and the prevalenceof anti-insulin autoantibodies in NOD mice [37]. Re-introduction of Ins2 expression in bone-marrow-derived cells,however, did not delay diabetes development in Ins2-KONOD mice [38]. On a non-autoimmune background, immu-nization with preproinsulin-2 (encoded by Ins2) led to a robustT cell response in Ins2-deficient mice, but not in wild-typemice [39]. These findings suggest that expression of Ins2 in

the thymus is required to establish tolerance to insulin [40,41].

In the human thymus, higher levels of PROINSULIN ex-pression lead to more efficient negative selection of insulin-specific T cells [42]. Increased thymic transcription ofPROINSULIN results from allelic variation in the variablenumber of tandem repeats (VNTR) in the promoter region ofthe INSULIN gene [42]. Individuals expressing the class IIIVNTR allele express higher thymic levels of proinsulin andare protected from T1D, while those who are homozygous forthe class I VNTR allele express lower thymic levels of proin-sulin and have a ~2- to 5-fold increased risk for T1D [42–44].The VNTR allele also regulates PROINSULIN expression inhuman peripheral myeloid cells that are thought to be involvedin promoting self-tolerance [28]. These human and NODmouse studies clearly show a correlation between PTA ex-pression levels and the maintenance of self-tolerance.

Since PTA expression in LNSCs and eTACs mediates thedeletional tolerance of autoreactive CD8+ and/or CD4+ Tcells, reduced expression of islet antigens in the NOD PLNmay result in the escape of islet-reactive Tcells that trigger thetransition from peri-insulitis to destructive insulitis. Loss ofPTA gene expression in the PLN may be the result of reducedDeaf1 function. Canonical Deaf1 gene expression is reduced,and alternative splicing of Deaf1 occurs in the PLN of 12-week-old NODmice. The alternatively spliced variant, Deaf1-Var1, lacks the nuclear localization signal and is retained in thecytoplasm, where it heterodimerizes with the canonical iso-form of Deaf1 and renders it nonfunctional. The Deaf1-Var1gene is most abundantly expressed in the LNSCs of the PLNand is significantly increased in the PLN of 12-week-old NODmice, as compared with age-matched NOD.B10 controls [30].Remarkably, a functionally similar isoform (DEAF1-VAR1)was also identified in human beings and was found to beupregulated in the PLN of T1D patients, as compared withnondiabetic controls. Increased DEAF1-VAR1 expression co-incided with loss of INSULIN gene expression, suggestingthat loss of DEAF1 function also results in reduced PTAexpression in the PLN of T1D patients [30]. Furthermore,reduced expression of the Deaf1-regulated Eif4g3 gene wasobserved in the PLN of both 12-week-old NODmice and T1Dpatients, as well as nondiabetic but islet autoantibody-positivesubjects, as compared with controls [18••]. This suggests thatalternative splicing of DEAF1 suppresses the transcription,processing, and presentation of PTAs in human LNSCs.

Since inflammation and hyperglycemia are hallmarks traitsof T1D, we recently examined whether alternative splicing ofDeaf1 could occur as an early consequence of inflammation orhyperglycemia in the PLN.We found thatDeaf1-Var1 expres-sion could be induced in the PLN of 12-week-old NOD.B10mice by intraperitoneal injection of activated splenocytes thathome to the PLN (unpublished data). Deaf1-Var1 expressionwas also increased in the PLN of 10-week-old NOD and

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NOD.B10 mice following hyperglycemia induced by treat-ment with S961, an insulin receptor antagonist (unpublisheddata). Deaf1-Var1 expression was drastically higher in hyper-glycemic NOD than in NOD.B10 mice, suggesting that in-flammation or hyperglycemia alone could drive the splicing ofDeaf1 but the combined effect of both results in the greatestamount of Deaf1 splicing. These data suggest that during theprogression of T1D, early inflammation in the PLN results inreduced canonical Deaf1 and increased Deaf1-Var1 expres-sion. This leads to diminished Deaf1 function and loss of PTAgene transcription, processing, and presentation.

Islet Inflammation May Generate UntolerizedNeoantigens

While ectopic expression of PTAs in mTECs, LNSCs, andeTACs can mediate self-tolerance to the vast majority of self-antigens, it might not protect against T cells that recognizeneoantigens generated during the inflammatory disease pro-cess. Inflammation can lead to splicing events that result in theexpression of untolerized epitopes. Exposure of human isletsor rat beta cells to interleukin-1β combined with interferongamma has been shown to induce differential splicing of 548and 2,651 genes, respectively [45, 46]. Within the humangenome, approximately 42 % of gene transcripts undergoalternative splicing, and 70 %–88 % of these splicing eventsalter the coding region [47, 48]. This may result in the expres-sion of additional exons or novel protein domains that lead tothe production of neoantigens. Studies have shown that splic-ing occurs more frequently in autoantigen genes than in ran-domly selected genes. Furthermore, among the alternativelyspliced transcripts, 92 % and 88 % contained isoform-specificregions that encode. MHC class I- and class II-restrictedT cell epitopes, respectively [49].

Differential splicing of several common autoimmune tar-gets of T1D, including IA-2 and IGRP, has been shown tooccur in the periphery and has been implicated in the devel-opment of T1D [28, 50–53]. The IA-2 gene encodes thetyrosine phosphatase-like protein in secretory granules ofislets and neuroendocrine cells and is differentially spliced inislets, as compared with the thymus and spleen. In islets, thefull-length IA-2 gene (PTPRN) and a variant transcript lackingexon 13 are both expressed, but in the thymus and spleen, onlythe variant transcript is detected [52]. Exon 13 encodes thetransmembrane and juxtamembrane domains of IA-2. Inter-estingly, epitopes from these domains are targeted during theautoimmune process [54], indicating that tolerance to certainepitopes is established only if they are expressed and present-ed in lymphoid organs. Differential splicing of the IGRP gene(G6PC2) has also been observed between the pancreas and thethymus and spleen. The full length G6PC2 gene consists of 5exons and can be spliced into at least five variant isoforms [50,

55]. Similar to PTPRN, the full-length isoform of G6PC2 isconsistently expressed only in the pancreas [50, 55]. Thethymus and spleen express other variants of G6PC2, butexpression of the full-length isoform is stochastic and, whenpresent, is expressed at extremely low levels [50, 55]. The lowquantity or absence of full-length G6PC2 in the thymus andspleen suggests that there may be incomplete tolerance toIGRP. This was confirmed by the detection of IGRP autoan-tibodies and cytotoxic T cells in both T1D and healthy indi-viduals [55].

In addition to these two common T1D antigens, we haveshown that the gene for adenosine A1 receptor (Adora1) isdifferentially spliced in the pancreas and PLN of NODmice at12 weeks of age. Adora1 inhibits glucagon secretion and isexpressed on all alpha cells and some beta cells in the islets ofhealthy NOD.B10 mice and young NOD mice (<12 weeks ofage) [56]. As NOD islets are infiltrated, the number of alphacells is reduced, and the remaining alpha cells lose Adora1expression [56]. Inflamed islets of 12-week-old NOD micealso express an alternatively spliced isoform of Adora1,Adora1-Var, which is not expressed in the PLN (Fig. 3a).Adora1-Var encodes a truncated protein that lacks theamino-terminal and first three transmembrane domains. Thus,a neoepitope may be present at the start site of Adora1-Var,where exon 2 is spliced out. To examine this, we tested theimmunoreactivity of NOD mouse sera at various ages againstthe recombinant Adora1-Var protein. The serum of 12-week-old NOD mice reacted with a product corresponding to thesize of Adora1-Var. This was not observed using the serum of12-week-old NOD.B10 mice or the serum from 8-week-oldNOD and NOD.B10 mice (Fig. 3b). Additional co-immunoprecipitation experiments confirmed that 12-week-old NOD serum bound specifically to Adora1-Var, suggestingthat autoantibodies against Adora1-Var may be present in theserum of 12-week-old NODmice (Fig. 3c). This age coincideswith the onset of destructive insulitis and the loss of Adora1/Adora1-Var-positive alpha cells surrounding the islets [56],suggesting that autoimmunity against Adora1-Var may con-tribute to the pathogenesis of T1D.

Posttranslational modifications of beta cell antigens mayalso produce neoepitopes that are not expressed in the thymusor secondary lymphoid organs [1, 57•, 58, 59]. These antigensare thought to be modified during the “initiation” phase ofdisease, when beta cells are first damaged by environmentalfactors such as viruses or cytokines released by nearbyantigen-presenting cells. The modified antigens are seen asforeign and trigger a subsequent lymphocyte-dependent auto-immune attack. This has already been shown to occur in otherautoimmune diseases, such as rheumatoid arthritis, wherecitrullinated proteins are targeted for destruction [60]. In thecase of T1D, posttranslational modifications can occur in twomajor autoantigens: GAD65 and insulin. Oxidative stress ofrat islets was found to modify native GAD65 to produce a

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high molecular weight, covalently linked aggregate of GAD,and serum of T1D patients bound with higher affinity to thehigh molecular weight form of GAD than to GAD65 [61]. Aposttranslationally modified form of the A-chain of humaninsulin has also been identified. This modified protein con-tains a disulphide bond between adjacent cysteine residues atA6 and A7 and is recognized by T cells isolated from T1Dpatients [62].

Therapeutic Approaches to Re-establish Deaf1 Functionand PTA Expression in the PLN

While it is difficult to manipulate the peripheral immunesystem to induce tolerance against all possibleneoantigens generated during the disease process, it

may be possible to prevent Deaf1 splicing to re-estab-lish adequate PTA expression in lymph nodes. Studieshave shown that aberrant alternative splicing could becorrected using target-specific antisense splice-switchingoligonucleotides to manipulate the selection of splicesites [63]. This approach has been used successfully tocorrect the splicing of multiple genes associated withdiseases such as Duchenne muscular dystrophy, cancer,various laminopathies, and autoimmune diseases, includ-ing rheumatoid arthritis [64–67]. Splice-switching oligo-nucleotides have been used to manipulate splicing of theCytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4)gene to modulate immune responses and alter diabetessusceptibility in NOD mice [68]. Antisense oligonucle-otides can mediate exon skipping or exon inclusiondepending on the site that is targeted. In the case of

Fig. 3 Expression of an untolerized Adora1 isoform in the pancreas ofNOD mice. a RT-PCR data showing expression of Adora1-Var in thepancreas but not PLN of 12-week-old NOD mice. Data was generatedusing a pool of cDNA obtained from 5 individual mice per group. b Left:An immunoblot showing expression of Adora1 and Adora1-Var in thelysate of transfected HEK293 cells. Membranes were probed with acommercially available Adora1 antibody (abcam #ab82477). Centerand right: Similar membranes were probed with IgG purified serumextracted from 8- and 12-week-old NOD and NOD.B10 mice. Thepooled purified serum of 5 mice per group was used. Only serum from

12-week-old NOD mice showed reactivity against a product that corre-spondswith the size of Adora1-Var (~25 kDa). cCo-immunoprecipitationexperiments were performed on the lysate of HEK293 cells transfectedwith a plasmid expressing Adora1-Var-EGFP using the IgG purifiedserum of 12-week-old NOD or NOD.B10 mice. Immunoblotting wasperformed using the Adora1 antibody (abcam). Only the NOD serumimmunoprecipitated a product corresponding to the size of Adora1-Var-EGFP (~55 kDa) (unpublished data; primer sequences used for RT-PCR,and generation of Adora1-Var and Adora1-Var-EGFP plasmids are de-scribed in [56])

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the mouse Deaf1-Var1, an antisense oligonucleotidecould be designed to bind to the intronic insertionbetween exon 6 and 7. This oligonucleotide wouldblock the splice site, prevent inclusion of the intron,and allow the transcript to skip to the next exon. Forthe human isoform of DEAF1-VAR1, which containsmissing exons, an antisense oligonucleotide can be de-signed to block the binding site of a repressor or alterthe inhibitory secondary structure of the transcript. Thiswould allow efficient recognition of the exon by thespliceosome and could potentially restore inclusion ofthe exon [69].

Since Deaf1 splicing is induced by inflammation in thePLN, immunomodulatory therapy may also be used torestore Deaf1 function. Previous work from this lab hasshown that that the expression of interleukin-4 (IL-4) issignificantly reduced in the PLN of NOD mice starting at12 weeks of age, when Deaf1 is spliced and the expres-sion of PTA genes is significantly reduced [70]. Weshowed that treatment of prediabetic NOD mice withbone-marrow-derived dendritic cells modified to expressIL-4 (DC/IL-4) could renormalize gene expression in thePLN to become more similar to that of NOD.B10 controlmice and prevent or delay disease progression [71, 72].The DC/IL4 cells preferentially home to the PLN andcould produce IL-4 for 24 h [71–73]. At the time thesestudies were performed, we had not yet identified Deaf1-Var1. Thus, we recently tested whether this treatmentcould prevent Deaf1 splicing. Remarkably, we showedthat Deaf1-Var1 expression was significantly lower inthe PLN of 12-week-old NOD mice 3 days after treatmentwith DC/IL-4, as compared with controls (unpublisheddata), indicating that the immunomodulatory effect ofIL-4 could prevent Deaf1 splicing and re-establish PTAexpression in the PLN.

Conclusion

While overlapping mechanisms are in place to establishself-tolerance, these mechanisms are not absolute. Someself-reactive T cells that recognize various islet antigenscan escape and destroy insulin-secreting beta cells.Here, we discussed how antigen presentation is nega-tively impacted by inflammation of the PLN and howneoantigens may be generated by inflammation of theislets. These can lead to the loss of self-tolerance andthe persistence and survival of autoreactive T cells.While we do not fully understand the complex periph-eral mechanisms involved in shaping the naive T cellrepertoire, we can still formulate ways to re-establishself-tolerance on the basis of the information that iscurrently available.

Acknowledgments This work was supported by grants from the Na-tional Institutes of Health (NIH). LindaYipwas supported by the JuvenileDiabetes Research Foundation (JDRF) TransitionAward.Work involvinglymph node specimens from T1D patients was supported by the JDRFnPOD (Network for the Pancreatic Organ Donor with Diabetes). Theauthors wish to thank C. Garrison Fathman (Stanford University) for hisuseful comments and R. J. Creusot (Columbia University) for helpgenerating the data shown in Fig. 2.

Compliance with Ethics Guidelines

Conflict of Interest Rebecca Fuhlbrigge and Linda Yip declare thatthey have no conflict of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

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