Impact of Islet Autoimmunity onthe Progressive b-Cell FunctionalDecline in Type 2 DiabetesDiabetes Care 2014;37:3286–3293 | DOI: 10.2337/dc14-0961

OBJECTIVE

Cross-sectional studies have suggested that islet autoimmunity may be moreprevalent in type 2 diabetes (T2D) than previously appreciated and may contrib-ute to the progressive decline in b-cell function. In this study, we longitudinallyevaluated the effect of islet autoimmune development on the progressive b-celldysfunction in T2D patients.

RESEARCH DESIGN AND METHODS

Twenty-three T2D patients negative for islet autoantibodies (GAD antibody andinsulinoma-associated protein 2) and islet-specific T cells were evaluated prospec-tively for up to 36 months. We investigated the percentage of patients who de-veloped islet autoantibodies (Ab+) and/or islet-reactive T cells (T+) and the effectof the islet autoimmunity on fasting and glucagon-stimulated C-peptideresponses. We defined positive islet autoimmunity as Ab+ and/or T+ for at leasttwo study visits.

RESULTS

Of the 23 patients, 6 (26%) remained negative for islet autoimmunity (Ab2T2), 14(61%) developed Ab+ and/or T+, and 3 (13%) were unclassifiable because theydeveloped islet autoimmunity at only one study visit. Islet Ab+was observed to beless stable than islet-specific T-cell responses. Development of islet autoimmunitywas significantly associated with a more rapid decline in fasting (P < 0.0001) andglucagon-stimulated (P < 0.05) C-peptide responses.

CONCLUSIONS

These pilot data suggest that the development of islet autoimmunity in T2D isassociated with a significantly more rapid b-cell functional decline.

Historically, type 2 diabetes (T2D) has not been considered to be immunemediated.However, many notable discoveries in recent years have provided evidence tosupport the concept of immune system involvement in T2D pathophysiology (1–5). Immune cells have been identified in the pancreases of phenotypic T2D patients(3–5). Moreover, treatment with interleukin-1 receptor agonist improves b-cellfunction in T2D patients (6–8). These studies suggest that b-cell damage/destruc-tion mediated by the immune systemmay be a component of T2D pathophysiology.Although the b-cell damage and destruction in autoimmune diabetes is most

likely T-cell mediated (T), immune markers of autoimmune diabetes have primarilycentered on the presence of circulating autoantibodies (Abs) to various islet anti-gens (9–15). Abs commonly positive in type 1 diabetes (T1D), especially GAD

VA Puget Sound Health Care System, Seattle,WA, and Department of Medicine, University ofWashington, Seattle, WA

Corresponding author: BarbaraM. Brooks-Worrell,bbrooks@u.washington.edu.

Received 15 April 2014 and accepted 1 Septem-ber 2014.

© 2014 by the American Diabetes Association.Readers may use this article as long as the workis properly cited, the use is educational and notfor profit, and the work is not altered.

Barbara M. Brooks-Worrell,

Edward J. Boyko, and Jerry P. Palmer

3286 Diabetes Care Volume 37, December 2014

PATH

OPHYS

IOLO

GY/COMPLICATIONS

antibody (GADA) and islet cell Abs (ICA),have been shown to bemore common inpatients with T2D than in nondiabeticcontrol populations, and the presenceof multiple islet Abs, such as GADA, ICA,and tyrosine phosphatase-2 (insulinoma-associated protein 2 [IA-2]), have beendemonstrated to be associated with anearlier need for insulin treatment inadult T2D patients (14,16–20).In 1996, our laboratory developed a

T-cell assay, cellular immunoblotting(CI), with excellent sensitivity and spec-ificity for measuring islet-specific T-cellresponses in T1D (21,22). We have usedCI to measure islet-reactive T cells intype 1 (T1D) patients (23–26) and inphenotypic T2D patients with and with-out islet Abs (26–30). In our previouscross-sectional studies, we demon-strated that T-cell reactivity to islet pro-teins in phenotypic T2D patients morestrongly correlated with impaired b-cellfunction compared with Ab positivity(27). Furthermore, we also demon-strated that attenuation of islet-reactiveT-cell responses was associated with im-provement in b-cell function in T2Dpatients (29). Longitudinal data are stilllacking on the percentage of nonau-toimmune T2D patients (Ab2T2) whodevelop Ab or T-cell positivity overtime and how this development of isletautoimmunity affects the progressiveb-cell dysfunction associated with T2Ddisease.In this pilot study, 23 phenotypic

Ab2T2 T2D patients were evaluatedprospectively for up to 36 months andtested every 3 months for the develop-ment of islet autoimmunity indicated byislet Abs (GADA and IA-2) and/or islet-specific T cells. Fasting C-peptide (FCP)levels and glucagon-stimulated C-peptide(SCP) responses were assessed to evalu-ate the interrelationship between isletautoimmune development in T2D andb-cell functional status.

RESEARCH DESIGN AND METHODS

SubjectsT2D patients were diagnosed by physi-cians in accordance with the AmericanDiabetes Association standards of medi-cal care in diabetes (31). T2D patientswere recruited from the general popula-tion through advertisements. Two bloodsamples, within 3months, were obtainedfrom consecutive T2Dpatients to confirmautoimmune status. Inclusion criteria

for T2D patients for this study were aBMI .25 kg/m2, no history of ketonuriaor ketoacidosis, onset of T2D betweenages 35 and 70 years, duration of diabe-tes#5 years, HbA1c levels between 7 and10%, FCP level$0.8 ng/mL, negative forAbs to GADA and IA-2, and negativefor islet-reactive T cells. Demographics,islet Ab and islet-reactive T-cell status,length of follow-up, baseline C-peptidedata, and duration of diabetes are re-ported in Table 1. T2D patients werenot requiring insulin treatment at diag-nosis and not treated with insulin or athiazolidinedione during follow-up. Pre-scribed medications for the enrolledpatients are listed in Table 2. Study visitswere scheduled at 3-month intervalsfor up to 36 months. Written informedconsent was obtained from each patientbefore participation. Type 1 associatedHLA data for the T2Dpatients are reportedin Table 3. This study was approved bythe University of Washington and the VAPuget Sound Health Care System Institu-tional Review Boards.

CI T-Cell AssayCI was performed on freshly isolated pe-ripheral blood mononuclear cells fromT2D patients, as previously described,to test for the presence of islet-specificT cells (21–30). Briefly, normal hu-man islet cell preparations underwentpreparative one-dimensional 10% SDS-PAGE, electroblotted onto nitrocellu-lose, and the nitrocellulose particlescontaining the islet proteins were pre-pared using carbonate/bicarbonate buf-fers. The nitrocellulose particles werethen added to freshly isolated periph-eral blood mononuclear cells frompatients, and the cultures were incu-bated for 5 days, pulsed with tritiatedthymidine for 18 h, and thymidine in-corporation was assessed using a bcounter. Human pancreatic islets wereobtained from the National Institutes ofHealth–supported Islet Cell ResourceCenters Administrative and Bioinfor-matics Coordinating Center. The speci-ficity of the T-cell responses to the isletproteins was demonstrated previously

Table 1—Characteristics of patients divided by islet Ab and islet-reactive T-cellpositivity

Patient category (N = 23)

Ab2T2 Ab+T2 Ab2T+ Ab+T+ Unknown*

Patients, n (%) 6 (26) 2 (9) 7 (30) 5 (22) 3 (13)

Age (years) (median 6 SE) 53 6 4 60 6 1 57 6 5 58 6 3 49 6 4

Sex (%)Males 17 100 71 80 67Females 83 0 29 20 33

BMI (kg/m2) (median 6 SE)Baseline 43 6 2 34 6 2 36 6 2 27 6 1 32 6 2Study end 43 6 2 34 6 1 34 6 2 29 6 2 31 6 2

Length of follow-up (months)(median 6 SE) 36 6 4 23 6 14 15 6 4 36 6 4 33 6 6

Baseline FCP (ng/mL) (median 6 SE) 5 6 0.4 5 6 0.2 3 6 0.4 3 6 0.2 4 6 1

Baseline glucagon-SCP (ng/mL)(median 6 SE) 9 6 1 9 6 2 5 6 1 5 6 0.4 7 6 2

Race (%)Caucasian 83 50 100 60 67African American 0 0 0 20 0Asian 17 50 0 20 33

T2D disease duration (%),1 year 17 100 14 20 01–2 years 33 0 43 40 67.2 years 50 0 43 40 33Mean 6 SD (years) 2 6 2 0.3 6 0.1 3 6 2 2 6 2 3 6 2Median 6 SE (years) 3 6 1 0.3 6 0.1 2 6 1 2 6 1 2 6 1

Positivity (%)GADA+ 0 50 0 100 33IA-2+ 0 100 0 20 33Islet T cells+ 0 0 100 100 67

*Autoimmune positive 1 time point only.

care.diabetesjournals.org Brooks-Worrell, Boyko, and Palmer 3287

(23). T-cell responses from T1D patientswere not hyperresponsive to controlantigens nor stimulated by proteinsfrom other control tissues (23).CI has been evaluated in two distinct

T-cell validation workshops. One valida-tion workshop was sponsored by theImmune Tolerance Network (ITN) andthe other by TrialNet (21,22). The work-shops were designed to test the abilityof several different assays, including CIperformed in our laboratory, to distin-guish T-cell responses to islet proteinsof T1D patients from control subjects.Both workshops used masked speci-mens, and CI was identified as an assaycapable of distinguishing T1D patientsfrom control subjects with excellent

sensitivity and specificity. A cutoffstimulation index $2.0 was used todetermine positive responses to eachblot section. This cutoff corresponds to3 SDs above the mean response of nor-mal control subjects to islet proteins(23). Overall T-cell positivity at eachtime point was set at four or more blots,the same cutoff used for T1D with thisassay (21,22,24). Patients were consid-ered positive for islet-reactive T cells ifthey were positive for four or more blotssections during at least two study visits.

C-Peptide AssaysFCP levels and glucagon-SCP responseswere used as measures of endogenousb-cell function. SCP wasmeasured 6min

after the intravenous injection of 1 mgglucagon. The C-peptide assay is a two-siteimmune-enzymatic assayperformedusingaTosoh 600 II autoanalyzer (TosohBioscienceInc., South San Francisco, CA) at the North-west Lipid Metabolism and Diabetes Re-search Laboratories (NWLMDRL), Seattle,WA (27). The interassay and intra-assay pre-cision analysis showed a coefficient of vari-ation of less than 10%. The assay has asensitivity level of 0.04 ng/mL.

GAD-65 Ab AssayGADA levelsweremeasured atNWLMDRLin a radiobinding immunoassay on codedserum samples, as previously described(32). The levels of GADA were expressedas a relative index (GADA index) using onepositive serum (JDF World Standard forICA) and three negative standard serafrom healthy subjects. Positivity was setat a GADA index $0.085, the 99th per-centile based on 200 normal control sub-jects. Positive and negative controls arerun in duplicate in each assay. NWLMDRLhas participated in IDS-sponsored Diabe-tes Antibody Standardization Programs(DASP). In the 2010 DASP workshop, thesensitivity of theGADA assaywas 82%andspecificity was 93.3% (33). NWLMDRL isactively participating in the National Insti-tutes of Health–sponsored Ab harmoniza-tion program. Patients were consideredpositive for GADA if they were positiveduring at least two study visits.

IA-2 Ab AssayIA-2 levels were measured at NWLMDRLunder conditions identical to those de-scribed for the GADA assay, using the plas-mid containing the cDNA coding for thecytoplasmic portion of IA-2. Positivity wasset at an IA-2 index of $0.017, the 99thpercentile based on a set of 200 normalcontrol subjects. In the 2010 DASP work-shop, this assay scored a sensitivity of 62%and specificity of 100% for its ability todistinguish T1D patients from normal

Table 2—Percentage of patients in each group receiving specified classes of drugsthroughout follow-up

Patient category

Ab2T2 Ab+T2 Ab2T+ Ab+T+ Unknown*n = 6 n = 2 n = 7 n = 5 n = 3

Diabetes medications†Sulfonylureas 83 50 57 80 67Biguanides 83 100 86 80 100Meglitinides 17 0 0 0 0a-Glucosidase inhibitors 33 0 14 0 33

Cholesterol medicationsStatins 33 100 29 60 67Fibrates 0 0 14 40 33Niacin 0 50 0 0 33

Blood pressure medicationACE inhibitors 50 100 14 20 67Angiotensin receptor blocker 0 0 14 0 0b-Blockers 50 50 29 20 100Calcium channel blockers 0 0 43 0 100Diuretics 33 0 43 0 67a-Blockers 17 0 43 60 0

Other medicationsLevothyroxine sodium 33 0 0 0 0Aspirin 17 50 14 40 67Antiepileptic agent 17 0 14 40 0Serotonin reuptake inhibitor 0 0 29 0 33b2-Adrenergic agonist 0 0 14 20 0

*Autoimmune positive 1 time point only. †No patients were treated with thiazolidinediones,dipeptidyl peptidase-4 inhibitors, or sodium-glucose linked transporter 2 inhibitors.

Table 3—Percentage of T1D-associated HLA class II alleles present in the T2D patients

Patient category

Ab2T2 Ab+T2 Ab2T+ Ab+T+ Unknown*n = 6 n = 2 n = 7 n = 5 n = 3

HLA class II (%)Susceptibility alleles (DRB1*03xx/04xx/*0101;

DQB1*0302/*0301/*0501) 50 (3/6) 0 86 (6/7) 40 (2/5) 67 (2/3)Protective alleles (DRB1*1501; DQB1*0602) 0 0 14 (1/7) 60 (3/5) 0

Other alleles 50 (3/6) 100 (2/2) 0 0 33 (1/3)

*Autoimmune positive 1 time point.

3288 Development of Islet Autoimmunity in T2D Diabetes Care Volume 37, December 2014

control subjects (33). Patientswere consid-ered positive for IA-2 if they were positiveduring at least two study visits.

StatisticsData were analyzed with nonparametrictests for comparison of medians andmeans, x2 and Fisher exact tests forcomparison of percentages, and multi-variable linear regression. The medianpercentage change was calculated asdescribed by Sosenko et al. (34). Themedianpercentage changewas comparedamong patients before and after develop-ment of autoimmunity and patients whodid not develop autoimmunity usingANOVA for three-group comparisonsand the Mann-Whitney U test for two-group comparisons. Two-sided P, 0.05was considered statistically significant.

RESULTS

Patient Categories and DemographicsWe identified 6 of 23 patients (26%)whoremained nonautoimmune (Ab2T2)during follow-up. In contrast, 14 (61%)demonstrated positivity for islet Abs(GADA and/or IA-2) and/or islet-specificT cells. We categorized the patients intothe following groups by their autoim-mune status: Ab2T2 (n = 6), Ab+T2(n = 2), Ab2T+ (n = 7), and Ab+T+ (n =5). We also observed three patients(13%) who were positive for Abs and/orT cells on only one study visit. The

responses of these 3 patients were keptseparate from the other nonautoimmunepatients and placed in a separate grouplabeled “unknown.” The “unknown” pa-tients were not used in further analysis.

Tables 1 and 2 report characteristics ofsubjects, including age, race, sex, BMI, di-abetes features, andmedication use by au-toimmune (Ab+T2, Ab2T+, and Ab+T+)and nonautoimmune (Ab2T2) T2D status.HLA alleles protective for T1D (DR*1501;DQB1*0602) were more frequent in theAb+T+ than in any other group, whereasthe T1D susceptibility alleles were morefrequent in the Ab2T+ patients (Table 3).

Autoimmune Responses to IsletProteinsSix of the 23 T2D patients (26%) re-mained negative for islet Abs and islet-reactive T cells (Ab2T2), whereas 7patients (30%) developed islet-reactiveT cells but remained Ab2T+. Figure 1illustrates the longitudinal T-cell re-sponses for the Ab2T2 (Fig. 1A) andAb2T+ (Fig. 1B) T2D patients. Figure 2shows the longitudinal islet Ab re-sponses for the two Ab+T2 T2D pa-tients. Both patients were positive forIA-2 (Fig. 2A) and one patient was alsopositive for GADA (Fig. 2B). The negativeislet-specific T-cell responses for thesepatients (Ab+T2) are not shown.

Islet Abs and islet-reactive T cells (Ab+T+)developed in 5 of the 23 patients (22%).

All five patients were positive for GADA,and one was positive for both GADA andIA-2. The T-cell results for the Ab+T+ pa-tients are shown in Fig. 3A, and theAb dataare shown in Fig. 3B (GADA) and Fig. 3C (IA-2). The patient who was positive for IA-2,developed IA-2+ and T+ positivity at 3months, became IA22 at 6 months, butremained T+ throughout the study (36months). This patient also becameGADA+at 9months,GADA2 at15months,then GADA+ again from 18 to 36 months.Another patient who developed T-cell pos-itivity at 33–36 months was GADA+ be-tween 6 and 24months and thenGADA2negative from 27 to 36 months. Thispatient was IA-22 throughout follow-up.

Three patients who developed posi-tivity for either islet Abs (GADA or IA-2)or islet-reactive T cells on only one studyvisit were classified as “unknown.” Thesepatients were observed to have a highpercentage of HLA T1D susceptibilityalleles (Table 3). Even though thesepatients were negative as defined forthis study, they appeared to bepotentiallydifferent from the Ab2T2 patients.Therefore, we did not include thesepatients in any further C-peptide analysis.

Two of the “unknown” patients devel-oped T-cell positivity at one time pointbut then became negative at subse-quent time points. In the remaining 12subjects (52%) in the study who de-veloped T-cell reactivity, the T-cell

Figure 1—Longitudinal islet-specific T-cell responses for six Ab2T2 T2D patients (A) and seven Ab2T+ T2D patients (B). The horizontal line at threeblot sections represents the cutoff for T-cell positivity.

care.diabetesjournals.org Brooks-Worrell, Boyko, and Palmer 3289

reactivity remained positive throughoutfollow-up. Islet Ab responses were ob-served to fluctuate between positivity

and negativity in most patients, al-though GADA positivity appeared to bemore stable than IA-2 positivity.

C-Peptide ResponsesThe median baseline FCP and glucagon-SCP responses for the five patientgroups are reported in Table 1. Thebaseline glucagon-SCP was observedto be lower in T+ patients (Ab2T+ andAb+T+) compared with the T2 patients(Ab2T2 and Ab+T2). These differenceswere present irrespective of age, BMI,diabetes duration, race, Abs, and sex.

To investigate the effect of islet auto-immune development on C-peptide re-sponses, we separated the responsesfor the groups developing islet auto-immunity (Ab+T2, Ab2T+, Ab+T+) cor-responding to the time interval beforeor after autoimmune development. Toaccount for the lower baseline FCP andSCP responses in the T+ patient groups,we compared the median percentagechange from baseline in FCP andglucagon-SCP. We then compared theseresponses with the patients who did notdevelop islet autoimmunity (Ab2T2).No significant differences were observedin median percentage change for FCPlevels between the nonautoimmune andthe autoimmune patients before auto-immune development (Fig. 4A). However,after autoimmune development, therewas a significant decline in FCP in theautoimmune patients compared withthe FCP of the nonautoimmune patients(P, 0.0001) and compared with the FCPbefore autoimmune development (P ,0.01, Fig. 4A).

Significant declines were also ob-served for the glucagon-SCP responsesbetween the autoimmune patients afterautoimmune development and the non-autoimmune patients (P , 0.05) andalso between the autoimmune patientsbefore autoimmune development andafter autoimmune development (P ,0.001, Fig. 4B). Interestingly, a signifi-cant difference (P , 0.02) was alsonoted in the SCP between the non-autoimmune and autoimmune patientsbefore autoimmune development(Fig. 4B).

CONCLUSIONS

The analysis of T-cell responses to isletproteins using the CI assay has allowedus to identify, in cross-sectional studies,cellular islet autoimmunity in T1D (21–23,25), in subjects at risk for T1D (24),and in phenotypic T2D patients (26–30).The presence of islet-specific cellular au-toimmunity in T2D patients was also

Figure 2—Longitudinal islet Ab responses for two Ab+T2 T2D patients. A: Both patients de-veloped IA-2 positivity, and one patient developed both IA-2 and GADA. B: GADA results. Thehorizontal lines show the cutoff for positive responses.

Figure 3—A: Five T2D patients developed both islet-specific T-cell reactivity and islet Abs duringfollow-up (Ab+T+). All five patients were GADA+ (B), and one patient was also IA-2+ (C). Thehorizontal lines show the cutoff for positive responses.

3290 Development of Islet Autoimmunity in T2D Diabetes Care Volume 37, December 2014

recently confirmed by an independentlaboratory (35). However, longitudinalstudies investigating islet autoimmunedevelopment in T2D patients havebeen lacking.In this study, we observed develop-

ment of islet autoimmunity, measuredby islet Abs and islet-specific T-cell re-sponses, in 61% of the phenotypic T2Dpatients. We also observed a significantassociation between positive islet-reactive T-cell responses and amore rapiddecline in b-cell function as assessedby FCP and glucagon-SCP responses.Moreover, the high percentage (30%)of Ab2T+ T2D patients observed in thisstudy supports our previously publishedcross-sectional observations and dem-onstrates the importance of identifyingislet autoimmunity by assaying both Absand T cells (27,28,36). This result is ofimportance, noting that historically,islet autoimmunity in T2D had beendetermined based solely on islet Ab pos-itivity (primarily GADA).In this study, we also observed that

islet-reactive T-cell responses appearedto be more stable compared with theislet Ab responses in autoimmune T2Dpatients. With regard to the islet Abs,GADA responses were more consis-tently positive over time comparedwith the IA-2 responses. In our previousstudies, we have observed very few T2Dpatients to be positive for insulin Abs(IAA) or zinc transporter Abs (ZnT8A).

Therefore, in our current study, we didnot test our patients for IAA or ZnT8A.Though T1D has been hypothesized tobe a “relapsing-remitting” disease, thestability of the positive islet-reactiveT-cell responses we observed in thisstudy do not support this concept inT2D autoimmune pathogenesis (37).The instability of the islet Abs may pos-sibly suggest that islet Abs specific forT2D are different from those associatedwith T1D or reflect a different patho-genesis of islet autoimmunity in T2Dversus T1D.

We also previously observed that T2Dpatients treated with rosiglitazone ex-perienced attenuation of islet-reactiveT cells and decreases in interferon-gand interleukin-12 responses, with asso-ciated improvement in b-cell function(29). On the basis of our previous data,we excluded from our current study T2Dpatients who were treated with thiazo-lidinediones. Assessing the prescribedmedications of the enrolled subjects inthis study did not demonstrate anyassociations between their currentmedications and development or atten-uation of islet autoimmunity (Table 2).We also did not evaluate cytokine re-sponse in this study, but this would beof interest in future work.

In the patients classified as Ab+T2,we observed a higher baseline C-peptideresponse compared with the T+ pa-tients (Table 1). The higher baseline C-

peptide values resulted in the significantdifferences observed in SCP responsesbetween Ab2T2 and the autoimmunepatients before autoimmune develop-ment may reflect the incorporation ofthe Ab+T2, who had higher baselineSCP, into the autoimmune patient group(Fig. 4B). We also observed a shorter T2Ddisease duration in the Ab+T2. Theseresults may indicate that these patientsare earlier in their disease pathogenesisand may be destined to develop islet-specific T-cell reactivity at a later date.Another hypothesis might be that thepresence of IA-2 in T2D patientsindicates a level of protection resultingin higher C-peptide preservation. Inter-estingly, these patients were found tolack both the HLA class II susceptibilityand protective alleles associated withdevelopment of T1D (Table 3) andpotentially islet autoimmunity in T2D.Follow-up of this group of patients andstudies in larger numbers of patients willhelp to address these questions.

Differences were also observed be-tween the baseline FCP and glucagon-SCP in the T+ T2D patients comparedwith the T2 T2D patients. These resultssuggest that the appearance of T+ in theperipheral bloodmay be a biomarker forpatients whose disease path includes is-let autoimmune damage. The associa-tion of a more rapid decline in b-cellfunction in T+ patients indeed suggeststhat the disease path for these patientsincludes islet autoimmune damagewithin the pancreas. Interestingly, wealso observed that 60% of our Ab+T+patients and 15% of our Ab2T+, com-pared with 0% of our Ab2T2 patients,were DR*1501 DQB1*0602 (Table 3).The results of our study may suggestthat DR*1501 DQB1*0602 is associatedwith the development of islet-reactive Tcells in adult T2D patients. If this is thecase, DR*1501 DQB1*0602 may provideprotection against development of isletautoimmunity and T1D in childhood butnot against development of islet auto-immunity in adult T2D. If confirmed, ourresults would be in contrast to Williamset al. (38), who observed that HLA-DRB1*02 was protective for T2D in full-heritage Pima Indians and may indicatethat racial or ethnic differences do existwith regards to development of islet auto-immunity in T2D patients.

The results of this pilot study led us tohypothesize that islet autoimmunity is

Figure 4—Median percentage change in FCP levels (A) and glucagon-SCP (B) responses for thenonautoimmune patients (Ab2T2) and patients developing islet autoimmunity (Ab+T2, Ab-T+,and Ab+T+) before and after autoimmune development.

care.diabetesjournals.org Brooks-Worrell, Boyko, and Palmer 3291

present or will develop in a large portionof phenotypic T2D patients and that thedevelopment of islet autoimmunity isassociated with a more rapid decline inb-cell function. Moreover, the preva-lence of islet autoimmunity in most pre-vious studies is grossly underestimatedbecause these studies have not testedfor islet-reactive T cells in T2D patientsbut have based the presence of autoim-munity on antibody testing alone. Pre-viously, the importance of quantifyingislet autoimmunity through the mea-surement of the islet-reactive T cells inT1D patients was emphasized bythe study of Wang et al. (36) demon-strating that ;20% of newly diagnosedautoimmune T1D patients were Ab2.The results of this pilot study suggestimportant changes to our understandingof T2D pathogenesis by demonstratingthat the prevalence of islet autoimmunedevelopment is not only more prevalentin T2D patients than previously esti-mated but may also play an importantrole in b-cell dysfunction in the T2D dis-ease process. The results of our pilotstudy await confirmation in future largerstudies.

Funding. This work was supported by resourcesfrom the VA Puget Sound Health Care System,Seattle, WA. In addition, partial support wasprovided by National Institutes of Health grantsP01-DK-053004 and P30-DK-017047.Duality of Interest. No potential conflicts ofinterest relevant to this article were reported.Author Contributions. B.M.B.-W. and J.P.P.contributed equally to the research, dataanalysis, and manuscript preparation. E.J.B.contributed to data analysis and manuscriptpreparation. J.P.P. is the guarantor of this workand, as such, had full access to all data in thestudy and takes responsibility for the integrityof the data and the accuracy of the dataanalysis.Prior Presentation. Parts of this study werepresented as an oral presentation at the 73rdScientific Sessions of the American DiabetesAssociation, Chicago, IL, 21–25 June 2013.

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34. Sosenko JM, Palmer JP, Rafkin-Mervis L, et al.Glucose and C-peptide changes in the perionsetperiod of type1diabetes in theDiabetes PreventionTrial-Type 1. Diabetes Care 2008;31:2188–219235. Sarikonda G, Pettus J, Phatak S, et al. CD8 Tcell reactivity to islet antigens is unique to type 1while CD4 T cell reactivity exists in both type 1

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