Extending Remission and Reversing New-Onset Type 1Diabetes by Targeted Ablation of Autoreactive T CellsKaitlin R. Carroll,1 Eileen E. Elfers,1 Joseph J. Stevens,1 Jonathan P. McNally,1 David A. Hildeman,1

Michael B. Jordan,1,2 and Jonathan D. Katz1,3

Diabetes 2018;67:2319–2328 | https://doi.org/10.2337/db18-0204

Preserving endogenous insulin production is clinicallyadvantageous and remains a vital unmet challenge inthe treatment and reversal of type 1 diabetes. Althoughbroad immunosuppression has had limited success inprolonging the so-called remission period, it comes atthe cost of compromising beneficial immunity. Here, weused a novel strategy to specifically deplete the activateddiabetogenic T cells that drive pathogenesis while pre-serving not only endogenous insulin production but alsoprotective immunity. Effector T (Teff) cells, such as di-abetogenic T cells, are naturally poised on the edge ofapoptosis because of activation-induced DNA damagethat stresses the p53 regulation of the cell cycle. We havefound that using small molecular inhibitors that furtherpotentiate p53while inhibiting the G2/M cell cycle check-point control drives apoptosis of activated T cells in vivo.When delivered at the onset of disease, these inhibitorssignificantly reduce diabetogenic Teff cells, prolongremission, preserve functional islets, and protect isletallografts while leaving naive, memory, and regulatoryT-cell populations functionally untouched. Thus, the tar-geted manipulation of p53 and cell cycle checkpointsrepresents a new therapeutic modality for the preserva-tion of islet b-cells in new-onset type 1 diabetes or afterislet transplant.

Type 1 diabetes develops silently and clinical presentationoccurs only after the destruction of a biologically sufficientmass of insulin-producing b-cells, yet an estimated 30% ofb-cell mass remains at disease onset (1). The preservation

of insulin and C-peptide, the portion of the proinsulinmolecule cleaved and secreted in equimolar concentrationto insulin, is known to have significant clinical benefits inpreventing severe hypoglycemia, retinopathy, nephropa-thy, and neuropathy (2–4). To preserve the b-cells, how-ever, subsequent autoimmune attack must be prevented.Although detectable insulin production can persist foryears or even decades in some patients (5), the functionalcapacity of b-cells decreases in response to continuedimmune-mediated damage by an average of 40% withina year after diagnosis (6). Thus, interventions that halt furtherimmune damage or induce the durable re-establishment ofimmune tolerance to b-cells remain both imperative andelusive. Moreover, without functional immune tolerance tob-cells, recent technical strides in restoring, regrowing, ortransplanting islets are likely to fail.

Although a durable means of controlling islet antigen–specific T cells remains to be elucidated, the salient qual-ities of an effective therapy are clear. Given the establishedefficacy of insulin replacement therapy, any direct controlor elimination of autoreactive T cells must be well toleratedwith minimal off-target effects, must be specific and sparenaive and memory T cells required for robust immunity topathogens, and must target the bulk of autoreactive T cells,even those for which the antigen specificity is unknown. Assuch, the ideal approach would target one or more intrinsictraits shared by all activated autoreactive effector cells atthe time of disease onset.

Recently, we identified such requisite properties inactivated effector T (Teff) cells that permit their

1Division of Immunology, Cincinnati Children’s Hospital Medical Center and De-partment of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH2Division of Bone Marrow Transplantation and Immune Deficiency, Department ofPediatrics, Cincinnati Children’s Hospital Medical Center, University of CincinnatiCollege of Medicine, Cincinnati, OH3Division of Endocrinology, Diabetes Research Center, Department of Pediatrics,Cincinnati Children’s Hospital Medical Center, University of Cincinnati College ofMedicine, Cincinnati, OH

Corresponding author: Jonathan D. Katz, jonathan.katz@cchmc.org, or Michael B.Jordan, michael.jordan@cchmc.org.

Received 20 February 2018 and accepted 29 July 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0204/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 67, November 2018 2319

IMMUNOLOGY

AND

TRANSPLANTATIO

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targeted elimination while sparing naive T-cell, regulatoryT (Treg) cell, and memory T-cell subsets (7). Teff cells areexceptional for their extraordinarily rapid rate of cell di-vision (8) and exhibit significant spontaneousDNA breakageand concomitant DNA damage response (DDR) upon acti-vation (7). Initiation of the DDR results in either cell cyclearrest, where progression through the cell cycle is pre-vented until DNA damage is repaired, or in apoptosis. Thiscell fate decision depends on the graded accumulation ofactivated p53 (phospho-Ser15) in the cell (9). Becauseactivated p53 accumulates, the tipping point is reached atwhich the cell undergoes apoptosis if the damage is toosevere to repair (9). We reasoned that since Teff cells balanceon this edge of apoptosis, by using small-molecule inhib-itors targeting distinct proteins in the DDR pathway suchas CHK1/2 and WEE1, which govern cell cycle arrest (inboth S phase and at the G2/M checkpoint), Teff cellswould enter mitosis prematurely (7,10–12), and wecould force the selective apoptosis of Teff cells with theiramplified p53 while sparing other T-cell subsets shielded byvirtue of their slower proliferative rate (13) and dimin-ished DDR (7). When these are combined with an in-hibitor of mouse double minute 2 homolog (MDM2; thenegative regulator of p53), the accumulation of activatedp53 is further potentiated (14), the net result of which isa synergistic increase in apoptosis. Importantly, cells notin cycle or without a concomitant DDR, such as naiveT cells, Treg cells, and quiescent memory T cells, wouldbe largely spared. We previously showed that the com-bination of these inhibitors, which we have termed“p53 potentiation with checkpoint abrogation” (PPCA),conferred significant therapeutic efficacy in treatingmouse models of multiple sclerosis (experimental auto-immune encephalomyelitis) and hemophagocytic lym-phohistiocytosis (7).

We hypothesized that PPCA administration at the firstonset of disease would specifically reduce or eliminatediabetogenic Teff cells, thus halting further damage tob-cells and sustaining residual endogenous insulinproduction. Herein, we present evidence that PPCA iseffective and well tolerated in the treatment of type 1diabetes in multiple clinically relevant circumstances byselectively targeting activated diabetogenic T cells, whileexhibiting minimal off-target effects on naive T-cell, Tregcell, and memory T-cell populations. Thus, by manipulat-ing p53 and cell cycle checkpoints, we can exploit theendogenous response of activated T cells in a novel andpromising new approach to re-establishing immune toler-ance in type 1 diabetes in a durable, selective, and non-harmful way.

RESEARCH DESIGN AND METHODS

MiceNOD/ShiLtJ (NOD), NOD.Cg-Tg(TcraBDC2.5, TcrbBDC2.5)1Doi/DoiJ (BDC2.5.NOD), NOD.129S7(B6)-Rag1tm1Mom/J(NOD.Rag2/2), NOD.B6-Ptprcb/6908MrkTacJ (CD45.2NOD), and C57BL/B6 (B6) mice were purchased from The

Jackson Laboratory; bred; and maintained under specificpathogen-free conditions in accordance with institutionalanimal care guidelines at Cincinnati Children’s HospitalMedical Center Vivarium.

Blood Glucose AssessmentPrediabetic NOD mice were monitored beginning at10 weeks of age for clinical signs of prediabetes (hyper-glycemia, assessed by nonfasting blood glucose [BG] test-ing). Diabetes onset was determined via two consecutiveBG readings of $200 mg/dL. Mice were monitored tri-weekly from this point until they developed end-stagedisease (BG $600 mg/dL); lost .20% body weight; estab-lished long-term normoglycemia for at least 60 days; ordied.

Drug TreatmentsAll chemotherapeutics were administered intraperitone-ally, and were dosed as follows: AZD1775 (WEE1 inhibitor[WEE1i]; ChemieTek), 40 mg/kg; AZD7762 (CHK1/2inhibitor [CHKi]; Selleck Chemicals), 25 mg/kg; andnutlin-3 (MDM2 inhibitor nutlin-3 [MDM2i]; CaymanChemical), 50 mg/kg. Vehicle was made as previouslydescribed (7).

MHC Tetramer Staining and Flow CytometrySingle-cell suspensions of spleen and pancreatic lymphnodes were separated by density gradient centrifugationusing Lympholyte-M and incubated with tetramer (Na-tional Institutes of Health Tetramer Core). Cells were thenstained for surface expression of the indicated antibodies.For intracellular staining, cells were fixed with the FoxP3fixation/permeabilization buffer and permeabilized withpermeabilization buffer. Flow data were collected using anLSR Fortessa System using FACS Diva software and ana-lyzed using FlowJo (Tree Star).

Isolation of Pancreatic LymphocytesSingle-cell suspension of pancreatic cells was preparedusing the Miltenyi Biotec gentleMACS Dissociator, filteredthrough a 70 mm cell strainer, and prepared according tothe Miltenyi Biotec Debris Removal Solution protocol. Iso-lated lymphocytes were then stained in accordance with themethods listed above.

Histological Scoring/ImmunofluorescencePancreas was formalin fixed and paraffin embedded. Sec-tions were stained with VectaFluor R.T.U. Antibody Kit(Vector Laboratories). Images were taken and analyzedusing NIS Elements Imaging Software. Insulitis was scoredas follows: 0, no visible infiltration; 1, peri-insulitis; 2,insulitis with,50% islet infiltration; 3, insulitis with.50%islet infiltration; and 4, islet scar.

T-Cell Isolation and EnrichmentT cells were prepared for adoptive transfer in accor-dance with the MojoSort Mouse CD3 T Cell Isolation Kit(BioLegend) protocol. A total of 107 cells were injected viaretro-orbital i.v. injection in 250 mL of DMEM.

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Next-Generation RNA Sequencing and AnalysisPolyA mRNA purification, quality control, and next-generation sequencing (NGS) were performed by theUniversity of Cincinnati Genomics, Epigenomics, and Se-quencing Core on an Illumina HiSEq 2500 System at50 million raw reads per sample, 150–base pair pairedends. AltAnalyze was used tomap reads (via built-in Kallistosoftware), create expression files, and perform initialGO-Elite functional annotation with reads per kilobase oftranscript per million mapped reads (less than one removed;GO-Elite filtered based on the minimum fold change of 2.0;P , 0.05, t test). Principal components analysis was per-formed within both AltAnalyze and MATLAB. Gene setenrichment analysis (GSEA) was run through the corre-sponding modules within GenePattern. Gene sets run onGSEA were collected from the Molecular Signatures Data-base version 6.1 and ImmGen. Additional comparisons ofupregulated and downregulated genes (2.0-fold, P , 0.05)were compared with annotated gene lists using ToppGene.Heat maps were generated in R.

Islet TransplantationIslet transplants were performed as described previously(15). Briefly, streptozotocin (STZ) (200 mg/kg) was

administered to NOD.Rag2/2 mice to induce diabetes,after which ;300 islets isolated from B6 mice weretransplanted under the left kidney capsule.

Statistical AnalysisAll statistical analyses were performed using one-way ortwo-way ANOVA, log-rank Mantel-Cox test, or multiplet test using GraphPad Prism version 6.07 for Windows.

RESULTS

Treatment With PPCA Significantly Extends theRemission Period in New-Onset Type 1 DiabetesAt the onset of type 1 diabetes, some residual b-cellsremain, yet, absent intervention, they will continue tobe destroyed by acutely activated diabetogenic T cells overtime. Therefore, we reasoned that since PPCA specificallytargets activated, proliferating Teff cells, the treatment ofnew-onset disease in NOD mice with PPCA should affectlong-term glycemic control. We used two different PPCAcombinations, a CHKi, and a WEE1i, each coupled with theMDM2i. Since inhibiting either the CHK1/2 or WEE1checkpoint kinase prevents the inhibitory phosphoryla-tion of CDKs, cycling Teff cells skirt the normal S and G2DNA damage regulatory checkpoints (10,11,16) and enter

Figure 1—Extension of diabetes remission and diabetes reversal with preservation of pancreatic b-cells after PPCA therapy in NODmicewithnew-onset diabetes. A and B: NOD mice developed spontaneous diabetes, defined as a BG concentration of 200–250 mg/dL, and weretreated with PPCA (either WEE1i+MDM2i [blue] or CHKi+MDM2i [green]) or were left untreated (red) on days 1–3 after development and asneeded thereafter and were monitored for disease development. A: BG measurements over time; the color inside symbol representsindividual mice.N = 5–11 mice/group. B: Percentage of mice with a BG concentration$600mg/dL over time.N = 5–11mice/group.C andD:NOD mice that were treated with WEE1i and MDM2i or vehicle after developing diabetes (BG .200 mg/dL) and harvested on day 60 post–disease development or at end-stage disease. C: Representative images of islets; pancreas was fixed in formalin and embedded in paraffin.Blue, DAPI; red, insulin; green, glucagon. D: Insulitis severity scores. At end-stage disease or at day 60 post–disease development, thepancreas was scored for insulitis (see RESEARCH DESIGN AND METHODS). **P . 0.01. Veh., vehicle.

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mitosis prematurely; the concurrent inhibition of p53 neg-ative regulator MDM2 (14) augments the accumulation ofphospho-p53, and, in the setting of mitotic dysregulation,drives a strong and synergistic increase in apoptosis ofthese Teff cells. To test this, we treated NOD mice imme-diately after the onset of spontaneous diabetes (200–250 mg/dL BG) for 3 successive days with PPCA andmonitored subsequent changes in BG levels. This treat-ment strategy was used because 3 days of treatment wasshown to be effective in reducing BG levels while still beingwell tolerated. Given that PPCA requires Teff cells to be inrapid cell cycle for maximum efficacy and that not all islet-reactive T cells are in the same synchronous activationstate, it was not surprising that we had to retreat mostnew-onset mice multiple times to maintain a BG lev-el ,200 mg/dL; the number of retreatments varied, butmice receiving WEE1i+MDM2i received on average 7.5courses of treatment. NODmice with untreated new-onsetdiabetes were used as a control group for comparison.The mice with new-onset diabetes treated with eitherPPCA combination therapy maintained glycemic control(BG ,200 mg/dL) for 51% (WEE1i+MDM2i) and 53%(CHKi+MDM2i) of the enrolled days (a period rangingfrom 21 to 234 days) (Fig. 1A). This is in stark contrastto the untreated mice, which were only able to maintainglycemic control for 5% of the days they were enrolled

(a period ranging from 34 to 72 days) (Fig. 1A). AlthoughPPCA ameliorated disease in all spontaneously diabetic NODmice, it was most efficacious when given to NOD micewith new-onset disease with BG levels between 200 and250 mg/dL at the time of disease onset, suggesting thatPPCA is most efficient when used early, at a time when sig-nificant b-cell function remains. In addition, PPCA-treatedmice had significantly decreased incidence of end-stagediabetes (BG $600 mg/dL), a metric used because it isindicative of a point at which endogenous insulin produc-tion, as measured by C-peptide levels (,150 ng/mL) (Sup-plementary Fig. 1), was minimal. Although all untreated miceprogressed to end-stage diabetes in an average of 25 days,71% of PPCA-treated mice never reached end-stage dis-ease (seven of nine mice treated with WEE1i+MDM2i andfive of eight mice treated with CHKi+MDM2i) (Fig. 1B).Both PPCA combinations yielded comparable results over-all, but WEE1i+MDM2i performed slightly better at pre-venting progression to end-stage disease; therefore, weopted to use this combination for subsequent experiments,unless otherwise noted. These results corresponded withsignificant preservation of functional b-cells at day 60or end-stage disease after onset and treatment. Micetreated with PPCA showed a marked preservation of isletmass, with comparable levels of insulitis and islet dam-age to prediabetic mice and significantly fewer severely

Figure 2—PPCA decreases islet antigen–specific T cells. New-onset diabetic NODmice were treated withWEE1i andMDM2i or vehicle days1–3 after developing spontaneous diabetes (BG 200–250 mg/dL) and harvested on day 4. Lymphocytes isolated from spleen were assessedfor total diabetogenic CD4+ and CD8+ T cells by I-Ag7 and H-2Kd tetramer (Tet) staining, respectively. A: Representative flow cytometricanalysis of Insulin B12–20 I-A

g7 tetramer stained CD4+ CD44hi T cells from spleen. B: Absolute number of CD4+ tetramer+ cells from diabeticNODmice treated with WEE1i and MDM2i (blue) or vehicle (red) and from prediabetic controls (black).C: Absolute number of CD8+ tetramer+

cells from diabetic NOD mice treated with WEE1i and MDM2i (blue) or vehicle (red) and from prediabetic controls (black); n = 20–43mice/group. *P . 0.05, **P . 0.01, ***P . 0.001, ****P . 0.0001.

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infiltrated or destroyed islets than vehicle-treated con-trols (Fig. 1C and D).

To determine the effects of PPCA therapy on immuneresponses, we quantified the T-cell response in mice trea-ted with or without PPCA. Lymphocytes were isolatedfrom the spleen on day 4 after disease development after3 days of treatment and were stained with tetramersfor insulin-specific T cells (Fig. 2A). After treatmentwith PPCA, the absolute numbers of both total activated(CD44+) CD4+ and CD8+ Teff cells as well as islet antigen–specific T cells were significantly decreased compared withthose isolated from the spleens of vehicle-treated andprediabetic controls (CD4+, Fig. 2B; CD8+, Fig. 2C), whichis consistent with the decreased number of activateddiabetogenic cells displaying increased levels of DNAdamage markers in PPCA-treated mice compared withvehicle-treated controls (Supplementary Fig. 2).

Treatment With PPCA Results in Decreased DiseaseTransferenceBecause insulin and islet-specific glucose-6-phosphatasecatalytic subunit-related protein (IGRP) tetramer reagentsonly identify a fraction of the total diabetogenic T cells, weassessed the overall pathogenicity of the T-cell compartmentafter PPCA in toto by adoptive transfer. As in previousexperiments, we treated NOD mice with new-onset diabetesfor 3 days with either PPCA or vehicle and harvestedlymphocytes 1 day after the last treatment.We then enrichedlymphocytes from the spleen and pancreatic lymph nodesfor CD3+ T cells, adoptively transferred 107 CD3+ T cells(CD45.1+) into CD45.2 NOD.Rag2/2 mice, and monitoredfor the development of disease. Mice that were injected withcells from PPCA-treated mice developed disease with a sig-nificantly lower incidence than those receiving cells fromvehicle-treated controls, demonstrating that PPCA ablatesdiabetogenic T cells such that they are significantly less ableto transfer disease (Fig. 3) and suggesting that PPCA reducedthe overall islet antigen–reactive pools of CD4+ and CD8+

T cells.

TreatmentWith PPCADecreases Islet Antigen–SpecificTeff Cells Without Compromising Other T-Cell SubsetsImportantly, the reduction in islet antigen–specific Teffcells in both the spleen and the pancreas occurs withoutsignificantly decreasing the absolute numbers of otherT-cell subsets, such as naive (CD44loCD62L+), regulatory(CD4+Foxp3+), and memory populations (Fig. 4A–C andSupplementary Fig. 3). This is consistent with data show-ing that only PPCA treatment significantly decreased cellswith DNA damage markers in activated diabetogenic cells,and not in naive or regulatory cells (Supplementary Fig. 4).Moreover, the diminution in Teff cells without significantloss in total and antigen-specific Treg cells resulted ina significant increase in the Treg/Teff ratio, both in thespleen and, more prominently, in the pancreas (Fig. 4Dand E). Interestingly, PPCA is significantly less effective inprolonging the remission in new-onset disease when dis-ease was induced using cyclophosphamide (Supplementary

Fig. 5), which is known to deplete Treg cells (17). Together,this suggests that, even without a total elimination of alldiabetogenic Teff cells, the clinical benefit resulting fromtheir significant reduction is likely due to the ability of thelargely unaltered islet-specific Treg-cell population to exertregulatory control and functional tolerance.

PPCA Results in Enriched Treg Cell Signature inResidual Activated CD4+ T CellsWe would predict from our mechanistic suppositions thatafter PPCA therapy the remaining activated T cells wouldmanifest an enriched Treg cell molecular signature, whileat the same time they would display reduced molecularevidence for cell cycle and DNA replication. To this end, weundertook NGS analysis of RNA from T cells sort purifiedfrom PPCA- or vehicle-treated new-onset diabetic NODmice, which were, as described above, treated with PPCA orvehicle for 3 days, and on day 4 CD4+ T cells were sortpurified into naive (CD442/lo), activated (CD44hi), andactivated insulin-specific (CD44hitet+) subsets; lysed; andsubjected to NGS. As expected, the naive CD4+ T cells fromboth groups had RNA expression signatures that differedfrom all the activated T-cell subsets but not from eachother, suggesting that PPCA does not alter the gene ex-pression profile of naive T cells (Supplementary Fig. 6).However, as predicted, both the activated and activated

Figure 3—Treatment with PPCA decreases disease transference inadoptive transfer. NOD mice were treated with WEE1i and MDM2i(blue) or vehicle (red) on days 1–3 after developing spontaneousdiabetes (BG $200 mg/dL) and were harvested on day 4. Lympho-cytes harvested from the spleen and pancreatic lymph nodes wereenriched for CD3+ T cells. CD45.2 NOD.RAG mice were adoptivelytransferred with 107 cells (i.v.) and were monitored for the develop-ment of diabetes (BG $200 mg/dL); n = 14–19 mice/group. Resultsare cumulative of two independent experiments. P = 0.0344.

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insulin antigen-specific subsets from PPCA- and vehicle-treated mice showed disparate expression profiles, whichstratified by drug treatment (Fig. 5A) but not tetramerreactivity, as both subsets of CD44hiCD4+ T cells (tetramerreactive and nonreactive) showed similar and highly re-lated expression profiles, and as such could be effectivelytreated as replicates rather than as distinct subpopulations(Fig. 5A and Supplementary Fig. 6). Using GSEA (18), weobserved the highly significant and predicted loss of G2/Mphase gene expression in the activated subsets of T cellsfrom PPCA-treated mice when compared with those fromvehicle-treated mice (Fig. 5B). Similar levels of both Ki67and gH2AX in the spleen and pancreas indicate that thisis also an accurate reflection of the apoptosis of activatedT cells in the islets (Supplementary Fig. 7). Importantly,PPCA preferentially augmented the Treg cell molecularsignature in activated CD4+ T cells (Fig. 5C); moreover, theleading-edge subset of Treg cell–expressed genes (Fig. 5D)included Ikzf4 (Eos), which is essential for inhibitory Tregcells (19,20). We also obtained results showing that 11 of200 annotated Treg cell genes were upregulated in thePPCA subsets (Supplementary Fig. 8). Together, thesedata, along with the preservation of Foxp3+ Treg cells(Fig. 4), provide the best evidence to date that PPCAtherapy largely spares natural and antigen-specific Tregcells, while dramatically reducing the numbers of acti-vated T cells in the cell cycle (specifically at the criticalG2/M border).

PPCA Preserves Islet TransplantsOne of the aims of reestablishing immune tolerance toislets is the ability to restore endogenous insulin pro-duction by islet transplantation. To determine whetherPPCA could protect islet grafts from autoreactive T cells,;300 islets from B6 mice were transplanted under thekidney capsules of NOD.Rag2/2 mice rendered diabetic byprior treatment with STZ. After transplantation, the NOD.Rag2/2 mice regained euglycemia within 24–48 h, wererested for.12 days to allow for graft vascularization, andwere then challenged with 106 diabetogenic T cells (i.v.)isolated from BDC2.5 TcR/NOD.Rag2/2 mice. Previously

Figure 4—PPCA spares naive cell and Treg cell subsets in thespleen and pancreas of NOD mice with new-onset type 1 diabetes.Treatment with PPCA does not decrease naive cell or regulatoryT-cell populations in the spleen. NOD mice were treated with WEE1iandMDM2i or vehicle days 1–3 after developing diabetes (BG = 200–250 mg/dL) and harvested on day 4. Lymphocytes isolated fromspleen and pancreas were assessed for naive T cells (CD4+ CD62L+)and Treg cells (CD4+ FoxP3+); n = 10–33 mice/group. Red, new-onset diabetes, vehicle treated; blue, new-onset diabetes, PPCA

treated; black, prediabetic. A: Percentage of naive and regulatoryT cells in spleen. B: Absolute numbers of naive and regulatory T cellsin spleen. C–E: Treatment with PPCA can decrease the ratio of islet-specific Teff cells to islet-specific Treg cells in the pancreas. NODmice were treated with WEE1i and MDM2i or vehicle days 1–3 afterdeveloping spontaneous diabetes (BG $200 mg/dL) and harvestedon day 4. Lymphocytes isolated from pancreas were assessed forthe percentage of insulin-specific CD4+ T cells by I-Ag7 tetramersspecific for ProInsulin47–84, Insulin A84–108, and Insulin B12–20. Teffcells are defined as CD4+CD44hi tetramer+; Treg cells are definedas CD4+FoxP3+tetramer+; n = 20–43 mice/group. C: Percentageof antigen-specific Teff and Treg cells in the pancreas. D: Ratio ofantigen-specific Treg/Teff cells in the pancreas. E: Comparison ofthe antigen-specific Treg/Teff cell ratio in the pancreas and spleen.**P . 0.01, ***P . 0.001, ****P . 0.0001. Panc, pancreas; Spl,spleen.

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published studies show that 106 T cells transfer diabeteswith near 100% efficacy in islet-engrafted NOD.Rag2/2

mice (15). After T-cell transfer, transplanted mice receivedeither PPCA or vehicle (day 2–3 post-transfer); all but onemouse receiving PPCA was protected from diabetes,whereas mice that received vehicle developed end-stagediabetes by day 30, with one exception likely the result of

suboptimal cell transfer (Fig. 6). Upon nephrectomy of theengrafted kidney, transplanted PPCA-treatedmice uniformlyand immediately became diabetic again (BG .600 mg/dL),indicating that the islet graft was functional and responsiblefor the normoglycemia observed (Fig. 6). The robustness ofPPCA in the transplant setting has the added advantage ofengendering a more synchronous reactivation of T cells, in

Figure 5—PPCA results in the enrichment of Treg versus Teff T-cell gene expression patterns. Splenocytes were harvested from NODmicewith new-onset diabetes on day 4 after in vivo treatment with PPCA or vehicle (day 1–3) and CD4+ T cells were isolated by cell sorting (FACSAria II) using antibodies to CD4, CD44, and I-Ag7 tetramers specific for Insulin-A chain, Insulin-B chain, and Pro-Insulin. T cells were sortedinto naive (CD4+CD442/lo), activated (CD4+CD44hi), and activated insulin-specific (CD4+CD44hitetramer+). A total of 1–7 3 105 cells fromeach group were isolated in each treatment group for NGS. A: Three-dimensional principal components analysis showing variance betweenactivated (CD44hi) CD4+ T cells based upon differentially expressed genes (fold change .1.5; P , 0.05) between PPCA-treated (blue) andvehicle-treated (red) mice. GSEA showing enrichment score of G2/M checkpoint genes (B) and Treg cell genes (C) among either vehicle- orPPCA-correlated genes. Both gene sets were gathered from the Molecular Signatures Database (M5901 and M3417, respectively); ad-ditionally relevant Treg cell genes were manually added to M3417 from gene sets obtained on ImmGen. Ranking was performed using thet test metric, and a weight of P = 2 was applied to appropriately account for gene interactions. D: Heat map showing the leading-edge sub-set of genes in Treg cell GSEA between vehicle- and PPCA-correlated genes (single genes from the leading edge are noted for relevance).

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this case islet-reactive T cells, making them more effectivelytargeted by PPCA therapy.

DISCUSSION

Many patients with type 1 diabetes retain b-cell functionand substantial endogenous insulin and C-peptide secretionat diagnosis (5), and, given the clinical significance ofpreserving this production and the rapidity with which itfalls within a year after diagnosis (2,6), a tractable inter-vention to halt disease progression early in the course ofdisease is imperative. We have demonstrated that PPCAis able to significantly preserve b-cell function by prevent-ing further lymphocytic infiltration through the ablationof activated diabetogenic Teff cells, which notably is notaccompanied by a decrease in the naive T-cell, Treg cell, ormemory T-cell populations, or by off-target effects on thegut, bone marrow, and thymocytes, or the ability to clearconcurrent viral infections (7). Our data suggest that themechanistic durability of PPCA lies in its reversal of thebalance of self-reactive Teff cells and their Treg cell counter-parts; the decrease in antigen-specific Teff cells with theattendant preservation of Treg cells resulted in a signifi-cantly enhanced Treg/Teff ratio in the spleen, and far moreextensively in the pancreas itself (Fig. 4). We also demon-strate a significant enrichment of Treg cell gene expressionin activated PPCA-treated cells (Fig. 5C). Although the ini-tial break in tolerance in type 1 diabetes seems to be due in

part to defects in the Treg cell compartment, there is alsoevidence that the Teff cells are resistant to regulation (21)and that even a minor reduction in Treg cells due toreduced interleukin-2Rb signaling is enough to acceleratediabetes onset (22), suggesting that reducing Teff cellsmay be the most effective strategy in attempting toreverse new-onset disease. This is supported by a recentstudy (23) showing protection from disease develop-ment in NOD mice due to gut metabolite-induced changes,either reducing the autoreactive Teff cell compartment oraugmenting the Treg cell compartment, although eachindependently provided protection from disease, the dietthat reduced the autoreactive Teff cells showed greaterefficacy.

Why some effector cells escape PPCA is unclear; how-ever, this may reflect the short temporal window in whichPPCA operates and the activation state of diabetogenicT cells at the time of treatment or it may also be explainedby the modulation of Bcl-2 family members in response togenomic stress. It is also possible that administeringadditional treatments after remission has been achievedand the Treg/Teff ratio has been altered, we can continueto ablate the surviving autoreactive cells, based on a recentstudy showing that Treg cells induce DNA damage in Teffcells via metabolic competition (24).

Although many similarities exist between the NODmodel and human type 1 diabetes, there are both phys-iological and environmental differences that may impactthe translation of PPCA treatment to human disease. TheNOD mouse model is a far more fulminant form of di-abetes, wherein mice lose insulin production with greaterrapidity than is generally seen in humans (25). In anothermarked difference from the situation that would occur inhuman diabetes, when mice developed clinical disease, wedid not administer exogenous insulin. The exposure toinsulin, whether from the higher levels of endogenous pro-duction or exogenous administration, would serve to fur-ther exacerbate the response of autoreactive cells specific toinsulin antigens, almost certainly increasing the percentageof autoreactive cells that are activated and the amount ofDDR signaling present. In addition, we have previouslyfound that human T cells have an even greater sensitivityto DDR manipulation than mouse T cells and exhibita greater selectivity to the inhibitors used (7). Together,this would suggest that PPCA is likely to be even moreeffective in humans with new-onset diabetes than in mice.

In addition to its implications for insulin and C-peptidepreservation in new-onset disease, PPCA has significanttranslational potential in islet transplantation. Througha variety of methodologies (regrowth of islets, autologousgraft through induced pluripotent stem cell differentia-tion, or allograft), islet restoration or transplantation hasbecome an increasingly technically feasible option; how-ever, to date the treatment showing the most efficacy islong-term therapy with broad immunosuppressive drugs(26). In contrast, PPCA has the potential to be equally ormore effective without the need for protracted use, and

Figure 6—Treatment with PPCA preserves islet transplant.NOD.RAGmice were treated with STZ (200mg/kg) to induce diabetes(BG 350–600 mg/dL), and 250–300 islets isolated from C57/B6mice were subsequently transplanted under the left kidney capsule.After stabilization of BG, 106 CD4+ T cells isolated from BDC2.5TcR/NOD/Rag mice were adoptively transferred i.v. Mice weretreated with CHKi and MDM2i (green) or vehicle (red) on days 2–3and when the BG concentration was .200 mg/dL post-transfer. Akidney containing transplanted islets was removed on day 40 post-transfer; n = 3–5/group.

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without any impact on protective immunity, including ondefenses against concurrent infections, and thus has thepotential to succeed as an immune intervention againstboth autoreactive and alloreactive T cells, providing dura-ble tolerance to transplanted islets. Moreover, our datamodel the coordinated activation that occurs when isletsare restored in a patient; by giving them recognizableantigen, the resulting temporally uniform recall responseprovides an optimal therapeutic window for maximalPPCA efficacy, potentially eliminating islet-reactive cellsfrom the T-cell repertoire entirely. Our data (Fig. 6) sug-gest that in the case of this concurrent cellular expansion,PPCA is so efficacious in most cases that there is no needto reset a regulatory balance; the adoptively transferreddiabetogenic T cells are from BDC2.5.TcR/NOD.Rag12/2

mice that lack Treg cells, underlining the extent of effectorcell ablation that can be achieved, although the possibilitythat some Teff cells may have converted to Treg cells hasnot been formally excluded. PPCA thus would be an ex-cellent candidate therapy adjunctive to, or even in replace-ment of, the current long-term immunomodulatory therapiesused to promote islet graft tolerance.

In conclusion, we have found that the targeted manip-ulation of p53 and cell cycle checkpoints selectively killsactivated autoreactive T cells in vivo; that this is an effectivetreatment strategy for type 1 diabetes in multiple clinicallyrelevant circumstances; and that it shows no effect on naiveT-cell, Treg cell, or memory T-cell populations, and signif-icantly increases the ratio of islet-specific Treg/Teff cells.These results provide the foundation for a promising newavenue of immune intervention in type 1 diabetes.

Acknowledgments. The authors thank the National Institutes of HealthTetramer Core for the Insulin A94–108 I-A

g7, Insulin B12–20 I-Ag7, Proinsulin47–64

I-Ag7, IGRP206–214 H-2Kd, and LCMV GP33–41 D

b tetramers. The authors also thankthe Cincinnati Children’s Hospital Medical Center (CCHMC) Pathology Core forassistance in processing histological samples; the University of CincinnatiGenomics, Epigenomics, and Sequencing Core for assistance with RNA sequenc-ing; as well as Dr. Emily Miraldi (CCHMC) and Dr. Harinder Singh (CCHMC) forinvaluable feedback and help with RNA sequencing analysis.Funding. This work was supported by National Institute of Health/NationalInstitute of Diabetes and Digestive and Kidney Diseases grants R01-DK-081175 (toD.A.H. and J.D.K.) and R01-AI-109810 (to D.A.H. and M.B.J.) and National Institute ofArthritis and Musculoskeletal and Skin Diseases grant P30-AR-047363 (to CCHMCCincinnati Rheumatic Diseases Center Animal Models of Inflammatory Disease Core).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. K.R.C. performed experiments and acquired data;interpreted and analyzed data; contributed to research design, analysis, and finalapproval; and wrote the manuscript. E.E.E. performed experiments and acquireddata. J.J.S. performed experiments and acquired data and interpreted andanalyzed data. J.P.M. interpreted and analyzed data and contributed to researchdesign, analysis, and final approval. D.A.H. interpreted and analyzed data;contributed to research design, analysis, and final approval; and reviewed andedited the manuscript. M.B.J. contributed to research design, analysis, and finalapproval and reviewed and edited the manuscript. J.D.K. interpreted and analyzeddata; contributed to research design, analysis, and final approval; wrote themanuscript; and reviewed and edited the manuscript. J.D.K. is the guarantor of

this work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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