Visualization of Activated T Cells by OX40-ImmunoPET as a ......anti-CD4 (clone GK1.5); BV605 anti-CD3 (clone 17A2); and BV650 anti-CD8 (clone 53–6.7). Samples were acquired on a

CANCER RESEARCH | TUMOR BIOLOGYAND IMMUNOLOGY

Visualization of Activated T Cells by OX40-ImmunoPETas a Strategy for Diagnosis of Acute Graft-versus-HostDisease A CIsrat S. Alam1, Federico Simonetta2, Lukas Scheller2, Aaron T. Mayer1,3, Surya Murty1,3, Ophir Vermesh1,Tomomi W. Nobashi1, Juliane K. Lohmeyer2, Toshihito Hirai2, Jeanette Baker2, Kenneth H. Lau1,Robert Negrin2, and Sanjiv S. Gambhir1,3†

ABSTRACT◥

Graft-versus-host disease (GvHD) is a major complication ofallogeneic hematopoietic cell transplantation (HCT), mediatedprimarily by donor T cells that become activated and attack hosttissues. Noninvasive strategies detecting T-cell activation wouldallow for early diagnosis and possibly more effective managementof HCT recipients. PET imaging is a sensitive and clinicallyrelevant modality ideal for GvHD diagnosis, and there is a strongrationale for the use of PET tracers that can monitor T-cellactivation and expansion with high specificity. The TNF receptorsuperfamily member OX40 (CD134) is a cell surface marker thatis highly specific for activated T cells, is upregulated duringGvHD, and mediates disease pathogenesis. We recently reportedthe development of an antibody-based activated T-cell imaging

agent targeting OX40. In the present study, we visualize thedynamics of OX40 expression in an MHC-mismatch mousemodel of acute GvHD using OX40-immunoPET. This approachenabled visualization of T-cell activation at early stages of disease,prior to overt clinical symptoms with high sensitivity and spec-ificity. This study highlights the potential utility of the OX40 PETimaging as a new strategy for GvHD diagnosis and therapymonitoring.

Significance: OX40-immunoPET imaging is a promising non-invasive strategy for early detection of GvHD, capable of detectingsigns of GvHD pathology even prior to the development of overtclinical symptoms.

IntroductionAllogeneic hematopoietic cell transplantation (HCT) is a well-

established curative therapy for a broad range of hematologic malig-nancies. Unfortunately, allogeneic HCT is still associated with signif-icant morbidity and mortality related to cancer relapse and transplantcomplications, namely graft-versus-host disease (GvHD). DuringGvHD, the interaction of donor-derived T lymphocytes with hosttissues induces their activation, proliferation, and migration to targettissues, notably skin, gastrointestinal tract, and hepatobiliary system,where theymediate cytolytic attack (1). Early diagnosis of acute GvHD

is essential to promptly establish appropriate treatment and preventdisease progression. At present, acuteGvHDdiagnosismainly relies onclinical manifestations combined with pathologic analysis of targetorgans by tissue biopsies. Unfortunately, these approaches lack spec-ificity and sensitivity and can result in iatrogenic complications.Further, these studies are only initiated after the development of overtclinical symptoms that occur later in the pathogenesis of GvHD.Diagnostic strategies allowing early and noninvasive identification ofpatients developing acute GvHD are therefore urgently needed formore effective intervention.

Standard radiological evaluation by ultrasound, contrast-enhancedCT or MRI is of limited utility for accurate diagnosis of acute GvHD.These anatomical imaging modalities report on gross morphologicchanges that lack specificity for GvHD (2). PET imaging is a nonin-vasive clinical imaging modality with high sensitivity and quantitativecapabilities, incentives that are ideal for the dynamic assessment ofinflammatory responses. Previous reports suggested that molecularimaging by 18F-fluorodeoxyglucose ([18F]FDG) PET can visualizetissue inflammation associated with acute gastrointestinal GVHD inboth murine models and in the clinic (3). Unfortunately, clinicalevaluation of [18F]FDG PET for acute GvHD diagnosis in largercohorts of patients revealed a lack of specificity and low positivepredictive value (4).

The use of alternative tracers designed to detect T-cell activationmore specifically has been reported. Previously we reported the abilityof 20-deoxy-20-[18F]fluoro-9-b-D-arabino-furanosylguanine ([18F]F-AraG), a small-molecule metabolic radiotracer preferentially accumu-lating in activated T cells to efficiently track T-cell activation andexpansion in a murine model of acute GvHD (5), an approachcurrently under clinical evaluation (NCT03367962). A major caveatof using small-molecule tracers is that the metabolic pathways theytarget may also be upregulated in other hematopoietic and nonhe-matopoietic tissues. ImmunoPET is a rapidly growing area of PET

1Department of Radiology, Molecular Imaging Program at Stanford (MIPS),Stanford University School of Medicine, Stanford, California. 2Division of Bloodand Marrow Transplantation, Department of Medicine, Stanford University,Stanford, California. 3Departments of Bioengineering and Materials Science andEngineering, Bio-X, Stanford University, Stanford, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

I.S. Alam and F. Simonetta contributed equally to this article.

L. Scheller and A.T. Mayer contributed equally to this article.

R. Negrin and S.S. Gambhir contributed equally to this article.

†In memory of the late Professor Sanjiv S. Gambhir.

Corresponding Authors: Israt S. Alam, Stanford University, 318 Campus Drive,Clark Center, Stanford, CA 94305. Phone: 650-725-3113; Fax: 650-724-4948;E-mail: [email protected]; and Robert Negrin, Center for Clinical SciencesResearch, 269 Campus Drive, Room 2205, Stanford, CA 94305. Phone: 650-723-0822; E-mail: [email protected]

Cancer Res 2020;80:4780–90

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imaging, leveraging the high specificity and high affinity of monoclo-nal antibodies and antibody fragments that are radiolabeled with PETisotopes, allowing for the targeted detection of cell surface markers onspecific subsets of cells (6).

We recently reported a novel immunoPET tracer developed using amurine OX40-specific monoclonal antibody (64Cu-OX40mAb) thatenables noninvasive imaging of murine OX40þ activated T cells (7, 8).OX40 (CD134), a member of the TNF receptor superfamily, is a T-cellcostimulatory molecule whose expression is highly restricted to acti-vated T cells (9). Previous studies have shown an increase in OX40expression at the T-cell surface during acute GvHD in rodents (10),nonhuman primates (11), and humans (12–15). Moreover, there isevidence that OX40 plays a role in acute GvHD pathogenesis whereOX40/OX40L blockade significantly attenuates disease progression inmurine (16–18) and nonhuman primate (11) models of disease.

In the present work, we evaluated the utility of OX40-targetedimmunoPET imaging and the sensitivity of this approach to image thekinetics and homing of activated T cells in vivo in a major MHC-mismatch murine model of acute GvHD.

Materials and MethodsAnimals

BALB/cJ (H-2kd) and C57Bl/6J (H-2kb) mice were purchased fromThe Jackson Laboratory. Firefly luciferase (Luc)þ transgenic C57BL/6L2G85 mice have been described previously (19) and were bred in ouranimal facility at Stanford University. B6.129S4-Tnfrsf4tm1Nik/JOX40 knockout mice (OX40�/� C57BL/6) were kindly provided byDr. Ronald Levy's laboratory at Stanford University.

Study approvalAll procedures performed on animals were approved by Stanford

University's Institutional Animal Care and Use Committee and werein compliance with the guidelines of humane care of laboratoryanimals.

Allogeneic bone marrow transplantation and acute GvHDinduction

Donor CD4þ and CD8þ conventional T cells (Tcon) wereisolated from splenocytes harvested from lucþ C57BL/6 mice andenriched with CD4 and CD8 MicroBeads (Miltenyi Biotec). Cellpurity was consistently >95%. T-cell–depleted bone marrow (BM)cells were prepared by first crushing bones followed by T-celldepletion using CD4 and CD8 MicroBeads (Miltenyi Biotec).BALB/c recipient mice were treated with lethal total body irradi-ation (TBI) consisting of 880 cGy in 2 doses administered 4 hoursapart. On the same day, 5 � 106 BM cells from C57BL/6 mice and1.0 � 106 Tcon from lucþ or OX40�/� C57BL/6 mice were injectedintravenously. Mice were monitored daily, and body weight andGvHD score were assessed at day 4, day 7, and weekly thereafter aspreviously described (20).

Flow cytometry analysisSingle-cell suspensions were prepared from cervical lymph

nodes (CLN), mesenteric lymph nodes (MLN), and spleen. Extracel-lular staining was preceded by incubation with purified FC blockingreagent (Miltenyi Biotech) to reduce nonspecific staining. Cells werestained with the following antibodies (Biolegend): FITC anti-CD45.1(clone A20); PE anti-OX40 (clone OX-86) or appropriate isotypecontrol (clone RTK2071); APC anti-Thy1.1 (clone OX-7); APC/Fire750 anti-CD19 (clone 6D5) and anti-CD45.2 (clone 104); BV421

anti-CD4 (clone GK1.5); BV605 anti-CD3 (clone 17A2); and BV650anti-CD8 (clone 53–6.7). Samples were acquired on a BD LSR II flowcytometer (BD Biosciences), and analysis was performed with FlowJo10.5.0 software (Tree Star).

ImmunofluorescenceIntestinal tissues were harvested from transplantedmice (day 7 after

transplant) and frozen in optimal cutting temperature embeddingcompound and stored at�80�C until further use. Tissue sections werecut longitudinally into 10 mm thick sections using a cryostat. Immu-nofluorescence staining was carried out using standard proceduresusing the following primary antibodies: anti-OX40 (clone OX86,BioXcell) and CD90.1-Biotin (Invitrogen). OX40 expression wasdetected using donkey anti-rat IgG-AF488 (Abcam) and Streptavi-din-AF647 (Invitrogen), respectively. The sections were finallymounted with VECTASHIELD Antifade Mounting Medium withDAPI (Vector Laboratories) and imaged with Nikon A1R-Si-MPmultiphoton confocal microscope using a Nikon CF160 Plan ApoLambda 10x/0.45 numerical aperture objective lens.

Bioluminescence imagingMice were injected with D-luciferin (10 mg/kg; intraperitoneally)

and anesthetized with isoflurane. Imaging was conducted using anIVIS Spectrum imaging system (Perkin Elmer), and datawere analyzedwith Living Image Software 4.1 (Perkin Elmer).

Radiolabeling of OX40-targeted monoclonal antibodyBioconjugation of murine-specific OX40mAb to DOTA-NHS

chelate (Macrocyclics) was performed using previously optimizedprotocols (7) and is described in Supplementary Methods. DOTA-OX40mAb was radiolabeled with 64CuCl2 (University of Wisconsin,Madison, WI, USA) to produce 64Cu-DOTA-OX40mAb (heronreferred to as 64Cu-OX40mAb) with final specific activity of 10 to 15mCi/mg, radiochemical purity >99%, and labeling efficiency of 95%to 99%. Radio-ITLC and radio-HPLC using size-exclusion liquidchromatography with a Phenomenex SEC 3000 column with sodi-um phosphate buffer (0.1 mol/L, pH 6.8) was performed to cor-roborate radiochemical purity. The final formulation was preparedin PBS.

Small animal PET/CT and ex vivo biodistribution studiesMice were anesthetized using isoflurane delivered by 100%

oxygen (2.0%–2.5% for induction and 1.5%–2.5% for maintenance).64Cu-OX40mAb (105 � 9 mCi, 8 � 3 mg) was administered i.v. viathe tail vein. Transplanted mice and TBI controls received tracer atday 4 or day 7 after transplant. Wild-type and OX40�/� mice werealso imaged for control studies. Static (10 minutes) PET scans wereacquired 24 hours after 64Cu-OX40mAb administration using asmall animal PET/CT hybrid scanner (Inveon, Siemens). CT imageswere acquired prior to each PET scan and within the same acqui-sition workflow to provide an anatomic reference for PET data andto allow for attenuation correction. PET image reconstruction andimage analysis were conducted as previously described and areoutlined in further details in Supplementary Methods (7).

Following the completion of the scan 24 hours after injection oftracer, ex vivo biodistribution studies were performed to measureblood- and tissue-associated radioactivity. Briefly, blood (�100 mL)was collected via cardiac puncture, and the following tissues werecollected: CLN andMLN, spleen, skin, lower part of the small intestine(above the ileocecal valve), large intestine, kidney, liver, muscle, andfemur. Tissues were placed in a tube, weighed, and radioactivity

OX40 as an Imaging Biomarker for GvHD

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measured using an automated gamma counter (Cobra II; Packard).Tissue-associated radioactivity was normalized to tissue weight andamount of radioactivity administered to each mouse, decay-correctedto the time of radiotracer injection. Data were expressed as percentageinjected dose per gram of tissue (%ID/g) values.

Statistical analysisStatistical analyses were performed using Prism 6 (GraphPad

Software) and R version 3.5.1 with R studio version 1.1.453.Heatmaps were generated using Pheatmap version 1.0.12. Principalcomponent analysis (PCA) was performed using the FactoMineRpackage version 1.41 and visualized using the factorextra packageversion 1.0.5. ROC curves were calculated and plotted using theplotROC version 2.2.1.

ResultsOX40 is significantly upregulated on T cells duringmurine acuteGvHD

Wefirst analyzed the dynamics ofOX40 expression on the surface ofCD4þ and CD8þ T cells recovered after adoptive transfer intoallogeneic (BALB/c) or syngeneic (C57BL/6) recipients at the time ofHCT. Donor-derived T cells were identified using the CD45.1þ andCD90.1þ congenicmarkers, andOX40 expressionwas assessed byflowcytometry (using a gating strategy and an isotype control outlined inSupplementary Fig. S1A and S1B, respectively). We detected a signif-icant upregulation of OX40 on CD4 (Fig. 1A, top plots) and to a lesserextent on CD8 T cells (Fig. 1A, bottom plots) recovered from the CLNand MLN, both at day 4 and day 7 after transplantation. CD4 T cellsisolated from the spleen significantly upregulated OX40 at day 4 aftertransplantation, whereas levels of expression at day 7 returned tobaseline (Fig. 1A, top right plot). Conversely, CD8 T cells recoveredfrom the spleen expressed higher levels ofOX40 both at day 4 and day 7compared with baseline (Fig. 1A, bottom right plot). Importantly,OX40 upregulation was uniquely observed after adoptive transfer intoallogeneic recipients, whereas cells recovered upon transfer intosyngeneic mice did not show any significant increase in OX40 expres-sion (Fig. 1A, green symbols and lines). We next investigated thespecificity of OX40 staining to identify activated donor-derived Tcells after unsupervised clustering of splenocytes recovered at day 7after transplantation from allogeneic recipients receiving BM alone(control group) or BM in combination with T cells (GvHD group).OX40 was selectively expressed on donor-derived activated T cells,whereas only minimal expression was detected within host-derivedor BM-derived donor cells (Fig. 1B). We finally assessed donor-derived T-cell infiltration identified by CD90.1 staining (Fig. 1C,red channel) and OX40 expression (green channel) in intestinaltissue, a prime site for GvHD pathology. Immunofluorescencestaining performed on small intestine samples harvested at day 7after transplantation revealed higher numbers of CD90.1þ andOX40þ cells infiltrating the small intestine in GvHD mice incomparison with control mice (Fig. 1C). Collectively, these resultsindicate that OX40 is significantly upregulated at the T-cell surfaceduring alloreactive responses in GvHD, allowing for the selectiveidentification of activated donor-derived T cells in both lymphoidorgans and the gastrointestinal tract.

OX40-targeted immunoPET enables visualization of activatedT cells in GvHD target organs

We next tested the ability of OX40-targeted immunoPET tocapture T-cell expansion and activation during acute GvHD using

64Cu-OX40mAb. Acute GvHD was induced using lucþ donorT cells enabling in vivo monitoring of donor T-cell expansion andaccumulation using bioluminescence imaging (BLI) as reference.In vivo BLI and 64Cu-OX40mAb injections were performed on day4 or day 7 after transplant. PET/CT images were acquired 24 hoursafter tracer injection (Fig. 2, Supplementary Fig. S2). Figure 2Ashows a reference atlas of a representative volume-rendered tech-nique (VRT) PET/CT image and axial PET/CT views with thelocation of key clearance, lymphoid. and GvHD target tissuesannotated. In agreement with previous reports (5, 19), in vivo BLIat day 4, a time point at which we could not detect any signs ofclinical disease according to our scoring system, revealed donor T-cell expansion and accumulation in the spleen and in MLN ofGvHD mice (Fig. 2B). PET/CT images of control mice acquired at24 hours after tracer injection showed that 64Cu-OX40mAb pri-marily accumulated in the heart and the liver, characteristic ofwhole antibody biodistribution, with minimal signal detected in thespleen and in the abdominal region (Fig. 2C, left plot). GvHD miceinjected with tracer on day 4 exhibited a pronounced 64Cu-OX40mAb-PET signal in the spleen, MLN, and lower abdomen(Fig. 2C) also seen in three-dimensional (3D) rotational images(Supplemental Video S1). We next performed the same analysis atday 7, a time point at which animals displayed overt signs of disease.In vivo BLI demonstrated an expansion of donor-derived T cellswith highest levels of signal originating from the abdominal region(Fig. 2D), indicating a dramatic intestinal infiltration at this timepoint in GvHDmice. PET imaging at day 7 (Fig. 2E, SupplementaryVideo S2) corroborated BLI and showed high accumulationof 64Cu-OX40mAb in the abdominal region and, to a lesserextent, in the splenic region, a finding compatible with the furthermigration of T cells to the intestinal tract at this later time point (19).Notably, compared with control mice, GvHD mice exhibitedlower PET signals in the heart (Fig. 2C and E; SupplementaryVideos S3 and S4).

To quantify the trends observed in the PET images, we performedregion of interest (ROI) analysis on multiple tissues and regions(Fig. 3A; Supplementary Methods). ROI analysis of CLN was avoideddue to contribution of high signal from surrounding blood vessels inthe cervical region at the relevant time points, instead we focused onlymphoid compartments in the abdomen. For day 4 tracer injections,ROI quantification of PET/CT images confirmed markedly increasedradiotracer uptake in GvHD versus control mice in lymphoid tissuesand GvHD sites (spleen: 15.42 � 2.29 vs. 8.04 � 1.10%ID/g; MLN:18.80� 3.41 vs. 10.82� 2.06%ID/g; abdominal region: 8.03� 2.52 vs.3.56 � 0.71%ID/g; P < 0.001; n ¼ 9–10 per group, Fig. 3B).Comparison of control mice with mice exposed to TBI withoutreceiving BM cells revealed a small but statistically significantcontribution of BM-derived cells in the background signals detectedin lymphoid organs, but not in the abdominal region (Supplemen-tary Fig. S3A and S3B), further supporting the specificity of thedetected 64Cu-OX40mAb-PET signal for GvHD-mediating T cells.ROI quantification of PET/CT images obtained 24 hours aftertracer injection (injection day 7, imaging day 8) revealed similartrends, with significantly higher radiotracer uptake in target tissuesin GvHD mice compared with controls (spleen:12.88 � 1.16 vs. 9.87� 1.26, P < 0.001; MLN: 14.62 � 3.54 vs. 10.45 � 1.55, P < 0.01 andabdominal region: 9.36 � 2.85 vs. 5.33 � 0.78, P < 0.001; n ¼ 12 pergroup, Fig. 3C). Importantly, the background 64Cu-OX40mAb-PETsignals in muscle showed no significant difference between thetwo groups. Comparison of control mice receiving BM with TBImice injected at day 7 confirmed the results obtained at day 4,

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Figure 1.

OX40 expression on T cells in a murine model of acute GvHD. A, Graphs represent percentages of OX40-expressing CD90.1þ CD45.1þ CD4þ (top plots) and CD8þ

(bottom plots) T cells recovered from CLN, MLN, and spleen 4 and 7 days after HCT and adoptive transfer of T cells isolated from C57Bl/6 donors into syngeneicC57Bl/6 (green symbols and lines) or allogeneic BALB/c (red symbols and lines) recipients. Results are pooled from two independent experiments with a total of10 to 11 mice per group. Day 4 and day 7 values were compared with day 0 values using a nonparametric Kruskal–Wallis test followed by Dunn's multiplecomparisons test. P values are shown when significant. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001. B, Representative t-distributed stochastic neighbor embedding(tSNE) clustering of live splenic cells from BALB/c mice at day 7 after HCT with C57BL/6 BM (CD45.2þ, H-2kbþ) with or without C57BL/6 T cells (CD45.1þ,Thy1.1þ, H-2kbþ). Clustering was performed on cells merged from control and GvHD mice based on CD45.1, CD45.2, Thy1.1, Thy1.2, H-2Kb, CD4, and CD8expression. Red, OX40-expressing cells. C, Representative confocal images of intestinal tissue from GvHD and control mice at day 7 after transplant (n ¼ 3 pergroup). High OX40 (green) and pan T-cell marker CD90.1 (red) staining were observed in the villi and crypts of the GvHD mice (bottom) in comparison withthe control mice (top). Merged views with DAPI staining of nuclei (blue) are shown. Scale bar, 200 mm.






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Figure 2.64Cu-DOTA-OX40mAb PET/CT imaging during acute GvHD. A, Reference atlas of a representative PET/CT image and axial PET/CT views with the location of keyclearance, lymphoid, and GvHD target tissues shown. H, heart; Li, liver; S, spleen; M, MLN; andAb, abdomen.B–E,Representative bioluminescence images (BLI;B andD) and 3D VRT images (C and E) of 64Cu-DOTA-OX40mAb PET/CT images acquired 24 hours after tracer administration at days 4 and 7 after HCT in control (left) orGvHD (right) mice. Images are representative of two independent experiments per time point, with 9 to 12 mice per group.

Alam et al.





with control mice specifically displaying higher 64Cu-OX40mAb-PET signal than TBI mice in the secondary lymphoid organs(Supplementary Fig. S3C and S3D).

Biodistribution analysis using ex vivo gamma counting of tissuesconfirmed PET results with significantly increased tracer uptakemeasured in lymphoid tissues of GvHD versus control mice: spleen(days 4 and 7; P < 0.01), MLN (P < 0.01), CLN (day 4; P < 0.05, day 7;

P < 0.01; Fig. 4A). Increased tracer uptake was also confirmed inGvHD target organs; small intestine (days 4 and 7;P< 0.05), colon (day7; P < 0.05), and skin (day 7; P < 0.01; day 4; Fig. 4A, day 7; Fig. 4B).MicewithGvHDdisplayed significantly reduced signals from the heartas shown by ROI quantification (Fig. 3) and biodistribution analysis ofheart and blood (Fig. 4) compared with control mice at day 4 and atday 7. A similar trend, reaching statistical significance only in ROIquantification at day 4, was observed for the liver (Fig. 3B). Suchbackground signals from heart, blood, and liver are likely nonspe-cific, due to the presence of antibody circulating in the bloodand were independent of any OX40-specific binding to circulatingcells as ROI quantification and in biodistribution analysis showedsimilar levels of signal in WT and OX40�/� mice (SupplementaryFig. S3E and S3F), suggesting the presence of a sink effect leading toa reduction in the concentration of circulating 64Cu-OX40mAbtracer in GvHD mice. Correlation analysis showed a significantpositive correlation between ROI quantification and biodistributionanalysis, especially in spleen (R2 ¼ 0.64, P ¼ 3.2e-08) and blood(R2 ¼ 0.61, P ¼ 2.8e-09; Supplementary Fig. S4), further confirmingthe ability of OX40-immunoPET to define the localization ofactivated T cells in murine acute GvHD. Collectively, these resultsdemonstrate the ability of 64Cu-OX40mAb immunoPET to detectT-cell expansion and activation during murine acute GvHD prior toand after clinical signs of GVHD are evident.

OX40-immunoPET signal correlates with clinical status duringmurine acute GvHD

We next analyzed the relationship between 64Cu-OX40mAbtracer uptake and clinical signs of GvHD. As expected, 64Cu-OX40mAb tracer uptake in spleen, MLN, and the abdomenshowed only poor if any correlation with body weight andGvHD score at day 4, when mice were essentially asymptomatic(Supplementary Fig. S5), highlighting the potential for early diseasedetection capabilities of OX40 imaging even before overt clinicalsigns of GvHD appear. Conversely, 64Cu-OX40mAb uptake inthe abdominal region at day 7 negatively correlated with bodyweight (R2 ¼ 0.44, P ¼ 0.0005) and positively correlatedwith the GvHD score (R2 ¼ 0.59, P ¼ 0.00001; Fig. 5). Moreover,64Cu-OX40mAb uptake in spleen and MLN at day 7 positivelycorrelated with the GvHD score (spleen: R2 ¼ 0.36, P ¼ 0.0018;MLN: R2 ¼ 0.18, P ¼ 0.042; Fig. 5). Collectively, these results showthat quantification of OX40-expressing activated T cells in lym-phoid organs and GvHD-target sites allows early detection of GvHDeven before the appearance of clinical signs and efficiently reflectsthe severity of the disease.

Early administration of OX40mAb at tracer doses significantlyexacerbates murine acute GvHD outcome

Previous reports have demonstrated a role for OX40 expression onT cells during murine acute GvHD induction, showing that spleno-cytes from OX40�/� C57BL/6 mice (H-2Kb) had reduced GvHDinduction potential upon transfer in MHC-mismatched B10.BR mice(H-2Kb) compared with their wild-type counterpart (17). In agree-ment, we observed similar results in our mouse model as adoptivetransfer of 1� 106 T cells from OX40�/� C57BL/6 mice (H-2Kb) intolethally irradiated BALB/c mice (H-2Kd) at time of transplantation,resulting in a significantly delayed kinetic of GvHD-related deathcompared with the adoptive transfer of identical numbers of cellsisolated fromWTC57BL/6 mice (Supplementary Fig. S6). In the samereport, Blazar and colleagues showed that the administration of high(200 mg) and repeated doses of the agonistic anti-OX40mAb (clone

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Quantification of 64Cu-DOTA-OX40mAb uptake using ROI analysis duringacute GvHD. A, Reference atlas for ROI definition. H, heart; Li, liver; S, spleen;M, MLN; Ab, abdomen; B, bladder; F, femur; and Mu, muscle. B and C,Quantitative ROI PET image analysis of spleen, MLN, abdomen, liver, muscle,femur, and heart at days 4 (B) and 7 (C) after HCT in controls (light blueand blue-filled boxes, respectively) or GvHD mice (orange and red-filledboxes, respectively). Values are summarized as box plots, representing therange, first quartile, median, third quartile, and eventual outliers. Traceruptake in control (day 4, n ¼ 9; day 7, n ¼ 12) and GvHD (day 4, n ¼ 10; day 7,n ¼ 12) groups was compared using the Mann–Whitney U test. � , P < 0.05;�� , P < 0.01; �� , P < 0.0001; ns, nonsignificant. Results are pooled from twoindependent experiments.






M5) significantly increased GvHD lethality in the same murinemodel (17). We therefore tested the safety of the agonistic anti-OX40mAb (clone OX86) we used for the imaging study in our murinemodel, administered at tracer doses (described in SupplementaryMethods). Administration of very low doses (15 mg—the maximalupper limit of antibody dose anticipated to ever be administeredduring PET imaging) of cold OX40mAb at day 4 after HCTsignificantly accelerated GvHD lethality compared with adminis-tration of isotype control (P < 0.0001; Fig. 6A). Conversely, we didnot detect any significant impact of administration of the same doseof cold OX40mAb at day 7 after HCT compared with mice thatreceived isotype control (Fig. 6B). Collectively, these results con-firm the role of OX40 in murine acute GvHD pathogenesis and

reveal that even very low doses of agonistic anti-OX40 mAb mightexacerbate GvHD lethality when administered at early phases ofGvHD induction.

Unsupervised analysis of OX40-immunoPET indicates thatsignals from abdomen, spleen, and MLN have high diagnosticpotential for murine acute GvHD detection

To assess the relative contribution of signals obtained from ROI ofdifferent tissues for the detection of murine acute GvHD, we firstperformed an unsupervised analysis of 64Cu-OX40mAb-PET signalsdetected by ROI quantification across all groups and mice (Fig. 7A).Unsupervised hierarchical clustering easily separated all GvHD miceinjected at day 4 afterHCT together with onemouse at day 7 afterHCT

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BioD (day 7) Control (n = 12) GvHD (n = 12)

* *

***** *** ns*** ns** ** **ns *ns ns

0

10

20

30

40

50

Splee

nCL

NML

N

Small

inte

stin

e

Colo

nSk

inLi

ver

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le

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ur

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Who

le bl

ood

Kidn

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cted

BioD (day 4) Control (n = 8) GvHD (n = 8)A

B

Figure 4.

Quantitative 64Cu-DOTA-OX40mAb tracer biodistribution during acute GvHD. A and B, Quantification of OX40-immunoPET signal (%ID/g) from ex vivobiodistribution analysis of spleen, CLN, MLN, small intestine, colon, skin, liver, muscle, femur, heart, whole blood, kidney, and tail 24 hours after tracer administrationon day 4 (A) and day 7 (B). Values are summarized as box plots, representing the range, first quartile, median, third quartile, and eventual outliers. Tracer uptake incontrol (day4, n¼9; day7, n¼ 12) andGvHD (day4, n¼ 10; day 7, n¼ 12) groupswas compared using theMann–WhitneyU test. � , P

(Fig. 7A). Within the second subgroup, hierarchical clustering iden-tified a second cluster containingmost of GvHDmice at day 7 with theexception of only 2 mice that clustered together with control mice(Fig. 7A). To identify the relative impact of different ROI in clustering,we performed a PCA of ROI data (Fig. 7B). The scree plot shownin Fig. 7C reveals that PC1 and PC2 combined accounted for 78.9% of

the variance of the data, supporting the feasibility of this approach.PC1 alone allowed the distinction of GvHDmice injected at day 4 fromall other mice, whereas a combination of PC1 and PC2 allowed theseparation of GvHD mice from controls independently of the timepoints (Fig. 7B). Mapping of the component loadings identifiedabdomen, spleen, and MLN ROIs as the variables most strongly

R2 = 0.12, P = 0.1 R2 = 0.084, P = 0.18 R2 = 0.44, P = 0.00052

Spleen MLN Abdomen

70

80

90

100

Wei

ght (

%)

Day 7 Control (n = 12) GvHD (n = 12)

R2 = 0.36, P = 0.0018 R2 = 0.18, P = 0.042 R2 = 0.59, P = 1.2e−05

50 10 15 20 50 10 15 20 50 10 15 20

0

2

4

6

%ID/g

GvH

D s

core

Figure 5.

Correlation of 64Cu-DOTA-OX40mAbPET results with clinical signs of acuteGvHD at day 7. Correlation of traceruptake at day 7 post HCT in spleen,MLN, and abdomen (determined byROI analysis of PET images) withbody weight (top plots) and GvHDscores (bottom plots). Correlationswere evaluated using a Spearmanrank correlation coefficient test.Results are pooled from two inde-pendent experiments with a total of9 to 12 mice per group per time point.P < 0.05 was considered statisticallysignificant.

0 20 40 600

50

100

Time after BMT (days)

Perc

ent s

urvi

val

TBI (n = 10)

BM (n = 10)

BM+ T cells + isotype ctrl (n = 10)

BM+ T cells + anti-OX40 (n = 10)

mAbday 4

P < 0.0001

A

0 20 40 600

50

100

Time after BMT (days)

Perc

ent s

urvi

val

TBI (n = 10)

BM (n = 10)

BM+ T cells + isotype ctrl (n = 10)

BM+ T cells + anti-OX40 (n = 10)

mAbday 7

ns

B

Figure 6.

Impact of dosing regimen of anti-OX40mAb administration on acute GvHD outcomes. Overall survival after HCTwith TBI (light gray) or BM alone (light blue and blueline) or BM and T cells (GvHD group, dotted orange and red lines). At day 4 (A) or 7 (B), mice were randomized to receive intravenous administration of AbOX40(continuous lines) or appropriate isotype control (dotted lines) at a dose similar to the ones employed for PET/CT studies (15 mg/mouse, representing upper limit oftracer dose). Results are pooled from two independent experiments, with a total of 10 mice per group. Survival curves were plotted using the Kaplan–Meier methodand compared by log-rank test. P < 0.05 was considered statistically significant.






contributing to the distinction between GvHD mice and control mice(Fig. 7B). We therefore assessed the diagnostic potential of abdomen,MLN, and spleen ROI data for detection of murine acute GvHD atboth day 4 and day 7 using ROC curve analysis. Muscle ROI wasused as a negative control. As shown in Fig. 7D, abdomen, spleen, andMLN PET ROIs had a perfect diagnostic power for murine acuteGvHDdetection at day 4 (abdomen: AUC¼ 1;MLN:AUC¼ 1; spleen:AUC¼ 1) and an excellent diagnostic power at day 7 (abdomen: AUC¼ 0.96; MLN: AUC ¼ 0.88; spleen: AUC ¼ 0.98). As expected, themuscle ROIs had no diagnostic potential for GvHD detection either atday 4 (AUC¼ 0.28) or day 7 (AUC¼ 0.43). These results indicate that64Cu-OX40mAb-PET signals detected in the abdomen, MLN, andspleen ROIs have excellent diagnostic potential for detection ofmurineacuteGvHDboth before and after the appearance of clinical symptomsof disease.

DiscussionIn the present report, we demonstrated the ability of OX40-

immunoPET to efficiently visualize T-cell activation, expansion,and tissue infiltration in a murine model of acute GvHD. As ourability to successfully prevent and treat acute GvHD depends on ourcapacity to efficiently detect disease before clinical manifestationsappear, we hypothesized that a noninvasive imaging strategyable to detect T-cell activation would be an extremely powerfulapproach for early diagnosis of acute GvHD. Toward this aim,the selection of the most appropriate activation marker appears tobe crucial. Previous studies in animal models (10, 11) as well as inhumans (12–15) have shown that expression of OX40 on T cellsincreases during GvHD, in particular at CD4þ T-cell surface (15).Importantly, OX40 outperformed other activation markers, includ-ing CD25 and CD69, for detection of alloreactive T-cell

GvH

D_D

4.8G

vHD

_D4.7

GvH

D_D

4.10G

vHD

_D4.4

GvH

D_D

4.5G

vHD

_D4.1

GvH

D_D

4.3G

vHD

_D4.6

GvH

D_D

7.7G

vHD

_D4.2

GvH

D_D

4.9G

vHD

_D7.4

GvH

D_D

7.5G

vHD

_D7.3

GvH

D_D

7.6G

vHD

_D7.11

GvH

D_D

7.1G

vHD

_D7.9

GvH

D_D

7.8G

vHD

_D7.10

Control_D

7.9C

ontrol_D7.12

Control_D

7.8C

ontrol_D7.10

Control_D

7.3C

ontrol_D4.8

Control_D

7.6C

ontrol_D7.7

Control_D

4.5C

ontrol_D4.3

Control_D

4.4C

ontrol_D4.1

Control_D

4.9C

ontrol_D4.2

Control_D

4.7G

vHD

_D7.12

Control_D

7.11C

ontrol_D7.1

GvH

D_D

7.2C

ontrol_D7.5

Control_D

4.6C

ontrol_D7.2

Control_D

7.4

Abdomen

Femur

Muscle

Heart

Liver

MLN

Spleen

Sample

Control day 4GvHD day 4

Control day 7GvHD day 7

5

10

15

20

25

A

Abdomen

MLN

Spleen

Femur

Heart

LiverMuscle

−2

0

2

−4 −2 0 4

Dim1 (52.8%)

Dim

2 (2

6.1%

)

Groups

Control day 4

Control day 7

GvHD day 4

GvHD day 7

2

52.8

26.1

9.26.6

2.6 1.5 1.20

20

40

Dimensions

Per

cent

age

of e

xpla

ined

var

ianc

e

1 2 3 4 5 6 7

B C

AUC = 1

0

0.25

0.50

0.75

1.00

Sen

sitiv

ity

Spleen MLN Abdomen Muscle

AUC = 1 AUC = 1 AUC = 0.28

AUC = 0.98

0

0.25

0.50

0.75

1.00

0 0.25 0.50 0.75 1.001 − Specificity

Sen

sitiv

ity

AUC = 0.88

0 0.25 0.50 0.75 1.001 − Specificity

AUC = 0.96

0 0.25 0.50 0.75 1.001 − Specificity

AUC = 0.43

0 0.25 0.50 0.75 1.001 − Specificity

Day 4

Day 7

D

Figure 7.

Unsupervised analysis of 64Cu-DOTA-OX40mAb PET in murine acute GvHD identi-fies diagnostic ROIs. A, Heatmap visualiza-tion of normalized OX40 PET tracer uptakevalues in ROIs (rows) from all transplantrecipient studies (columns). Column labelsbelow the heatmap indicate “Group_Day_.replicate#” for each individual mouse. Datashown are pooled from two independentexperiments per time point. B, PCA per-formed using normalized OX40 PET traceruptake values from ROI analysis. The relativecontribution of each ROI to the clusteringis depicted as an arrow. C, Screen plot show-ing the percentage of the variance explainedby each principal component. D, ROC show-ing sensitivity against 1-specificity for distin-guishing control mice from GvHD miceusing OX40 PET tracer uptake values fromROIs identified in the PCA (MLN, spleen,abdomen) plus muscle as negative control.AUC is indicated.

Alam et al.





responses (14, 15, 21). After confirming OX40 upregulation at theT-cell surface during alloreactive responses in both secondarylymphoid organs and target tissues, we demonstrate that OX40-immunoPET was able to visualize T-cell activation even prior to thedevelopment of overt clinical symptoms in a murine model ofGvHD. Importantly, OX40-immunoPET was able to distinguishclearly early intestinal GvHD from toxicities resulting from theconditioning regimen, which represents a major limitation to theradiologic diagnosis of GvHD (2). However, the preclinical natureof our study limits the number of confounding factors oftenencountered in the differential diagnosis of GvHD, notably infec-tions. Although OX40 specificity for the T-cell compartment makesfalse positive originating from bacterial infections unlikely, wecannot exclude that viral infections, notably cytomegalovirus colitis,could result in positive signals in OX40-immunoPET. For inter-pretation of imaging results, it will therefore be critical to integrateclinical (donor/recipient serostatus) and biological (viral loads)elements for the differential diagnosis between these two entities.

The need for specific visualization of T-cell responses in vivo hasmotivated the development of several immunoPET agents in recentyears. To date there have been a few candidates reported, targeted tolineage-defining cell surface markers expressed on T cells such asCD3 (22–25). Although these phenotypic-targeted probes can capturethe dynamics of T cells, they fail to directly report on T-cell activation.Recently Pektor and colleagues reported a CD3-targeted immunoPETapproach for imaging GvHD progression in a humanized mousemodel (23). It is well-documented that T-cell activation mediates thedownregulation of the CD3/T-cell receptor (26), a potential limitationin the context of inflammation. Another approach reported for PETimaging of GvHD progression in a humanized mouse model has beento image human class II MHC (HLA-DR) using a camelid-derivedsingle-domain antibody (27). AlthoughHLA-DR expression increasesduringT-cell activation, it is also expressed at high levels in themyeloidcompartment and thus lacks specificity as an activated T-cell imagingtarget compared with imaging of OX40.

Our preclinical data indicate that, owing to its high sensitivity,OX40-immunoPET could detect signs of GvHD even before clinicalsymptomsmanifest. However, given the complexity and relatively highcosts of immunoPET, it is unlikely that this approach will be applied asa screening strategy for all allogeneic HCT recipients. For clinicaltranslation of this type of imaging, it will therefore be critical tocarefully establish patient selection criteria. We can imagine a scenarioin which investigation by immunoPET will be triggered by clinicalsuspicion, for example to investigate possible gut GvHD after theappearance of skin GvHD, or after initial screening using bloodbiomarkers like REG3a and ST2 (28). Alternatively, immunoPET ofactivated T cells could also be employed as a screening strategy inpatients at high risk of developing GvHD (e.g., posttransplant fromMHC-mismatched and/or unrelated donors). Results of early clinicaltrials, like the one ongoing at our institution using the small-moleculePET tracer [18F]F-AraG to detect T-cell activation for GvHDdiagnosis(NCT03367962), will lay the groundwork for defining the patientselection criteria.

A major limitation of our study comes from the exacerbation ofGvHD we observed upon administration of tracer doses of agonisticanti-OX40 antibody, when given at early time points of disease (day 4).The anti-OX40 antibody significantly exacerbated GvHD similar toprevious reports, although in that study repeated administration ofhigher doses of anti-OX40 mAbs was studied (17). These resultsfurther confirm the role of OX40 in the GvHD pathogenesis andstress the importance of the selection of appropriate clones for each

application. Although agonistic anti-OX40 antibodies can be appro-priate for imaging of T-cell activation in cancer immunotherapysettings (7, 8), immunoPET in immunopathologic settings targetingcostimulatorymolecules will probably require the use of nonagonist oreven antagonist antibody clones. Although the generation of anantagonistic murine anti-OX40 clone is beyond the scope of thepresent report, clinical translation of OX40-immunoPET for GvHDdiagnosis would probably benefit from the use of anti–human-OX40antagonist clones such as the GBR830 clone, currently under clinicalinvestigation for atopic dermatitis (NCT03568162; ref. 29) and recent-ly reported to suppress xenogeneic GvHD when administered incombination with Cyclosporine A (30). Alternative approaches tominimize adverse biological effects may also include the generation ofantibody fragments lacking the potency of a full-length antibody andthe Fc region known to engage other immune cells, or engineeredbinders (31). Perturbations in the biology of immune cells by immu-noPET tracers have previously been noted (32, 33) and may be furtherminimized by improving the specific activity of the radiolabeled probeso that significantly less mass is administered.

In summary, this study demonstrates the utility of OX40 as asensitive imaging biomarker for the early and specific visualizationof activated T cells in GvHD. Integrated with tissue biopsies andendoscopic evaluation, we anticipate that this whole-body imagingapproach of T-cell activation by immunoPET could significantlyimprove upon current GvHD diagnosis and provide earlier diagnosiswhere interventions may be more effective for improved clinical care.

Disclosure of Potential Conflicts of InterestI.S. Alam reports grants from Ben & Catherine Ivy Foundation, grants from The

Canary Foundation, grants fromNational Cancer Institute (R01 1CA201719-02), andgrants from Parker Institute for Cancer Immunotherapy during the conduct of thestudy. R. Negrin reports grants fromNational institutes of Health (R01 CA23158201)and grants from Parker Institute for Cancer Immunotherapy (P01 CA49605) duringthe conduct of the study. S.S. Gambhir reports grants from National Cancer Institute(R01 1 CA201719-02) and grants from Parker Institute for Cancer Immunotherapy(P01CA49605) during the conduct of the study. No potential conflicts of interest weredisclosed by the other authors.

Authors’ ContributionsI.S. Alam: Conceptualization, data curation, software, formal analysis, validation,

investigation, methodology, writing-original draft, project administration, writing-review and editing. F. Simonetta: Conceptualization, data curation, software, formalanalysis, funding acquisition, validation, investigation, visualization, methodology,writing-original draft, project administration, writing-review and editing. L. Scheller:Data curation, methodology, conducted experiments. A.T. Mayer: Conductedexperiments. S. Murty: Conducted experiments. O. Vermesh: Conductedexperiments. T.W. Nobashi: Conducted experiments. J.K. Lohmeyer: Conductedexperiments. T. Hirai: Conducted experiments. J. Baker: Generated the Lucþtransgenic C57bl/6 L2g85 mouse strain. K.H. Lau: Data curation, methodology.R. Negrin: Conceptualization, resources, supervision, funding acquisition, writing-review and editing. S.S. Gambhir:Conceptualization, resources, supervision, fundingacquisition, writing-review and editing.

AcknowledgmentsThe authors would like to acknowledge the Stanford Center for Innovation in

In-Vivo Imaging (SCI3) and, in particular, Drs. Timothy Doyle and Frezghi Habtefor supporting the preclinical imaging experiments. We also thank the Stanfordshared FACS facility for their support. In addition, we are extremely grateful tothe following: Dr. Idit Sagiv-Barfi for supporting in vivo studies, Drs. CorinneBeinat and Michelle James for their helpful advice on histology, and Dr. MartinSchneider for supporting microscopy.

This work was supported in part by funding from the Ben & Catherine IvyFoundation (S.S. Gambhir), the Canary Foundation (S.S. Gambhir), NCI R01 1CA201719-02 (S.S. Gambhir), R01 CA23158201 (R. Negrin), P01 CA49605, theParker Institute for Cancer Immunotherapy (S.S. Gambhir and R. Negrin), theGeneva University Hospitals Fellowship to F. Simonetta, the Swiss Cancer League






(BIL KLS 3806-02-2016 to F. Simonetta), the Fondation de Bienfaisance ValeriaRossi di Montelera (Eugenio Litta Fellowship to F. Simonetta), and the AmericanSociety for Blood and Marrow Transplantation (New Investigator Award 2018 toF. Simonetta).

We dedicate this paper to the loving memory of the late Professor Sanjiv SamGambhir. His extraordinary impact, vision, and humanity live on andwill continue toguide us for years to come.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 10, 2020; revised July 9, 2020; accepted August 27, 2020;published first September 8, 2020.

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Alam et al.




2020;80:4780-4790. Published OnlineFirst September 8, 2020.Cancer Res Israt S. Alam, Federico Simonetta, Lukas Scheller, et al. Strategy for Diagnosis of Acute Graft-versus-Host DiseaseVisualization of Activated T Cells by OX40-ImmunoPET as a

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Visualization of Activated T Cells by OX40-ImmunoPET as a ......anti-CD4 (clone GK1.5); BV605 anti-CD3 (clone 17A2); and BV650 anti-CD8 (clone 53–6.7). Samples were acquired on a