Article
Reference
Visualization of activated T cells by OX40-immunoPET as a strategy for diagnosis of acute Graft-versus-Host-Disease
ALAM, Israt S, et al.
Abstract
Graft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT), mediated primarily by donor T cells that become activated and attack host tissues. Non-invasive strategies detecting T cell activation would allow for early diagnosis and possibly more effective management of HCT recipients. Positron emission tomography (PET) imaging is a sensitive and clinically relevant modality ideal for GvHD diagnosis and there is a strong rationale for the use of PET tracers that can monitor T cell activation and expansion with high specificity. The tumor necrosis factor (TNF) receptor superfamily member OX40 (CD134) is a cell surface marker that is highly specific for activated T cells, is upregulated during GvHD, and mediates disease pathogenesis. We recently reported the development of an antibody-based activated T cell imaging agent targeting OX40. In the present study, we visualize the dynamics of OX40 expression in a major histocompatibility complex (MHC)-mismatch mouse model of acute GvHD using OX40-immunoPET. This approach enabled visualization of T cell activation at early stages [...]
ALAM, Israt S, et al . Visualization of activated T cells by OX40-immunoPET as a strategy for diagnosis of acute Graft-versus-Host-Disease. Cancer Research , 2020, vol. 80, no. 21, p.
4780-4790
DOI : 10.1158/0008-5472.CAN-20-1149 PMID : 32900772
Available at:
http://archive-ouverte.unige.ch/unige:153229
Disclaimer: layout of this document may differ from the published version.
Visualization of activated T cells by OX40-immunoPET as a strategy for diagnosis of acute Graft-versus-Host-Disease
Israt 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,#, Sanjiv S. Gambhir1,3,#,ϕ
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA, 94305, USA
2Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, Stanford, CA, USA
3Departments of Bioengineering and Materials Science & Engineering, Bio-X, Stanford University, Stanford, CA 943065, USA
Abstract
Graft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT), mediated primarily by donor T cells that become activated and attack host tissues. Non-invasive strategies detecting T cell activation would allow for early diagnosis and possibly more effective management of HCT recipients. Positron emission tomography (PET) imaging is a sensitive and clinically relevant modality ideal for GvHD diagnosis and there is a strong rationale for the use of PET tracers that can monitor T cell activation and expansion with high specificity. The tumor necrosis factor (TNF) receptor superfamily member OX40 (CD134) is a cell surface marker that is highly specific for activated T cells, is upregulated during GvHD, and mediates disease pathogenesis. We recently reported the development of an antibody-based activated T cell imaging agent targeting OX40. In the present study, we visualize the dynamics of OX40 expression in a major histocompatibility complex (MHC)-mismatch mouse model of acute GvHD using OX40-immunoPET. This approach enabled visualization of T cell activation at early stages of disease, prior to overt clinical symptoms with high sensitivity and specificity. This study
Co-corresponding authors: Israt S. Alam, The James H. Clark Center, 318 Campus Drive, Room E150A, Stanford, CA, 94305, USA. Phone: 650-725-3113; israt@stanford.edu; Robert Negrin, Center for Clinical Sciences Research, 269 Campus Drive, Room 2205, Stanford, CA 94305, USA. Phone: (650) 723-0822; negrs@stanford.edu.
*ISA and FS contributed equally to this work
§LS and ATM contributed equally to this work
#RN and SSG contributed equally to this work ϕIn memory of the late Professor Sanjiv Sam Gambhir Authors’ contributions
Contribution: ISA and FS conceived and designed research studies, developed methodology, conducted experiments, acquired and analyzed data, and prepared the manuscript. LS and KHL developed methodology, conducted experiments and acquired data. ATM, SM, OV, TN, JL, TH conducted experiments. JB generated the Luc+ transgenic C57BL/6 L2G85 mouse strain. RN and SSG conceived and designed research studies and prepared the manuscript.
Conflict-of-interest disclosure: SSG is the founder and equity holder of CellSight Inc. that develops and translates strategies for imaging cell trafficking/transplantation. All other authors have declared that no conflict of interest exists.
HHS Public Access
Author manuscript
Cancer Res. Author manuscript; available in PMC 2021 June 24.
Published in final edited form as:
Cancer Res. 2020 November 01; 80(21): 4780–4790. doi:10.1158/0008-5472.CAN-20-1149.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
highlights the potential utility of the OX40 PET imaging as a new strategy for GvHD diagnosis and therapy monitoring.
Keywords
Hematopoietic cell transplant (HCT); Graft-versus-Host-Disease (GvHD); T cell activation;
OX40; positron emission tomography (PET); immunoPET
Introduction
Allogeneic hematopoietic cell transplantation (HCT) is a well-established curative therapy for a broad range of hematologic malignancies. Unfortunately, allogeneic HCT is still associated with significant morbidity and mortality related to cancer relapse and transplant complications, namely Graft-versus-Host Disease (GvHD). During GvHD, the interaction of donor-derived T lymphocytes with host tissues induces their activation, proliferation and migration to target tissues, notably skin, gastrointestinal tract and hepatobiliary system, where they mediate cytolytic attack (1). Early diagnosis of acute GvHD is essential to promptly establish appropriate treatment and prevent disease progression. At present, acute GvHD diagnosis mainly relies on clinical manifestations combined with pathological analysis of target organs by tissue biopsies. Unfortunately, these approaches lack specificity and sensitivity and can result in iatrogenic complications. Further, these studies are only initiated after the development of overt clinical symptoms that occur later in the
pathogenesis of GvHD. Diagnostic strategies allowing early and non-invasive identification of patients developing acute GvHD are therefore urgently needed for more effective intervention.
Standard radiological evaluation by ultrasound, contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) is of limited utility for accurate diagnosis of acute GvHD. These anatomical imaging modalities report on gross morphological changes that lack specificity for GvHD (2). Positron emission tomography (PET) imaging is a non- invasive clinical imaging modality with high sensitivity and quantitative capabilities, incentives that are ideal for the dynamic assessment of inflammatory responses. Previous reports suggested that molecular imaging by 18F-fluorodeoxyglucose ([18F]FDG) PET can visualize tissue inflammation associated with acute gastrointestinal GVHD in both murine models and in the clinic (3). Unfortunately, clinical evaluation of [18F]FDG PET for acute GvHD diagnosis in larger cohorts of patients revealed a lack of specificity and low positive predictive value (4).
The use of alternative tracers designed to detect T cell activation more specifically have been reported. Previously we reported the ability of 2’-deoxy-2’-[18F]fluoro-9-β-D-arabino- furanosylguanine ([18F]F-AraG), a small molecule metabolic radiotracer preferentially accumulating in activated T cells to efficiently track T cell activation and expansion in a murine model of acute GvHD (5), an approach currently under clinical evaluation (NCT03367962). A major caveat of using small molecule tracers is that the metabolic pathways they target may also be upregulated in other hematopoietic and non-hematopoietic tissues. ImmunoPET is a rapidly growing area of PET imaging, leveraging the high
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
specificity and high affinity of monoclonal antibodies and antibody fragments that are radiolabelled with PET isotopes, allowing for the targeted detection of cell surface markers on specific subsets of cells (6).
We recently reported a novel immunoPET tracer developed using a murine OX40-specific monoclonal antibody (64Cu-OX40mAb) that enables non-invasive imaging of murine OX40+ activated T cells (7,8). OX40 (CD134), a member of the tumor necrosis factor (TNF) receptor superfamily, is a T cell costimulatory molecule whose expression is highly
restricted to activated T cells (9). Previous studies have shown an increase in OX40 expression at the T cell surface during acute GvHD in rodents (10), non-human primates (11) and humans (12–15) Moreover, there is evidence that OX40 plays a role in acute GvHD pathogenesis where OX40/OX40L blockade significantly attenuates disease progression in murine (16–18) and non-human primate (11) models of disease.
In the present work, we evaluated the utility of OX40-targeted immunoPET imaging and the sensitivity of this approach to image the kinetics and homing of activated T cells in vivo in a major MHC-mismatch murine model of acute GvHD.
Materials and methods
Animals
BALB/cJ (H-2kd) and C57Bl/6J (H-2kb) mice were purchased from the Jackson Laboratory (Sacramento, CA). Firefly Luciferase (Luc)+ transgenic C57BL/6 L2G85 mice have been described previously (19) and were bred in our animal facility at Stanford University.
B6.129S4-Tnfrsf4tm1Nik/J OX40 knockout mice (OX40−/− C57BL/6) were kindly provided by Dr. Ronald Levy’s laboratory at Stanford University.
Study approval
All procedures performed on animals were approved by Stanford University’s Institutional Animal Care and Use Committee (Stanford, CA, USA) and were in compliance with the guidelines of humane care of laboratory animals.
Allogeneic bone marrow transplantation and acute GvHD induction
Donor CD4+ and CD8+ conventional T cells (Tcon) were isolated from splenocytes harvested from luc+ C57BL/6 mice and enriched with CD4 and CD8 MicroBeads (Miltenyi Biotec). Cell purity was consistently >95%. T-cell–depleted bone marrow (BM) cells were prepared by first crushing bones followed by T-cell depletion using CD4 and CD8 MicroBeads (Miltenyi Biotec). BALB/c recipient mice were treated with lethal total body irradiation (TBI) consisting of 880 cGy in 2 doses administered 4 hours apart. On the same day, 5 × 106 BM cells from C57BL/6 mice and 1.0 × 106 Tcon from luc+ or OX40−/−
C57BL/6 mice were injected intravenously. Mice were monitored daily and body weight and GvHD score was assessed at day 4, day 7 and weekly thereafter as previously described (20).
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
Flow cytometry analysis
Single cell suspensions were prepared from cervical lymph nodes (CLN), mesenteric lymph nodes (MLN) and spleen. Extracellular staining was preceded by incubation with purified FC blocking reagent (Miltenyi Biotech) to reduce non-specific staining. Cells were stained with the following antibodies (Biolegend): FITC anti-CD45.1 (clone A20); PE anti-OX40 (clone OX-86) or appropriate isotype control (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); BV650 anti-CD8 (clone 53–6.7). Samples were acquired on a BD LSR II flow cytometer (BD Biosciences), and analysis was performed with FlowJo 10.5.0 software (Tree Star).
Immunofluorescence
Intestinal tissues were harvested from transplanted mice (day 7 post-transplant) and frozen in Optimal Cutting Temperature (OCT) embedding compound and stored at −80°C until further use. Tissue sections were cut longitudinally into 10 μm thick sections using a cryostat. Immunofluorescence staining was carried out using standard procedures using the following primary antibodies: anti-OX40 (clone OX86, BioXcell) and CD90.1-Biotin (Invitrogen). OX40 expression was detected using donkey anti-rat IgG-AF488 (Abcam) and Streptavidin-AF647 (Invitrogen) respectively. The sections were finally mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and imaged with Nikon A1R-Si-MP multiphoton confocal microscope using a Nikon CF160 Plan Apo Lambda 10x/0.45 numerical aperture objective lens.
Bioluminescence imaging (BLI)
Mice were injected with D-luciferin (10 mg/kg; intraperitoneally) and anesthetized with isoflurane. Imaging was conducted using an IVIS Spectrum imaging system (Perkin Elmer) and data were analysed with Living Image Software 4.1 (Perkin Elmer).
Radiolabeling of OX40-targeted monoclonal antibody
Bioconjugation of murine specific OX40mAb to DOTA-NHS chelate (Macrocyclics) was performed using previously optimized protocols (7) and is described in supplemental methods. DOTA-OX40mAb was radiolabeled with 64CuCl2 (University of Wisconsin, Madison, WI, USA) to produce 64Cu-DOTA-OX40mAb (heron referred to as 64Cu- OX40mAb) with final specific activity of 10–15μCi/μg, radiochemical purity >99%, and labelling efficiency of 95–99%. Radio-ITLC and radio-HPLC using size-exclusion liquid chromatography with a Phenomenex SEC 3000 column (Torrance, CA, USA) with sodium phosphate buffer [0.1 mol/L, pH 6.8)] was performed to corroborate radiochemical purity.
The final formulation was prepared in PBS.
Small animal PET/CT and ex vivo biodistribution studies
Mice 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 μCi, 8 ± 3μg) was administered intravenously (i.v.) via the tail vein. Transplanted mice and total body irradiation (TBI) controls received tracer at day 4 or day 7 post-transplant. Wild-type and OX40−/− mice were
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
also imaged for control studies. Static (10 min) PET scans were acquired 24 hours after
64Cu-OX40mAb administration using a small animal PET/CT hybrid scanner (Inveon, Siemens). CT images were acquired prior to each PET scan and within the same acquisition workflow to provide an anatomic reference for PET data and to allow for attenuation correction. PET image reconstruction and image analysis were conducted as previously described and is outlined in further details in supplemental methods (7).
Following the completion of the scan 24 hours post injection of tracer, ex vivo
biodistribution studies were performed to measure blood- and tissue-associated radioactivity.
Briefly, blood (~100μl) was collected via cardiac puncture and the following tissues were collected; cervical and mesenteric lymph nodes, spleen, skin, lower part of the small intestine (above the ileocecal valve), large intestine, kidney, liver, muscle and femur. Tissues were placed in a tube, weighed and radioactivity measured using an automated gamma counter (Cobra II; Packard). Tissue-associated radioactivity was normalized to tissue weight and amount of radioactivity administered to each mouse, decay-corrected to the time of radiotracer injection. Data were expressed as percentage injected dose per gram of tissue (%ID/g) values.
Statistical Analysis
Statistical analyses were performed using Prism 6 (GraphPad Software, La Jolla, CA) and R version 3.5.1 with R studio version 1.1.453. Heatmaps were generated using Pheatmap version 1.0.12. Principal component analysis was performed using the FactoMineR package version 1.41 and visualized using the factorextra package version 1.0.5. Receiver operating characteristic (ROC) curves where calculated and plotted using the plotROC version 2.2.1.
Results
OX40 is significantly upregulated on T cells during murine acute GvHD
We first analysed the dynamics of OX40 expression on the surface of CD4+ and CD8+ T cells recovered after adoptive transfer into allogeneic (BALB/c) or syngeneic (C57BL/6) recipients at the time of HCT. Donor-derived T cells were identified using the CD45.1+ and CD90.1+ congenic markers and OX40 expression was assessed by flow cytometry (using a gating strategy and an isotype control outlined in Supplemental Figure 1A and Supplemental Figure 1B respectively). We detected a significant upregulation of OX40 on CD4 (Figure 1A, upper panels) and to a lesser extent on CD8 T cells (Figure 1A, lower panels) recovered from the cervical (CLN) and mesenteric lymph nodes (MLN), both at day 4 and day 7 after transplantation. CD4 T cells isolated from the spleen significantly upregulated OX40 at day 4 after transplantation, while levels of expression at day 7 returned to baseline (Figure 1A, upper right panel). Conversely, CD8 T cells recovered from the spleen expressed higher levels of OX40 both at day 4 and day 7 compared to baseline (Figure 1A, lower right panel).
Importantly, OX40 upregulation was uniquely observed after adoptive transfer into allogeneic recipients, while cells recovered upon transfer into syngeneic mice did not show any significant increase in OX40 expression (Figure 1A, green symbols and lines). We next investigated the specificity of OX40 staining to identify activated donor-derived T cells after unsupervised clustering of splenocytes recovered at day 7 post-transplantation from
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
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, while only minimal expression was detected within host-derived or bone marrow-derived donor cells (Figure 1B). We finally assessed donor-derived T cell infiltration identified by CD90.1 staining (Figure 1C, red channel) and OX40 expression (green channel) in intestinal tissue, a prime site for GvHD pathology. Immunofluorescence staining performed on small intestine samples harvested at day 7 post-transplantation revealed higher numbers of CD90.1+ and OX40+ cells infiltrating the small intestine in GvHD mice in comparison to control mice (Figure 1C). Collectively these results indicate that OX40 is significantly upregulated at the T cell surface during alloreactive responses in GvHD, allowing for the selective
identification of activated donor-derived T cells in both lymphoid organs and the gastrointestinal tract.
OX40 targeted immunoPET enables visualization of activated T cells in GvHD target organs We next tested the ability of OX40-targeted immunoPET to capture T cell expansion and activation during acute GvHD using 64Cu-OX40mAb. Acute GvHD was induced using luc+
donor T cells enabling in vivo monitoring of donor T-cell expansion and accumulation using bioluminescence imaging (BLI) as reference. In vivo BLI and 64Cu-OX40mAb injections were performed on day 4 or day 7 post-transplant. PET/CT images were acquired 24 hours after tracer injection (Figure 2, Supplemental Figure 2). Figure 2A shows a reference atlas of a representative volume rendered technique (VRT) PET/CT image and axial PET/CT views with the location of key clearance, lymphoid and GvHD target tissues annotated. In
agreement with previous reports (5,19), in vivo BLI at day 4, a time point at which we could not detect any signs of clinical disease according to our scoring system, revealed donor T cell expansion and accumulation in the spleen and in MLN of GvHD mice (Figure 2B).
PET/CT images of control mice acquired at 24 hours after tracer injection showed that 64Cu- OX40mAb primarily accumulated in the heart and the liver, characteristic of whole antibody biodistribution, with minimal signal detected in the spleen and in the abdominal region (Figure 2C, left panel). GvHD mice injected with tracer on day 4 exhibited a pronounced
64Cu-OX40mAb-PET signal in the spleen, MLN and lower abdomen (Figure 2C) also seen in 3D-rotational images (Supplemental Video 1). We next performed the same analysis at day 7, a time point at which animals displayed overt signs of disease. In vivo BLI
demonstrated an expansion of donor derived T cells with highest levels of signal originating from the abdominal region (Figure 2D) indicating a dramatic intestinal infiltration at this time point in GvHD mice. PET imaging at day 7 (Figure 2E, Supplemental Video 2) corroborated BLI and showed high accumulation of 64Cu-OX40mAb in the abdominal region and, to a lesser extent, in the splenic region, a finding compatible with the further migration of T cells to the intestinal tract at this later time point (19). Notably, compared to control mice, GvHD mice exhibited lower PET signals in the heart (Figure 2C, 2E, Supplemental Videos 3, 4).
To quantify the trends observed in the PET images, we performed region of interest (ROI) analysis on multiple tissues and regions (Figure 3A; supplementary methods). ROI analysis of CLN was avoided due to contribution of high signal from surrounding blood vessels in the cervical region at the relevant time points, instead we focused on lymphoid
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
compartments in the abdomen. For day 4 tracer injections, ROI quantification of PET/CT images confirmed markedly increased radiotracer uptake in GvHD vs. control mice in lymphoid tissues and 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=9–10 per group, Figure 3B). Comparison of control mice with mice exposed to TBI without receiving bone marrow cells revealed a small but statistically significant contribution of BM-derived cells in the background signals detected in lymphoid organs, but not in the abdominal region (Supplemental Figure 3A and 3B), further supporting the specificity of the detected 64Cu-OX40mAb-PET signal for GvHD-mediating T cells. ROI quantification of PET/CT images obtained 24 hours after tracer injection (injection day 7, imaging day 8) revealed similar trends, with significantly higher radiotracer uptake in target tissues in 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 and abdominal region: 9.36±2.85 vs. 5.33±0.78, p<0.001; n=12 per group, Figure 3C). Importantly, the background 64Cu-OX40mAb-PET signals in muscle showed no significant difference between the two groups. Comparison of control mice receiving BM with TBI mice injected at day 7 confirmed the results obtained at day 4, with control mice specifically displaying higher 64Cu-OX40mAb-PET signal than TBI mice in the secondary lymphoid organs (Supplemental Figure 3C and 3D).
Biodistribution analysis using ex vivo gamma counting of tissues confirmed PET results with significantly increased tracer uptake measured in lymphoid tissues of GvHD vs. control mice; spleen (day 4 and 7; p<0.01), MLN (p<0.01), CLN (day 4; p<0.05, day 7; p<0.01;
Figure 4A). Increased tracer uptake was also confirmed in GvHD target organs; small intestine (day 4 and 7; p<0.05), colon (day 7; p<0.05) and skin (day 7; p<0.01) (Day 4;
Figure 4A, Day 7; Figure 4B). Mice with GvHD displayed significantly reduced signals from the heart as shown by ROI quantification (Figure 3) and biodistribution analysis of heart and blood (Figure 4), compared to control mice at day 4 and at day 7. A similar trend, reaching statistical significance only in ROI quantification at day 4, was observed for the liver (Figure 3B). Such background signals from heart, blood and liver are likely non- specific, due to the presence of antibody circulating in the blood and were independent of any OX40-specific binding to circulating cells as ROI quantification and in biodistribution analysis showed similar levels of signal in WT and OX40−/− mice (Supplemental Figure 3E and 3F), suggesting the presence of a sink effect leading to a reduction in the concentration of circulating 64Cu-OX40mAb tracer in GvHD mice. Correlation analysis showed a significant positive correlation between ROI quantification and biodistribution analysis, especially in spleen (R2 = 0.64, p=3.2e-08) and blood (R2 = 0.61, p=2.8e-09) (Supplemental Figure 4), further confirming the ability of OX40-immunoPET to define the localization of activated T cells in murine acute GvHD. Collectively, these results demonstrate the ability of
64Cu-OX40mAb immunoPET to detect T cell expansion and activation during murine acute GvHD prior to and after clinical signs of GVHD are evident.
OX40-immunoPET signal correlates with clinical status during murine acute GvHD
We next analyzed the relationship between 64Cu-OX40mAb tracer uptake and clinical signs of GvHD. As expected, 64Cu-OX40mAb tracer uptake in spleen, mesenteric lymph node (MLN) and the abdomen showed only poor if any correlation with body weight and GvHD
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
score at day 4, when mice were essentially asymptomatic (Supplemental Figure 5), highlighting the potential for early disease detection capabilities of OX40 imaging even before overt clinical signs of GvHD appear. Conversely, 64Cu-OX40mAb uptake in the abdominal region at day 7 negatively correlated with body weight (R2=0.44, p=0.0005) and positively correlated with the GvHD score (R2=0.59, p=0.00001; Figure 5). Moreover, 64Cu- OX40mAb uptake in spleen and MLN at day 7 positively correlated with the GvHD score (spleen: R2=0.36, p=0.0018; MLN: R2=0.18, p=0.042; Figure 5). Collectively, these results show that quantification of OX40-expressing activated T cells in lymphoid organs and GvHD-target sites allow early detection of GvHD even before the appearance of clinical signs and efficiently reflects the severity of the disease.
Early administration of OX40mAb at tracer doses significantly exacerbates murine acute GvHD outcome
Previous reports have demonstrated a role for OX40 expression on T cells during murine acute GvHD induction, showing that splenocytes from OX40−/− C57BL/6 mice (H-2Kb) had reduced GvHD induction potential upon transfer in MHC-mismatched B10.BR mice (H-2Kb) compared to their wild type counterpart (17). In agreement, we observed similar results in our mouse model as adoptive transfer of 1×106 T cells from OX40−/− C57BL/6 mice (H-2Kb) into lethally irradiated BALB/c mice (H-2Kd) at time of transplantation, resulted in a significantly delayed kinetic of GvHD-related death compared with the adoptive transfer of identical numbers of cells isolated from WT C57BL/6 mice
(Supplemental Figure 6). In the same report Blazar et al. showed that the administration of high (200μg) and repeated doses of the agonistic anti-OX40mAb (clone M5) significantly increased GvHD lethality in the same murine model (17). We therefore tested the safety of the agonistic anti-OX40mAb (clone OX86) we used for the imaging study in our murine model, administered at tracer doses (described in supplemental methods). Administration of very low doses (15μg-the maximal upper limit of antibody dose anticipated to ever be administered during PET imaging) of cold OX40mAb at day 4 after HCT, significantly accelerated GvHD lethality compared to administration of isotype control (p<0.0001; Figure 6A). Conversely, we did not detect any significant impact of administration of the same dose of cold OX40mAb at day 7 after HCT compared with mice that received isotype control (Figure 6B). Collectively, these results confirm the role of OX40 in murine acute GvHD pathogenesis and reveal that even very low doses of agonistic anti-OX40 mAb might exacerbate GvHD lethality when administered at early phases of GvHD induction.
Unsupervised analysis of OX40-immunoPET indicates that signals from abdomen, spleen and mesenteric lymph nodes have high diagnostic potential for murine acute GvHD detection
To assess the relative contribution of signals obtained from ROI of different tissues for the detection of murine acute GvHD, we first performed an unsupervised analysis of 64Cu- OX40mAb-PET signals detected by ROI quantification across all groups and mice (Figure 7A). Unsupervised hierarchical clustering easily separated all GvHD mice injected at day 4 after HCT together with one mouse at day 7 after HCT (Figure 7A). Within the second subgroup, hierarchical clustering identified a second cluster containing most of GvHD mice at day 7 with the exception of only two mice that clustered together with control mice
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
(Figure 7A). To identify the relative impact of different ROI in clustering we performed a principal component analysis (PCA) of ROI data (Figure 7B). The scree plot shown in Figure 7C reveal 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 GvHD mice injected at day 4 from all other mice, while a combination of PC1 and PC2 allowed the separation of GvHD mice from controls independently of the time points (Figure 7B).
Mapping of the component loadings identified abdomen, spleen and MLN ROIs as the variables most strongly contributing to the distinction between GvHD mice and control mice (Figure 7B). We therefore assessed the diagnostic potential of abdomen, MLN and spleen ROIs data for detection of murine acute GvHD at both day 4 and day 7 using receiver operating characteristic (ROC) curve analysis. Muscle ROI was used as a negative control.
As shown in Figure 7D, abdomen, spleen and MLN PET ROIs had a perfect diagnostic power for murine acute GvHD detection at day 4 [abdomen: area under the curve (AUC)=1;
MLN: AUC=1; spleen: AUC=1] and an excellent diagnostic power at day 7 (abdomen:
AUC=0.96; MLN: AUC=0.88; spleen: AUC=0.98). As expected, the muscle ROIs had no diagnostic potential for GvHD detection either at day 4 (AUC=0.28) or day 7 (AUC=0.43).
These results indicate that 64Cu-OX40mAb-PET signals detected in the abdomen, MLN and spleen ROIs have excellent diagnostic potential for detection of murine acute GvHD both before and after the appearance of clinical symptoms of disease.
Discussion
In 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 our ability to successfully prevent and treat acute GvHD depends on our capacity to efficiently detect disease before clinical manifestations appear, we hypothesized that a non-invasive imaging strategy able to detect T-cell activation would be an extremely powerful approach for early diagnosis of acute GvHD. Towards this aim, the selection of the most appropriate activation marker appears to be crucial. Previous studies in animal models (10,11) as well as in humans (12–15) have shown that expression of OX40 on T cells increases during GvHD, in particular at CD4+ T cell surface (15). Importantly, OX40 outperformed other activation markers, including CD25 and CD69, for detection of alloreactive T cell responses (14,15,21). After confirming OX40 upregulation at the T cell surface during alloreactive responses in both secondary lymphoid organs and target tissues, we demonstrate that OX40-immunoPET was able to visualize T cell activation even prior to the development of overt clinical symptoms in a murine model of GvHD. Importantly, OX40-immunoPET was able to distinguish clearly early intestinal GvHD from toxicities resulting from the conditioning regimen, which represents a major limitation to the radiological diagnosis of GvHD (2). However, the preclinical nature of our study limits the number of confounding factors often encountered in the differential diagnosis of GvHD, notably infections. Although OX40 specificity for the T cell compartment makes false positive originating from bacterial infections unlikely, we cannot exclude that viral
infections, notably cytomegalovirus (CMV) colitis, could result in positive signals in OX40- immunoPET. For interpretation of imaging results, it will therefore be critical to integrate
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
clinical (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 has motivated the
development of several immunoPET agents in recent years. To date there have been a few candidates reported, targeted to lineage-defining cell surface markers expressed on T cells such as CD3 (22–25). While these phenotypic-targeted probes can capture the dynamics of T cells, they fail to directly report on T cell activation. Recently Pektor et al. reported a CD3- targeted immunoPET approach for imaging GvHD progression in a humanized mouse model (23). It is well-documented that T cell activation mediates the downregulation of the CD3/T-cell receptor (26) a potential limitation in the context of inflammation. Another approach reported for PET imaging of GvHD progression in a humanized mouse model has been to image human class II major histocompatibility complex (HLA-DR) using a camelid derived single-domain antibody (27). Although HLA-DR expression increases during T cell activation, it is also expressed at high levels in the myeloid compartment and thus lacks specificity as an activated T cell imaging target compared to imaging of OX40.
Our preclinical data indicate that, owing to its high sensitivity, OX40-immunoPET could detect signs of GvHD even before clinical symptoms manifest. However, given the
complexity and relatively high costs of immunoPET, it is unlikely that this approach will be applied as a screening strategy for all allogeneic HCT recipients. For clinical translation of this type of imaging it will therefore be critical to carefully establish patient selection criteria. We can imagine a scenario in which investigation by immunoPET will be triggered by clinical suspicion, for example to investigate possible gut GvHD after the appearance of skin GvHD, or after initial screening using blood biomarkers like REG3α and ST2(28).
Alternatively, immunoPET of activated T cells could also be employed as a screening strategy in patients at high risk of developing GvHD (for example post-transplant from MHC-mismatched and/or unrelated donors). Results of early clinical trials, like the one ongoing at our institution using the small molecule PET tracer [18F]F-AraG to detect T cell activation for GvHD diagnosis (NCT03367962), will lay the groundwork for defining the patient selection criteria.
A major limitation of our study comes from the exacerbation of GvHD we observed upon administration of tracer-doses of agonistic anti-OX40 antibody, when given at early time points of disease (day 4). The anti-OX40 antibody significantly exacerbated GvHD similar to previous reports although in that study repeated administration of higher doses of anti- OX40 MAbs were studied (17). These results further confirm the role of OX40 in the GvHD pathogenesis and stress the importance of the selection of appropriate clones for each application. While agonistic anti-OX40 antibodies can be appropriate for imaging of T-cell activation in cancer immunotherapy settings (7,8), immunoPET in immunopathological settings targeting costimulatory molecules will probably require the use of non-agonist or even antagonist antibody clones. Although the generation of an antagonistic murine anti- OX40 clone is beyond the scope of the present report, clinical translation of OX40-
immunoPET for GvHD diagnosis would probably benefit from the use of anti-human-OX40 antagonist clones such as the GBR830 clone, currently under clinical investigation for atopic dermatitis (29) (NCT03568162) and recently reported to suppress xenogeneic GvHD when
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
administered in combination with Cyclosporine A (30). Alternative approaches to minimize adverse biological effects may also include the generation of antibody fragments lacking the potency of a full-length antibody and the Fc region known to engage other immune cells, or engineered binders (31). Perturbations in the biology of immune cells by immunoPET tracers has previously been noted (32,33) and may be further minimized by improving the specific activity of the radiolabelled probe so that significantly less mass is administered.
In summary, this study demonstrates the utility of OX40 as a sensitive imaging biomarker for the early and specific visualization of activated T cells in GvHD. Integrated with tissue biopsies and endoscopic evaluation, we anticipate that this whole-body imaging approach of T cell activation by immunoPET could significantly improve upon current GvHD diagnosis and provide earlier diagnosis where interventions may be more effective for improved clinical care.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
The authors would like to acknowledge the Stanford Center for Innovation in In-Vivo Imaging (SCI3) and in particular, Drs. Timothy Doyle and Frezghi Habte for supporting the preclinical imaging experiments. We also thank the Stanford shared FACS facility for their support. In addition, we are extremely grateful to the following; Dr Idit Sagiv-Barfi for supporting in vivo studies, Drs Corinne Beinat and Michelle James for their helpful advice on histology and Dr Martin Schneider for supporting microscopy.
This work was supported in part from funding from the Ben & Catherine Ivy Foundation (SSG), the Canary Foundation (SSG), NCI R01 1 CA201719–02 (SSG), R01 CA23158201 (RN), P01 CA49605 the Parker Institute for Cancer Immunotherapy (SSG and RN), the Geneva University Hospitals Fellowship to FS, the Swiss Cancer League (BIL KLS 3806-02-2016 to FS), the Fondation de Bienfaisance Valeria Rossi di Montelera (Eugenio Litta Fellowship to FS), the American Society for Blood and Marrow Transplantation (New Investigator Award 2018 to FS).
References
1. Zeiser R, Blazar BR. Acute Graft-versus-Host Disease - Biologic Process, Prevention, and Therapy.
N Engl J Med 2017;377:2167–79 [PubMed: 29171820]
2. Lubner MG, Menias CO, Agrons M, Alhalabi K, Katabathina VS, Elsayes KM, et al. Imaging of Abdominal and Pelvic Manifestations of Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplant. AJR Am J Roentgenol 2017;209:33–45 [PubMed: 28463600]
3. Stelljes M, Hermann S, Albring J, Kohler G, Loffler M, Franzius C, et al. Clinical molecular imaging in intestinal graft-versus-host disease: mapping of disease activity, prediction, and monitoring of treatment efficiency by positron emission tomography. Blood 2008;111:2909–18 [PubMed: 18057227]
4. Bodet-Milin C, Lacombe M, Malard F, Lestang E, Cahu X, Chevallier P, et al. 18F-FDG PET/CT for the assessment of gastrointestinal GVHD: results of a pilot study. Bone Marrow Transplant 2014;49:131–7 [PubMed: 24076550]
5. Ronald JA, Kim BS, Gowrishankar G, Namavari M, Alam IS, D’Souza A, et al. A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant. Cancer Res 2017;77:2893–902 [PubMed: 28572504]
6. Freise AC, Wu AM. In vivo imaging with antibodies and engineered fragments. Mol Immunol 2015;67:142–52 [PubMed: 25934435]
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
7. Alam IS, Mayer AT, Sagiv-Barfi I, Wang K, Vermesh O, Czerwinski DK, et al. Imaging activated T cells predicts response to cancer vaccines. J Clin Invest 2018;128:2569–80 [PubMed: 29596062]
8. Sagiv-Barfi I, Czerwinski DK, Levy S, Alam IS, Mayer AT, Gambhir SS, et al. Eradication of spontaneous malignancy by local immunotherapy. Sci Transl Med 2018;10
9. Croft M Control of immunity by the TNFR-related molecule OX40 (CD134). Annu Rev Immunol 2010;28:57–78 [PubMed: 20307208]
10. Tittle TV, Weinberg AD, Steinkeler CN, Maziarz RT. Expression of the T-cell activation antigen, OX-40, identifies alloreactive T cells in acute graft-versus-host disease. Blood 1997;89:4652–8 [PubMed: 9192792]
11. Tkachev V, Furlan SN, Watkins B, Hunt DJ, Zheng HB, Panoskaltsis-Mortari A, et al. Combined OX40L and mTOR blockade controls effector T cell activation while preserving Treg
reconstitution after transplant. Sci Transl Med 2017;9
12. Lamb LS Jr., Abhyankar SA, Hazlett L, O’Neal W, Folk RS, Vogt S, et al. Expression of CD134 (0X-40) on T cells during the first 100 days following allogeneic bone marrow transplantation as a marker for lymphocyte activation and therapy-resistant graft-versus-host disease. Cytometry 1999;38:238–43 [PubMed: 10516610]
13. Kotani A, Ishikawa T, Matsumura Y, Ichinohe T, Ohno H, Hori T, et al. Correlation of peripheral blood OX40+(CD134+) T cells with chronic graft-versus-host disease in patients who underwent allogeneic hematopoietic stem cell transplantation. Blood 2001;98:3162–4 [PubMed: 11698307]
14. Sanchez J, Casano J, Alvarez MA, Roman-Gomez J, Martin C, Martinez F, et al. Kinetic of regulatory CD25high and activated CD134+ (OX40) T lymphocytes during acute and chronic graft-versus-host disease after allogeneic bone marrow transplantation. Br J Haematol 2004;126:697–703 [PubMed: 15327522]
15. Paz Morante M, Briones J, Canto E, Sabzevari H, Martino R, Sierra J, et al. Activation-associated phenotype of CD3 T cells in acute graft-versus-host disease. Clin Exp Immunol 2006;145:36–43 [PubMed: 16792671]
16. Stuber E, Von Freier A, Marinescu D, Folsch UR. Involvement of OX40-OX40L interactions in the intestinal manifestations of the murine acute graft-versus-host disease. Gastroenterology
1998;115:1205–15 [PubMed: 9797376]
17. Blazar BR, Sharpe AH, Chen AI, Panoskaltsis-Mortari A, Lees C, Akiba H, et al. Ligation of OX40 (CD134) regulates graft-versus-host disease (GVHD) and graft rejection in allogeneic bone marrow transplant recipients. Blood 2003;101:3741–8 [PubMed: 12521997]
18. Tripathi T, Yin W, Xue Y, Zurawski S, Fujita H, Hanabuchi S, et al. Central Roles of OX40L-OX40 Interaction in the Induction and Progression of Human T Cell-Driven Acute Graft-versus-Host Disease. Immunohorizons 2019;3:110–20 [PubMed: 31240276]
19. Beilhack A, Schulz S, Baker J, Beilhack GF, Wieland CB, Herman EI, et al. In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood 2005;106:1113–22 [PubMed: 15855275]
20. Cooke KR, Kobzik L, Martin TR, Brewer J, Delmonte J Jr., Crawford JM, et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood 1996;88:3230–9 [PubMed: 8963063]
21. Ge X, Brown J, Sykes M, Boussiotis VA. CD134-allodepletion allows selective elimination of alloreactive human T cells without loss of virus-specific and leukemia-specific effectors. Biol Blood Marrow Transplant 2008;14:518–30 [PubMed: 18410894]
22. Beckford Vera DR, Smith CC, Bixby LM, Glatt DM, Dunn SS, Saito R, et al. Immuno-PET imaging of tumor-infiltrating lymphocytes using zirconium-89 radiolabeled anti-CD3 antibody in immune-competent mice bearing syngeneic tumors. PLoS One 2018;13:e0193832 [PubMed:
29513764]
23. Pektor S, Schloder J, Klasen B, Bausbacher N, Wagner DC, Schreckenberger M, et al. Using immuno-PET imaging to monitor kinetics of T cell-mediated inflammation and treatment efficiency in a humanized mouse model for GvHD. Eur J Nucl Med Mol Imaging 2019
24. Freise AC, Zettlitz KA, Salazar FB, Tavare R, Tsai WK, Chatziioannou AF, et al. Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis. J Nucl Med 2018;59:980–5 [PubMed: 29326360]
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
25. Tavare R, Escuin-Ordinas H, Mok S, McCracken MN, Zettlitz KA, Salazar FB, et al. An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy. Cancer Res 2016;76:73–82 [PubMed: 26573799]
26. San Jose E, Borroto A, Niedergang F, Alcover A, Alarcon B. Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism.
Immunity 2000;12:161–70 [PubMed: 10714682]
27. Van Elssen C, Rashidian M, Vrbanac V, Wucherpfennig KW, Habre ZE, Sticht J, et al. Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease. J Nucl Med 2017;58:1003–8 [PubMed: 28209904]
28. Hartwell MJ, Ozbek U, Holler E, Renteria AS, Major-Monfried H, Reddy P, et al. An early- biomarker algorithm predicts lethal graft-versus-host disease and survival. JCI Insight 2017;2:e89798 [PubMed: 28194439]
29. Guttman-Yassky E, Pavel AB, Zhou L, Estrada YD, Zhang N, Xu H, et al. GBR 830, an anti- OX40, improves skin gene signatures and clinical scores in patients with atopic dermatitis. J Allergy Clin Immunol 2019;144:482–93 e7 [PubMed: 30738171]
30. Macoin JBS, Monney T, Sancheti P, Reddy V, Back J. GBR830, a True OX40 Antagonist Antibody with Potent Suppressive Effects on T Cell-Mediated Pathological Responses. ACR/ARHP Annual Meeting: Arthiritis Rheumatol; 2018.
31. Tavare R, McCracken MN, Zettlitz KA, Knowles SM, Salazar FB, Olafsen T, et al. Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo. Proc Natl Acad Sci U S A 2014;111:1108–13 [PubMed: 24390540]
32. James ML, Hoehne A, Mayer AT, Lechtenberg K, Moreno M, Gowrishankar G, et al. Imaging B cells in a mouse model of multiple sclerosis using 64Cu-Rituximab-PET. J Nucl Med 2017 33. Freise AC, Zettlitz KA, Salazar FB, Lu X, Tavare R, Wu AM. ImmunoPET Imaging of Murine
CD4(+) T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging. Mol Imaging Biol 2017;19:599–609 [PubMed: 27966069]
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
Statement of significance
OX40-immunoPET imaging is a promising non-invasive strategy for early detection of GvHD, capable of detecting signs of GvHD pathology even prior to the development of overt clinical symptoms.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
FIGURE 1. OX40 expression on T cells in a murine model of acute GvHD.
(A) Graphs represent percentages of OX40-expressing CD90.1+ CD45.1+ CD4+ (upper panels) and CD8+ (lower panels) T cells recovered from cervical lymph nodes (CLN), mesenteric lymph nodes (MLN) and spleen 4 and 7 days after HCT and adoptive transfer of T cells isolated from C57Bl/6 donors into syngeneic C57Bl/6 (green symbols and lines) or allogeneic BALB/c (red symbols and lines) recipients. Results are pooled from two
independent experiments with a total of 10–11 mice per group. Day 4 and day 7 values were compared to day 0 values using a nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test. P values are shown when significant (****p < 0.0001, ***p <
0.001, **p < 0.01, *p < 0.05). (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 (CD45.2+, H-2kb+) with or without C57BL/6 T cells (CD45.1+, Thy1.1+, H-2kb+). 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 CD8 expression. Red indicates OX40-expressing cells. (C) Representative confocal images of intestinal tissue from GvHD and control mice at day 7 post-transplant (n=3 per group). High OX40 (green) and pan T cell marker CD90.1 (red) staining are observed in the villi and crypts of the GvHD mice (lower panel) in comparison to the control mice (upper panel). Merged views with DAPI staining of nuclei (blue) are shown; scale bar, 200μm.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
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 key clearance, lymphoid and GvHD target tissues shown (H; heart, Li; liver, S;
spleen, M; mesenteric lymph node and Ab; abdomen). (B, D) Representative
bioluminescence images (BLI) and (C, E) 3D volume rendered technique (VRT) images of
64Cu-DOTA-OX40mAb PET/CT images acquired 24 hours post-tracer administration at day 4 and 7 after HCT in control (left panels) or GvHD (right panels) mice. Images are
representative of two independent experiments per time point with 9–12 mice per group.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
FIGURE 3. Quantification of 64Cu-DOTA-OX40mAb uptake using region of interest (ROI) analysis during acute GvHD.
(A) Reference atlas for ROI definition (H; heart, Li; liver, S; spleen, M; mesenteric lymph node, Ab; abdomen, B; bladder, F; femur and Mu; muscle). (B) Quantitative region of interest (ROI) PET image analysis of spleen, mesenteric lymph nodes (MLN), abdomen, liver, muscle, femur and heart at day 4 (B) and 7 (C) post-HCT in controls (light blue and blue filled boxes, respectively) or GvHD mice (orange and red filled boxes, respectively).
Values are summarized as box plots, representing the range, first quartile, median, third quartile, and eventual outliers. Tracer uptake in control (day 4: n=9; day 7: n=12) and GvHD (day 4: n=10; day 7: n=12) groups were compared using the Mann–Whitney U test (****p <
0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). Results are pooled from two independent experiments.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
FIGURE 4. Quantitative 64Cu-DOTA-OX40mAb tracer biodistribution during acute GvHD.
Quantification of OX40-immunoPET signal (%ID/g) from ex vivo biodistribution analysis of spleen, cervical lymph nodes (CLN), mesenteric lymph nodes (MLN), small intestine, colon, skin, liver, muscle, femur, heart, whole blood, kidney and tail 24 hours after tracer
administration on 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 in control (day 4: n=9; day 7: n=12) and GvHD (day 4: n=10; day 7: n=12) groups were compared using the Mann–Whitney U test (**p < 0.01, *p < 0.05, n=12 per group). Results are pooled from two independent experiments.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
FIGURE 5. Correlation of 64Cu-DOTA-OX40mAb PET results with clinical signs of acute GvHD at day 7.
Correlation of tracer uptakes at day 4 (A) and day 7 (B) after HCT in spleen, mesenteric lymph nodes (MLN) and abdomen ROI with body weight (upper panels) and GvHD scores (lower panels). Correlations were evaluated using a Spearman rank correlation coefficient test. Results are pooled from two independent experiments with a total of 9–12 mice per group per time point. P ˂ 0.05 was considered statistically significant.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
FIGURE 6. Impact of dosing regimen of anti-OX40mAb administration on acute GvHD outcomes.
Overall survival after HCT with TBI (light grey) or BM alone (light blue and blue line) 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 μg/mouse, representing upper limit of tracer dose). Results are pooled from two independent experiments with a total of 10 mice per group. Survival curves were plotted using the Kaplan-Meier method and compared by log-rank test. P ˂ 0.05 was considered statistically significant.
A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt A uthor Man uscr ipt
FIGURE 7. Unsupervised analysis of 64Cu-DOTA-OX40mAb PET in murine acute GvHD identifies diagnostic ROIs.
(A) Heatmap visualization of normalized OX40 PET tracer uptake values in regions of interest (rows) from all transplant recipient studies (columns). Column labels below the heatmap indicate “Group_Day_.replicate#” for each individual mouse. Data shown are pooled from two independent experiments per time point. (B) Principal component analysis (PCA) performed using normalized OX40 PET tracer uptake values from ROI analysis. The relative contribution of each ROI to the clustering is depicted as an arrow. (C) Screen plot showing the percentage of the variance explained by each principal component. (D) Receiver operator curves (ROC) showing sensitivity against 1-specificity for distinguishing control mice from GvHD mice using OX40 PET tracer uptake values from ROIs identified in the PCA (MLN, spleen, abdomen) plus muscle as negative control. Area under the curve (AUC) is indicated.
