A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant

Abstract

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

Conflict of interest statement

Conflict of Interest: Dr. Yaghoubi and Dr. Gambhir are founders of CellSight Technologies Inc. that has licensed the rights to [18F]AraG from Stanford University.

©2017 American Association for Cancer Research.

Figures

Figure 1. [18F]F-AraG Accumulates in Cells via dCK and dGK Activity and at Increased Levels in Activated Versus Resting Primary Human T Cells

a) Uptake and b) retention of [18F]F-AraG across wild-type CCRF-CEM T lymphoblasts, mutant CCRF-CEM dCK- cells (dCK-), and dCK- cells overexpressing either dCK (dCK+) or dGK (dGK+) (n=4 per cell type per time point). Significantly less uptake was seen due to the loss of dCK in wild-type cells (CCRF-CEM vs. dCK-), (p<0.001). There was a trend towards higher uptake in dCK+ versus dCK- cells, whereas dGK+ cells had significantly higher uptake compared to dCK- cells and equivalent uptake compared to wild-type cells. No differences in retention were seen across cell types. c) Activated primary human T cells had significantly higher [18F]F-AraG uptake compared to resting T cells at all time points examined (***p<0.001; n=4 per cell state per time point). Data in all graphs are expressed as mean ± SD.

Figure 2. Bioluminescence and [18F]F-AraG PET Imaging of Donor T Cell Dynamics During the Initiation of T Cell Activation in a Mouse Model of acute GVHD

a) Bioluminescence imaging of control and GVHD mice 3 days after HCT revealed homing of luciferase-positive donor T cells to secondary lymphoid organs such as the spleen, cervical lymph nodes (CLN), and mesenteric lymph nodes (MLN) in GVHD mice. b) Representative [18F]F-AraG PET/CT images (10 minute static scan; 60-70 minutes post-tracer administration; ∼200 μCi) at this time point showed visibly higher tracer uptake in the CLN of GVHD mice versus control mice (n=3 per group). c) Quantitative region of interest PET image analysis of the CLN corroborated our qualitative assessments, demonstrating significantly (*p<0.05) higher tracer uptake in the CLN of GVHD mice versus control mice (%ID/g; percentage injected dose per gram of tissue). d) Biodistribution studies (90 minutes after tracer administration) also supported our imaging findings showing significantly (*p<0.05) higher tracer uptake in CLNs in GVHD versus control mice. Significantly (*p<0.05) higher tracer uptake was also apparent in the T-cells harboring MLN and a trend (p=0.12) towards higher uptake was seen in the spleen. Data in all graphs are expressed as mean ± SD.

Figure 3. Bioluminescence and [18F]F-AraG PET Imaging of Donor T Cell Dynamics During the Peak of T Cell Activation in a Mouse Model of acute GVHD

a) Bioluminescence imaging of control and GVHD mice 10 days after HCT revealed more widespread distribution of luciferase-positive donor T cells in GVHD mice but still localized accumulation within the cervical lymph nodes (CLN). Note the order of magnitude difference in scale between the images shown here and those in Fig. 2a (Day 3 after cell transplantation). b) Representative [18F]F-AraG PET/CT images (10 minute static scans, 60-70 post-tracer administration; ∼200 μCi) at this time point showed visibly higher tracer uptake in the CLN of GVHD (n=4) versus control mice (n=3). c) Quantitative region of interest PET image analysis of the CLN corroborated our qualitative assessments, demonstrating significantly higher tracer uptake in the CLN of GVHD versus control mice (*p<0.05) (%ID/g; percentage injected dose per gram of tissue). d) Biodistribution studies (90 minutes after tracer administration) also supported our imaging findings showing significantly higher tracer uptake in CLNs in GVHD versus control mice. Significantly higher tracer uptake was also apparent in the MLN (*p<0.05). At this time point, significantly lower tracer uptake was noted within the spleen (*p<0.05). e) Comparison of Day 3 and Day 10 bioluminescence and PET image analysis of CLNs shows both significantly higher BLI and PET signals on Day 10 compared to Day 3. Data in all graphs are expressed as mean ± SD.

Figure 4. Whole body PET images of [18F]F-AraG in a healthy human volunteer

Frontal (top panel) and dorsal (bottom panel) coronal PET images taken immediately after intra-venous injection of the tracer and at serial time points in one of the 6 humans imaged. Highest activity is observed in the liver, kidneys and bladder, associated with [18F]F-AraG clearance from the body. Uptake is also observed in the heart and spleen while relatively low background activity is observed in all other tissues. Similar images were obtained from the other 5 subjects.