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Background:
Myeloid cells play an essential role in the progression and remission of multiple sclerosis (MS)1-5. Activated macrophages and microglia are the predominant immune cell within acute and chronic-active MS lesions, where they contribute to tissue injury and repair6. The abundance and spatiotemporal dynamics of activated myeloid cells represent attractive targets for molecular imaging of MS disease activity, however, current imaging methods for specific and functional tracking of myeloid cells in vivo is limited. Recent studies have identified the upregulation of C-type lectin receptor a (CLEC7a) expression on the surface of activated myeloid cells in MS patients7.
Hypothesis/Objective:
Here, we evaluate CLEC7a expression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and report the development and application of CLEC7a-targeted PET for non-invasive imaging of activated myeloid cells.
Methods:
Animals: EAE was induced in female C57BL/6 mice (9-11 weeks) with myelin oligodendrocyte glycoprotein (MOG)35-55 emulsified in Complete Freund’s Adjuvant. Flow Cytometry: Anti-CD45-FITC was retro-orbitally injected 5 minutes prior to perfusion to label intravenous immune cells and discriminate tissue-based populations. Spinal cords were harvested and single cell suspensions were stained to assess CLEC7a expression on myeloid populations. Radiochemistry: CLEC7a monoclonal antibody was conjugated to DOTA with an average ratio of 1.6. CLEC7a-mAb-DOTA was 64Cu-radiolabeled at an efficiency of 75 – 100% and final radiochemical purity of >99%. 64Cu-Isotype-mAb was synthesized using the same methods to assess tracer specificity. PET/CT Imaging: 15-minute static PET/CT images were acquired 20 hours post-tracer injection (70-80mCi) on a dual micro-PET/CT scanner (Inveon). PET data was analyzed using VivoQuant v5.0. Following PET, tissues were harvested for biodistribution and ex vivo autoradiography to confirm in PET findings.
Results:
Flow cytometry revealed significantly increased proportions of CLEC7a-expressing myeloid cell populations in the lumbar and thoracic spinal cords of EAE mice compared to naïve and adjuvant-only controls (Fig. 1A). Notably, infiltrating CD45hiCD11b+CD49d+Ly6C+ monocytes showed elevated CLEC7a expression, where CD49d serves as a peripheral marker that distinguishes infiltrating from resident cells. Among resident microglial subpopulations, CD45hiCD11b+CD49d- cells (likely activated microglia) demonstrated significantly increased CLEC7a expression. Interestingly Clec7a+CD45midCD11b+CD49d- populations (resident microglia) were highest in adjuvant control, intermediate in EAE, and lowest in naïve mice. This pattern suggests resident microglia may upregulate CD45 expression upon activation and demonstrate that CLEC7a is broadly expressed across infiltrating and resident myeloid subsets during neuroinflammation, establishing it as an ideal target for imaging disease-associated inflammation.
Stability studies in mouse plasma (37°C) demonstrated 64Cu-CLEC7a-mAb stability up to 48 hours. In vivo CLEC7a-PET in EAE and naïve mice and revealed significantly increased 64Cu-CLEC7a-mAb signal in the lumbar and thoracic spinal cords of EAE versus naïve mice (Fig. 1B), with a positive correlation found between tracer signal and EAE score. Comparable results were seen when expressed as SUV (not shown). These findings were confirmed by ex vivo autoradiography (Fig. 1B) and biodistribution (gamma counting, Fig. 1D). 64Cu-Isotype-mAb-PET in EAE mice revealed significantly lower signal in the lumbar and thoracic spinal cords compared to 64Cu-CLEC7a (Fig. 1C), supporting the specificity of our CLEC7a-PET approach. Reduced 64Cu-Isotype-mAb signal was also confirmed by ex vivo autoradiography and biodistribution.
Conclusion:
CLEC7a represents a promising PET imaging biomarker for imaging myeloid activation in neuroinflammatory disease. Elevated CLEC7a expression is observed on both resident microglia and infiltrating monocytes in EAE spinal cords. CLEC7a-PET signal was significantly increased in the spinal cords of EAE mice compared to naïve and correlated with disease severity. 64Cu-Isotype-mAb-PET in EAE mice confirmed 64Cu-CLEC7a-mAb specificity. These results highlight the potential of CLEC7a-PET for non-invasive detection innate immune responses in MS and other neuroinflammatory conditions. Ongoing work includes PET imaging of adjuvant-only control mice as well as low and high EAE disease states.
Image/Figure:
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Image/Figure Caption:
Fig. 1: A) Percentage of CLEC7a positive microglia (CD45midCD11b+CD49d-), activated microglia (CD45hiCD11b+CD49d-), and infiltrating monocytes (CD45hiCD11b+CD49d+Ly6C+) in the lumbar and cervical + thoracic spinal cord. B) Representative CLEC7a-PET/CT images of whole-body signal in Naïve and EAE mice and representative ex vivo autoradiography images of naïve and EAE spinal cords. CLEC7a-PET CT-based quantification of the spinal cord and correlation between EAE score and CLEC7a-tracer uptake is shown as %ID/g. Quanification of ex vivo autoradiography is shown as mean pixel intensity (MPI) per dose. C) Representative PET/CT images of 64Cu-CLEC7a-mAb and 64Cu-Isotype-mAb in EAE mice and representative ex vivo autoradiography images of the spinal cords. CLEC7a-PET CT-based quantification of the spinal cord is shown as %ID/g. Quantification of ex vivo autoradiography is shown as mean pixel intensity (MPI) per dose D) Quantification of ex vivo biodistribution (gamma counting) of organs of interest expressed as %ID/g. For flow cytometry analysis: naïve n = 4, control n = 4, EAE n = 6. For PET and ARG analysis, naïve = 4, 64Cu-CLEC7a EAE = 7, 64Cu-Isotype EAE = 3. For biodistribution analysis naïve n = 5, EAE n = 7.
Author
Elizabeth A. Kras
Washington University in St. Louis