Longitudinal Imaging of T Cells and Inflammatory Demyelination in a Preclinical Model of Multiple Sclerosis Using 18F-FAraG PET and MRI

Abstract

Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease-modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T cell levels, which may have important implications for patient stratification and choice of DMT. Although MRI has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2'-deoxy-2'-¹⁸F-fluoro-9-β-d-arabinofuranosylguanine (¹⁸F-FAraG) PET imaging to noninvasively assess infiltrating T cells and to provide, in combination with MRI, a novel tool to determine lesion types. Methods: We used a novel MS mouse model that combines cuprizone and experimental autoimmune encephalomyelitis to reproducibly induce 2 brain inflammatory lesion types, differentiated by their T cell content. ¹⁸F-FAraG PET imaging, T2-weighted MRI, and T1-weighted contrast-enhanced MRI were performed before disease induction, during demyelination with high levels of innate immune cells, and after T cell infiltration. Fingolimod immunotherapy was used to evaluate the ability of PET and MRI to detect therapy response. Ex vivo immunofluorescence analyses for T cells, microglia/macrophages, myelin, and blood-brain barrier (BBB) integrity were performed to validate the in vivo findings. Results:¹⁸F-FAraG signal was significantly increased in the brain and spinal cord at the time point of T cell infiltration. ¹⁸F-FAraG signal from white matter (corpus callosum) and gray matter (cortex, hippocampus) further correlated with T cell density. T2-weighted MRI detected white matter lesions independently of T cells. T1-weighted contrast-enhanced MRI indicated BBB disruption at the time point of T cell infiltration. Fingolimod treatment prevented motor deficits and decreased T cell and microglia/macrophage levels. In agreement, ¹⁸F-FAraG signal was decreased in the brain and spinal cord of fingolimod-treated mice; T1-weighted contrast-enhanced MRI revealed intact BBB, whereas T2-weighted MRI findings remained unchanged. Conclusion: The combination of MRI and ¹⁸F-FAraG PET enables detection of inflammatory demyelination and T cell infiltration in an MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and after DMT. On clinical translation, these methods hold great potential for stratifying patients, monitoring MS progression, and determining therapy responses.

Keywords: 18F-FAraG PET imaging; MRI; T cells; central nervous system; multiple sclerosis.

Graphical abstract

FIGURE 1.

(A) ¹⁸F-FAraG PET/CT images and ¹⁸F-FAraG PET/CT overlaid on T2-weighted (T₂w) MR images at baseline, W3, and W7–14dpi. Graphs show ¹⁸F-FAraG uptake in entire brain, corpus callosum, hippocampus, and cortex. (B) Immunofluorescence images of CD3 T cells (green) from corpus callosum, hippocampus, and cortex at W7–14dpi. Quantification of CD3 T cells in corpus callosum, hippocampus, and cortex at baseline, W3, and W7–14dpi. (C) Correlation of ¹⁸F-FAraG signal with CD3 T cells at W7–14dpi. ID = injected dose. **P ≤ 0.01. ***P ≤ 0.001. ****P ≤ 0.0001.

FIGURE 2.

(A) T1-weighted (T₁w) MR images after injection of gadolinium and T2-weighted (T₂w) MR images at baseline, W3, and W7–14dpi. Arrows indicate corpus callosum. (B) Corresponding quantification of T1-enhancing lesions and normalized T2-weighted signal intensity from corpus callosum, hippocampus, and cortex. *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001.

FIGURE 3.

(A) Immunofluorescence images from corpus callosum (dashed lines) for microglia/macrophages (Iba1, red), myelin (MBP, orange), and fibrinogen (magenta). (B) Corresponding quantitative analyses in control animals, at W3 and W7–14dpi. *P ≤ 0.05. **P ≤ 0.01. ****P ≤ 0.0001.

FIGURE 4.

(A) ¹⁸F-FAraG PET/CT sagittal images at baseline, W3, and W7–14dpi. Arrows point to lumbar spinal cord. Graphs show corresponding quantification of ¹⁸F-FAraG signal in cervical/thoracic and lumbar spinal cord. (B) Immunofluorescence images of CD3 T cells (green) in cervical/thoracic and lumbar spinal cord at W7–14dpi, and quantification of CD3 immunostaining at baseline, W3, and W7–14dpi. (C) Correlation of ¹⁸F-FAraG signal with CD3 T cells at W7–14dpi. ID = injected dose. **P ≤ 0.01. ****P ≤ 0.0001.

FIGURE 5.

¹⁸F-FAraG PET/CT images showing subiliac lymph nodes and quantification of ¹⁸F-FAraG uptake at baseline, W3, and W7–14dpi. ID = injected dose. **P ≤ 0.01. ****P ≤ 0.0001.

FIGURE 6.

(A) ¹⁸F-FAraG PET/CT and ¹⁸F-FAraG PET/CT overlaid on T2-weighted (T₂w) MR images from untreated and fingolimod-treated animals at W7–14dpi. Graph shows quantification of ¹⁸F-FAraG signal in entire brain, corpus callosum, hippocampus, and cortex. (B) T1-weighted (T₁w) and T2-weighted MR images of fingolimod-treated and untreated mice and corresponding quantification of T1-enhancing lesions and normalized T2-weighted signal intensity of corpus callosum, hippocampus and cortex. (C) Immunofluorescence images of corpus callosum for CD3 T cells (green), microglia/macrophages (Iba1, red), and myelin (MBP, orange) from untreated and fingolimod-treated mice at W7–14dpi. Graphs show quantification of immunofluorescence images from corpus callosum, hippocampus, and cortex. ID = injected dose. *P ≤ 0.05. **P ≤ 0.01. ****P ≤ 0.0001.