PET analysis and quantitation

CT and PET files were co-registered and analyzed using the VivoQuant 4 (inviCRO) and Inveon Research Workspace 2.5 (Siemens) software to quantify tracer uptake in specific regions and to generate images, respectively. Brain regions of interest (ROIs) were chosen on the basis of our immunohistochemistry staining results (Fig. ​(Fig.2),2), as well as previous reports of tertiary lymphoid organs in brainstem and meninges of EAE mice [14]. For brain region quantitation, a 3D mouse brain atlas with ROIs for medulla, pons, and white matter was fitted to the PET/CT images via alignment of the atlas with the skull of each mouse (as determined by CT). Quantification of PET signal in all 15 brain regions that are part of the atlas are shown in Suppl. Fig. 3A. PET signal in the femur was quantified to assess levels of CD19⁺ B cells in bone marrow, whereby signal from bone was identified using Otsu thresholding following our previously published procedures [13], after which manual ROIs were drawn for bone marrow. Values were displayed as percentage of injected dose per gram (%ID/g), allowing comparison with gamma counting results.

B cell infiltrates are present in brainstem, meninges, and white matter of EAE mice. Representative B220 immunostaining of naïve and EAE mouse brain tissue (n = 7 EAE, n = 5 naïve mice, average of 4 slices per animal). Scale bars are 5 mm in low magnification (×1) sagittal brain images and 100 μm in high magnification (×20) images of the brainstem, meninges, and cerebellar white matter