Fig 2: CD8 T cell–mediated limbic encephalitis is associated with hippocampal T2-signal and volume increase as well as significantly increased TSPO-radiotracer uptake.(A) Representative [18F]DPA-714 PET-MRI images coregistered with the corresponding MRI of a SIINFEKL-CASAC immunized CV (left)– and OVAV (right)–injected BL6 mouse 1 week after vector-based neuronal antigen transfer (top row). T2-weighted images with volume enlargement and generalized edema (bright) as signs of inflammation in the hippocampus in the BL6-OVAV group versus the BL6-CV group. Blood remnants (dark) are detectable on the cortex surface due to bilateral intrahippocampal injection in both experimental groups. (B) The mean radiotracer uptake within the right (R) and left (L) hippocampus expressed as percentage of the injected dose per milliliter (%ID/ml) indicated a significant increase in radiotracer uptake in the BL6-OVAV-group compared to the BL6-CV group (n = 12, P = 0.003). (C) Analysis of the MRI-based relative hippocampus volume revealed slight but significant volume enlargement in the BL6-OVAV group compared to the BL6-CV group (n = 16, P = 0.009). (D) Cross-validation of [18F]DPA-714 PET imaging by TSPO immunohistochemistry. (E) The percentage of TSPO+ area was significantly higher in the BL6-OVAV group compared to the BL6-CV-group (n = 18, P < 0.001). (F) TSPO was up-regulated in Iba-1+ microglia, while minor expression was detected in GFAP-positive astrocytes in the hippocampus. Nuclei were counterstained with DAPI (blue). Statistical significance between groups was determined by Student’s t test or Mann-Whitney U test.

Fig 3: Contribution of TSPO expression on immune cells and immune cell landscape on [18F]FEPPA activity in the blood. (a) Translocator protein (TSPO) expression of various immune cells, and (b) Absolute counts of immune cells in the blood at various stages of Zika virus (ZIKV) disease. Immune cells were identified from n = 4 mice at each disease stage by flow cytometry using fluorophore-tagged antibodies for specific immune cell markers, which are shown in the legend. Data are presented as mean ± SD, and individual points represent data from individual mice. Means were compared by Mann–Whitney test. p values are displayed accordingly. *p < 0.05. (c–e) Correlation between ex vivo [18F]FEPPA activity in the blood and absolute counts of immune cells. (c) Total immune CD45+ cells, (d) myeloid cells, and (e) lymphoid cell subsets were identified by flow cytometry. Analysis for Spearman correlation (?) was performed on scatter plots with given best-fit linear regression model (R2)

Fig 4: Contribution of TSPO expression on immune cells to [18F]FEPPA uptake in whole brains during ZIKV disease. (a) Study timeline and experimental procedures performed on the acute Zika virus (ZIKV) mouse infection model. (b) Translocator protein (TSPO) expression profile of various immune cell subsets in whole brains at various stages of Zika virus (ZIKV) disease. Immune cells were identified from n = 6 mice at each disease stage by flow cytometry using fluorophore-tagged antibodies for specific immune cell markers, which are shown in the legend. Data are presented as mean ± SD, and individual points represent data from individual mice. Means were compared by Kruskal–Wallis test with Dunn’s post-hoc correction. p values are displayed accordingly. *p < 0.05, **p < 0.005. (c–f) Correlation between TSPO expression of immune cells in the brain and ex vivo [18F]FEPPA uptake in whole brains determined by gamma counting. TSPO expression of either (c) total CD45+ immune cells, (d) microglia, (e) monocytes, and (f) granulocytes isolated from whole brains was determined by flow cytometry. Data points from pre-infection are shown as circles, and those from late disease are shown in squares. Analysis for Spearman correlation (?) was performed on scatter plots with given best-fit linear regression model (R2). The 95% confidence interval of the best-fitted regression lines is shown in dashed lines

Fig 5: TSPO colocalizes with activated microglia and reactive astrocytes in seropositive and seronegative CD8 T cell–mediated ALE.(A) Overview of the TSPO expression pattern in human brain specimen derived from healthy brain, temporal lobe epilepsy without any inflammation, ALE with anti-Hu AABs, and acute and chronic ALE with anti-GAD65 AABs. In healthy brain, TSPO can be found at low levels not only in Iba-1+ microglia but also in other cell types such as endothelial cells, oligodendrocyrtes and astrocytes. Moreover, in TLE without inflammation and without neurodegeneration, Iba-1+ microglial cells are only weakly TSPO reactive. In anti-Hu ALE, TSPO is strongly up-regulated in areas with extensive T cell infiltration, especially mirroring Iba-1+ microglia activation. During acute inflammation and neurodegeneration, TSPO reflects mainly reactive gliosis in anti-GAD65 ALE. In the chronic stage of anti-GAD65 where T cell inflammation subsided, TSPO shows moderate activity in microglia and astrocytes. (B) Exemplary costaining of TSPO with Iba-1 in CD8 T cell–mediated human seropositive (anti-Hu and anti-GAD65 ALE) and seronegative specimen. In particular, in seropositive anti-Hu ALE and anti-GAD65 ALE, the presence of CD8 T cells is associated with strong TSPO reactivity in surrounding microglia. In seronegative ALE, TSPO+ microglia and astrocytes (yellow arrowhead) can be seen together with TSPO-negative microglia (red arrowhead) and TSPO-negative neurons (white arrowhead).